Sample records for cloning animals
-
Ethical issues in animal cloning.
Fiester, Autumn
2005-01-01
The issue of human reproductive cloning has recently received a great deal attention in public discourse. Bioethicists, policy makers, and the media have been quick to identify the key ethical issues involved in human reproductive cloning and to argue, almost unanimously, for an international ban on such attempts. Meanwhile, scientists have proceeded with extensive research agendas in the cloning of animals. Despite this research, there has been little public discussion of the ethical issues raised by animal cloning projects. Polling data show that the public is decidedly against the cloning of animals. To understand the public's reaction and fill the void of reasoned debate about the issue, we need to review the possible objections to animal cloning and assess the merits of the anti-animal cloning stance. Some objections to animal cloning (e.g., the impact of cloning on the population of unwanted animals) can be easily addressed, while others (e.g., the health of cloned animals) require more serious attention by the public and policy makers.
-
Cloned animal products in the human food chain: FDA should protect American consumers.
Butler, Jennifer E F
2009-01-01
Animal cloning is "complex process that lets one exactly copy the genetic, or inherited, traits of an animal." In 1997, Dolly the sheep was the first animal cloned and since then "scientists have used animal cloning to breed dairy cows, beef cattle, poultry, hogs and other species of livestock." Cloned animals are highly attractive to livestock breeders because "cloning essentially produces an identical copy of an animal with superior traits." The main purpose of cloning livestock is "more focused on efficiency and economic benefits of the producer rather than the overall effect of cloning on an animal's physical and mental welfare." The focus of this article is threefold. First, the science behind animal cloning is explained and some potential uses and risks of this technology are explored. Second, FDA's historical evolution, current regulatory authority, and limitations of that authority, is described. Lastly, a new regulatory vision recognizes the realities of 21st century global markets and the dynamic evolution of scientific discovery and technology.
-
Aging of Cloned Animals: A Mini-Review.
Burgstaller, Jörg Patrick; Brem, Gottfried
2017-01-01
The number of species for which somatic cell nuclear transfer (SCNT) protocols are established is still increasing. Due to the high number of cloned farm, companion, and sport animals, the topic of animal cloning never ceases to be of public interest. Numerous studies cover the health status of SCNT-derived animals, but very few cover the effects of SCNT on aging. However, only cloned animals that reach the full extent of the species-specific lifespan, doing so with only the normal age-related afflictions and diseases, would prove that SCNT can produce completely healthy offspring. Here, we review the available literature and own data to answer the question whether the aging process of cloned animals is qualitatively different from normal animals. We focus on 4 main factors that were proposed to influence aging in these animals: epigenetic (dys)regulation, accumulation of damaged macromolecules, shortened telomeres, and (nuclear donor-derived) age-related DNA damage. We find that at least some cloned animals can reach the species-specific maximum age with a performance that matches that of normal animals. However, for most species, only anecdotal evidence of cloned animals reaching high age is available. We therefore encourage reports on the aging of cloned animals to make further analysis on the performance of SCNT possible. © 2016 S. Karger AG, Basel.
-
Hur, Sun Jin
2017-10-01
The author widely surveyed and analyzed the food safety issues, ethical issues, permits, and approval of animal products from animals cloned by somatic cell nuclear transfer worldwide. As a result of a 2-year survey, the author found that there is no evidence that meat and milk derived from cloned animals or their progeny pose a risk to food safety in terms of genotoxicity, adverse reproductive effects, or allergic reactions. Most countries have not approved meat and milk derived from cloned animals, and their progeny are entering the food supply. To establish the guidelines, the author suggests four principles of safety assessment for meat and milk derived from cloned animals. The four main principles for safety assessment are similarities of chemical composition, adverse reproductive effects, genotoxicity, and allergic reactions under the influence of meat and milk from cloned animals and noncloned counterparts. This principle means that meat and milk derived from a cloned animal are safe if there are no differences in the four safety assessments of meat and milk between cloned animal's progeny and noncloned counterparts. Copyright © 2017 Elsevier Ltd. All rights reserved.
-
U.S. consumers attitudes toward farm animal cloning.
Brooks, Kathleen R; Lusk, Jayson L
2011-10-01
In January 2008, the United States Food and Drug Administration concluded "meat and milk from cattle, swine, and goat clones or their offspring are as safe to eat as food we eat from those species now" (U.S. FDA, 2010). However, cloning remains a very controversial topic. A web-based survey administered by Knowledge Networks was used to determine U.S. consumers' awareness of and attitudes toward meat and milk from cloned cattle. Findings reveal consumers do not differentiate much between products from cloned animals and products from non-cloned animals. Overall consumers are concerned that animal cloning is an unnatural process and that it will lead to human cloning. Copyright © 2011 Elsevier Ltd. All rights reserved.
-
Science and technology of farm animal cloning: state of the art.
Vajta, Gábor; Gjerris, Mickey
2006-05-01
Details of the first mammal born after nuclear transfer cloning were published by Steen Malte Willadsen in 1986. In spite of its enormous scientific significance, this discovery failed to trigger much public concern, possibly because the donor cells were derived from pre-implantation stage embryos. The major breakthrough in terms of public recognition has happened when Ian Wilmut et al. [Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J., Campbell, K.H., 1997. Viable offspring derived from fetal és adult mammalian cells. Nature 385, 810-813] described the successful application of almost exactly the same method, but using the nuclei of somatic cells from an adult mammal, to create Dolly the sheep. It has become theoretically possible to produce an unlimited number of genetic replicates from an adult animal or a post-implantation foetus. Since 1997 a number of different species including pigs, goats, horses, cats, etc. have been cloned with the somatic cell nuclear transfer technique. Although the technology still has relatively low success rates and there seems to be substantial problems with the welfare of some of the cloned animals, cloning is used both within basic research and the biomedical sector. The next step seems to be to implement cloning in the agricultural production system and several animals have been developed in this direction. This article reviews the current state of the art of farm animal cloning from a scientific and technological perspective, describes the animal welfare problems and critically assess different applications of farm animal cloning. The scope is confined to animal biotechnologies in which the use of cell nuclear transfer is an essential part and extends to both biomedical and agricultural applications of farm animal cloning. These applications include the production of genetically identical animals for research purposes, and also the creation of genetically modified animals. In the agricultural sector, cloning can be used as a tool within farm animal breeding. We do not intend to give an exhaustive review of the all the literature available; instead we pinpoint issues and events pivotal to the development of current farm animal cloning practices and their possible applications.
-
Aizaki, Hideo; Sawada, Manabu; Sato, Kazuo
2011-10-01
Novel food technologies, such as cloning, have been introduced into the meat production sector; however, their use is not widely supported by many consumers. This study was designed to assess whether Japanese consumers' attitudes toward consumption of cloned beef (specifically, beef derived from bovine embryo and somatic cell-cloned cattle) would change after they were provided with technological information on animal cloning through a web-based survey. The results revealed that most respondents did not discriminate between their attitudes toward the consumption of the two types of cloned beef, and that most respondents did not change their attitudes toward cloned beef after receiving the technological information. The respondents' individual characteristics, including their knowledge about the food safety of cloned beef and their basic knowledge about animal cloning, influenced the likelihood of a change in their attitudes after they received the information. In conclusion, some consumers might become less uncomfortable about the consumption of cloned beef by the straightforward provision of technological information about animal cloning; however, most consumers are likely to maintain their attitudes. Copyright © 2011 Elsevier Ltd. All rights reserved.
-
Ethical issues in livestock cloning.
Thompson, P B
1999-01-01
Although cloning may eventually become an important technology for livestock production, four ethical issues must be addressed before the practice becomes widespread. First, researchers must establish that the procedure is not detrimental to the health or well-being of affected animals. Second, animal research institutions should evaluate the net social benefits to livestock producers by weighing the benefits to producers against the opportunity cost of research capacity lost to biomedical projects. Third, scientists should consider the indirect effects of cloning research on the larger ethical issues surrounding human cloning. Finally, the market structure for products of cloned animals should protect individual choice, and should recognize that many individuals find the prospect of cloning (or consuming cloned animals) repugnant. Analysis of these four issues is complicated by spurious arguments alleging that cloning will have a negative impact on environment and genetic diversity.
-
Food consumption risks associated with animal clones: what should be investigated?
Rudenko, Larisa; Matheson, John C; Adams, Amey L; Dubbin, Eric S; Greenlees, Kevin J
2004-01-01
Somatic Cell Nuclear Transfer (SCNT), or cloning, is likely to be used for the expansion of elite breeding stock of agronomically important livestock used for food. The Center for Veterinary Medicine at the US Food and Drug Administration has been developing a risk assessment to identify hazards and characterize food consumption risks that may result from cloning. The risk assessment is comprised of two prongs. The first evaluates the health of animal clones, and is referred to as the Critical Biological Systems Approach. The second considers the composition of meat and milk from animal clones. Assessing the safety of food products from animal clones and their progeny, at least during these early stages of the development of the technology, is best accomplished by using both approaches: prospectively drawing on our knowledge of biological systems in development and maturation, and in retrograde, from an analysis of food products. Subtle hazards and potential risks that may be posed by animal clones must, however, be considered in the context of other mutations and epigenetic changes that occur in all food animal populations.
-
Animal cloning: problems and prospects.
Wells, D N
2005-04-01
An efficient animal cloning technology would provide many new opportunities for livestock agriculture, human medicine, and animal conservation. Nuclear cloning involves the production of animals that are genetically identical to the donor cells used in a technique known as nuclear transfer (NT). However, at present it is an inefficient process: in cattle, only around 6% of the embryos transferred to the reproductive tracts of recipient cows result in healthy, longterm surviving clones. Of concern are the high losses throughout gestation, during birth and in the post-natal period through to adulthood. Many of the pregnancy losses relate to failure of the placenta to develop and function correctly. Placental dysfunction may also have an adverse influence on postnatal health. These anomalies are probably due to incorrect epigenetic reprogramming of the donor genome following NT, leading to inappropriate patterns of gene expression during the development of clones. Whilst some physiological tests on surviving clones suggest normality, other reports indicate a variety of post-natal clone-associated abnormalities. This variability in outcome may reflect species-specific and/or cloning methodological differences. Importantly, to date it appears that these clone-associated phenotypes are not transmitted to offspring following sexual reproduction. This indicates that they represent epigenetic errors, rather than genetic errors, which are corrected during gametogenesis. Whilst this needs confirmation at the molecular level, it provides initial confidence in the first application of NT in agriculture, namely, the production of small numbers of cloned sires from genetically elite bulls, for natural mating, to effectively disseminate genetic gain. In addition to the animal welfare concerns with the technology, the underlying health of the animals and the consequential effect on food safety are critical aspects that require investigation to gain regulatory and consumer acceptance. Future improvements in animal cloning will largely arise from a greater understanding of the molecular mechanisms of reprogramming.
-
Proper reprogramming of imprinted and non-imprinted genes in cloned cattle gametogenesis.
Kaneda, Masahiro; Watanabe, Shinya; Akagi, Satoshi; Inaba, Yasushi; Geshi, Masaya; Nagai, Takashi
2017-11-01
Epigenetic abnormalities in cloned animals are caused by incomplete reprogramming of the donor nucleus during the nuclear transfer step (first reprogramming). However, during the second reprogramming step that occurs only in the germline cells, epigenetic errors not corrected during the first step are repaired. Consequently, epigenetic abnormalities in the somatic cells of cloned animals should be erased in their spermatozoa or oocytes. This is supported by the fact that offspring from cloned animals do not exhibit defects at birth or during postnatal development. To test this hypothesis in cloned cattle, we compared the DNA methylation level of two imprinted genes (H19 and PEG3) and three non-imprinted genes (XIST, OCT4 and NANOG) and two repetitive elements (Satellite I and Satellite II) in blood and sperm DNAs from cloned and non-cloned bulls. We found no differences between cloned and non-cloned bulls. We also analyzed the DNA methylation levels of four repetitive elements (Satellite I, Satellite II, Alpha-satellite and Art2) in oocytes recovered from cloned and non-cloned cows. Again, no significant differences were observed between clones and non-clones. These results suggested that imprinted and non-imprinted genes and repetitive elements were properly reprogramed during gametogenesis in cloned cattle; therefore, they contributed to the soundness of cloned cattle offspring. © 2017 Japanese Society of Animal Science.
-
Somatic cell nuclear transfer cloning: practical applications and current legislation.
Niemann, H; Lucas-Hahn, A
2012-08-01
Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved. © 2012 Blackwell Verlag GmbH.
-
The US FDA and animal cloning: risk and regulatory approach.
Rudenko, Larisa; Matheson, John C
2007-01-01
The Food and Drug Administration's (FDA's) Center for Veterinary Medicine issued a voluntary request to producers of livestock clones not to introduce food from clones or their progeny into commerce until the agency had assessed whether production of cattle, swine, sheep, or goats by somatic cell nuclear transfer (SCNT) posed any unique risks to the animal(s) involved in the process, humans, or other animals by consuming food from those animals, compared with any other assisted reproductive technology (ART) currently in use. Following a comprehensive review, no anomalies were observed in animals produced by cloning that have not also been observed in animals produced by other ARTs and natural mating. Further systematic review on the health of, and composition of meat and milk from, cattle, swine, and goat clones and the progeny of cattle and sheep did not result in the identification of any food-consumption hazards. The agency therefore concluded that food from cattle, swine, and goat clones was as safe to eat as food from animals of those species derived by conventional means. The agency also concluded that food from the progeny of the clone of any species normally consumed for food is as safe to eat as those animals. The article also describes the methodology used by the agency to analyze data and draw these conclusions, the plans the agency has proposed to manage any identified risks, and the risk communication approaches the agency has used.
-
Meat and milk compositions of bovine clones
Tian, X. Cindy; Kubota, Chikara; Sakashita, Kunihito; Izaike, Yoshiaki; Okano, Ryoichi; Tabara, Norio; Curchoe, Carol; Jacob, Lavina; Zhang, Yuqin; Smith, Sadie; Bormann, Charles; Xu, Jie; Sato, Masumi; Andrew, Sheila; Yang, Xiangzhong
2005-01-01
The technology is now available for commercial cloning of farm animals for food production, but is the food safe for consumers? Here, we provide data on >100 parameters that compare the composition of meat and milk from beef and dairy cattle derived from cloning to those of genetic- and breed-matched control animals from conventional reproduction. The cloned animals and the comparators were managed under the same conditions and received the same diet. The composition of the meat and milk from the clones were largely not statistically different from those of matched comparators, and all parameters examined were within the normal industry standards or previously reported values. The data generated from our match-controlled experiments provide science-based information desired by regulatory agencies to address public concerns about the safety of meat and milk from somatic animal clones. PMID:15829585
-
Yamanaka, Ken-Ichi; Kaneda, Masahiro; Inaba, Yasushi; Saito, Koji; Kubota, Kaiyu; Sakatani, Miki; Sugimura, Satoshi; Imai, Kei; Watanabe, Shinya; Takahashi, Masashi
2011-08-01
Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non-cloned or cloned dams using semen from non-cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non-cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non-cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle. 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.
-
Creating Fido's twin: can pet cloning be ethically justified?
Fiester, Autumn
2005-01-01
Taken at face value, pet cloning may seem at best a frivolous practice, costly both to the cloned pet's health and its owner's pocket. At worst, its critics say, it is misguided and unhealthy--a way of exploiting grief to the detriment of the animal, its owner, and perhaps even animal welfare in general. But if the great pains we are willing to take to clone Fido raise the status of companion animals in the public eye, then the practice might be defensible.
-
Artificial cloning of domestic animals
Keefer, Carol L.
2015-01-01
Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research. PMID:26195770
-
Artificial cloning of domestic animals.
Keefer, Carol L
2015-07-21
Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research.
-
Mehndiratta, P L; Bhalla, P
2014-09-01
Widespread use of antibiotics in human, veterinary medicine and agricultural settings has played a significant role in the emergence of resistant MRSA clones due to selection pressure. MRSA has now become established in human population as well as in various animal species. An animal associated clone, MRSA ST 398 has been reported from animal foods and also from human infections in the community as well as from the health care associated infections. Clonal relationship between strains of animal and human origins are indicators of interspecies transmission of clones. Spread of these organisms may pose a great impact on public health if animal associated strains enter into the community and health care settings. Surveillance is important to correlate the genetic changes associated with their epidemiological shift and expansion to predict its impact on public health. Strict regulations on the use of antibiotics in humans as well as in animal food production are required to control the emergence of drug resistant clones. t0 his article reviews the information available on the role of antibiotics in emergence of MRSA strains, their epidemiological shift between humans and animals and its impact on the public health.
-
Agency perspectives on food safety for the products of animal biotechnology.
Howard, H J; Jones, K M; Rudenko, L
2012-08-01
Animal biotechnology represents one subset of tools among a larger set of technologies for potential use to meet increasing world demands for food. Assisted reproductive technologies (ART) such as artificial insemination and embryo transfer continue to make positive contributions in food animal production. The US Food and Drug Administration (FDA) performed a comprehensive risk assessment to identify potential food consumption or animal health risks associated with animal cloning, an emerging ART. At that time, FDA concluded that animal cloning posed no unique risks either to animal health or to food consumption, and food from animal clones and their sexually reproduced offspring required no additional federal regulation beyond that applicable to conventionally bred animals of the species examined. At this time, no new information has arisen that would necessitate a change in FDA's conclusions on food from animal clones or their sexually reproduced offspring. Use of recombinant DNA technologies to produce genetically engineered (GE) animals represents another emerging technology with potential to impact food animal production. In its regulation of GE animals, FDA follows a cumulative, risk-based approach to address scientific questions related to the GE animals. FDA evaluates data and information on the safety, effectiveness and stability of the GE event. FDA carries out its review at several levels (e.g. molecular biology, animal safety, food safety, environmental safety and claim validation). GE animal sponsors provide data to address risk questions for each level. This manuscript discusses FDA's role in evaluation of animal cloning and GE animals. © 2012 Blackwell Verlag GmbH.
-
Metabolomic phenotyping of a cloned pig model
2011-01-01
Background Pigs are widely used as models for human physiological changes in intervention studies, because of the close resemblance between human and porcine physiology and the high degree of experimental control when using an animal model. Cloned animals have, in principle, identical genotypes and possibly also phenotypes and this offer an extra level of experimental control which could possibly make them a desirable tool for intervention studies. Therefore, in the present study, we address how phenotype and phenotypic variation is affected by cloning, through comparison of cloned pigs and normal outbred pigs. Results The metabolic phenotype of cloned pigs (n = 5) was for the first time elucidated by nuclear magnetic resonance (NMR)-based metabolomic analysis of multiple bio-fluids including plasma, bile and urine. The metabolic phenotype of the cloned pigs was compared with normal outbred pigs (n = 6) by multivariate data analysis, which revealed differences in the metabolic phenotypes. Plasma lactate was higher for cloned vs control pigs, while multiple metabolites were altered in the bile. However a lower inter-individual variability for cloned pigs compared with control pigs could not be established. Conclusions From the present study we conclude that cloned and normal outbred pigs are phenotypically different. However, it cannot be concluded that the use of cloned animals will reduce the inter-individual variation in intervention studies, though this is based on a limited number of animals. PMID:21859467
-
Makoschey, Birgit; van Kilsdonk, Emma; Hubers, Willem R; Vrijenhoek, Mieke P; Smit, Marianne; Wichgers Schreur, Paul J; Kortekaas, Jeroen; Moulin, Véronique
2016-03-01
Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that affects domesticated ruminants and occasionally humans. Classical RVF vaccines are based on formalin-inactivated virus or the live-attenuated Smithburn strain. The inactivated vaccine is highly safe but requires multiple administrations and yearly re-vaccinations. Although the Smithburn vaccine provides solid protection after a single vaccination, this vaccine is not safe for pregnant animals. An alternative live-attenuated vaccine, named Clone 13, carries a large natural deletion in the NSs gene which encodes the major virulence factor of the virus. The Clone 13 vaccine was previously shown to be safe for young lambs and calves. Moreover, a study in pregnant ewes suggested that the vaccine could also be applied safely during gestation. To anticipate on a possible future incursion of RVFV in Europe, we have evaluated the safety of Clone 13 for young lambs and pregnant ewes. In line with the guidelines from the World Organisation for Animal health (Office International des Epizooties, OIE) and regulations of the European Pharmacopeia (EP), these studies were performed with an overdose. Our studies with lambs showed that Clone 13 dissemination within vaccinated animals is very limited. Moreover, the Clone 13 vaccine virus was not shed nor spread to in-contact sentinels and did not revert to virulence upon animal-to-animal passage. Importantly, a large experiment with pregnant ewes demonstrated that the Clone 13 virus is able to spread to the fetus, resulting in malformations and stillbirths. Altogether, our results suggest that Clone 13 can be applied safely in lambs, but that caution should be taken when Clone 13 is used in pregnant animals, particularly during the first trimester of gestation.
-
Cloning of endangered mammalian species: any progress?
Loi, Pasqualino; Galli, Cesare; Ptak, Grazyna
2007-05-01
Attempts through somatic cell nuclear transfer to expand wild populations that have shrunk to critical numbers is a logical extension of the successful cloning of mammals. However, although the first mammal was cloned 10 years ago, nuclear reprogramming remains phenomenological, with abnormal gene expression and epigenetic deregulation being associated with the cloning process. In addition, although cloning of wild animals using host oocytes from different species has been successful, little is known about the implication of partial or total mitochondrial DNA heteroplasmy in cloned embryos, fetuses and offspring. Finally, there is a need for suitable foster mothers for inter-intra specific cloned embryos. Considering these issues, the limited success achieved in cloning endangered animals is not surprising. However, optimism comes from the rapid gain in the understanding of the molecular clues underlying nuclear reprogramming. If it is possible to achieve a controlled reversal of the differentiated state of a cell then it is probable that other issues that impair the cloning of endangered animals, such as the inter-intra species oocyte or womb donor, will be overcome in the medium term.
-
Coulon, M; Baudoin, C; Abdi, H; Heyman, Y; Deputte, B L
2010-12-01
For more than ten years, reproductive biotechnologies using somatic cell nuclear transfer have made possible the production of cloned animals in various domestic and laboratory species. The influence of the cloning process on offspring characteristics has been studied in various developmental aspects, however, it has not yet been documented in detail for behavioral traits. Behavioral studies of cloned animals have failed to show clear inter-individual differences associated with the cloning process. Preliminary results showed that clones favor each other's company. Preferential social interactions were observed among cloned heifers from the same donor in a mixed herd that also included cloned heifers and control heifers produced by artificial insemination (AI). These results suggest behavioral differences between cloned and non-cloned animals and similarities between clones from the same donor. The aim of the present study was to replicate and to extend these previous results and to study behavioral and cognitive mechanisms of this preferential grouping. We studied a group composed of five cloned heifers derived from the same donor cow, two cloned heifers derived from another donor cow, and AI heifers. Cloned heifers from the same donor were more spatially associated and interacted more between themselves than with heifers derived from another donor or with the AI individuals. This pattern indicates a possible kin discrimination in clones. To study this process, we performed an experiment (using an instrumental conditioning procedure with food reward) of visual discrimination between images of heads of familiar heifers, either related to the subjects or not. The results showed that all subjects (AI and cloned heifers) discriminated between images of familiar cloned heifers produced from the same donor and images of familiar unrelated heifers. Cattle discriminated well between images and used morphological similarities characteristic of cloned related heifers. Our results suggest similar cognitive capacities of kin and non kin discrimination in AI and cloned animals. Kinship may be a common factor in determining the social grouping within a herd. Copyright © 2010 Elsevier Inc. All rights reserved.
-
Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells.
Mizutani, Eiji; Torikai, Kohei; Wakayama, Sayaka; Nagatomo, Hiroaki; Ohinata, Yasuhide; Kishigami, Satoshi; Wakayama, Teruhiko
2016-04-01
Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38-77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal.
-
Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells
Mizutani, Eiji; Torikai, Kohei; Wakayama, Sayaka; Nagatomo, Hiroaki; Ohinata, Yasuhide; Kishigami, Satoshi; Wakayama, Teruhiko
2016-01-01
Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38–77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal. PMID:27033801
-
Transplantation and differentiation of donor cells in the cloned pigs
DOE Office of Scientific and Technical Information (OSTI.GOV)
Shimada, Arata; Tomii, Ryo; Kano, Koichiro
2006-06-02
The application of nuclear transfer technology is an interesting approach to investigate stem and progenitor cell transplantation therapy. If stem cells are used as a nuclear donor, donor cells can engraft into cloned animals without histocompatible problems. However, it is still uncertain whether donor cells can engraft to cloned animal and differentiate in vivo. To address this problem, we transplanted donor cells to dermal tissues of cloned pigs developed by using preadipocytes as donor cells. Preadipocytes are adipocytic progenitor which can differentiate to mature adipocytes in vitro. We showed that the donor preadipocytes were successfully transplanted into the cloned pigsmore » without immune rejection and they differentiated into mature adipocytes in vivo 3 weeks after transplantation. In contrast, allogenic control preadipocytes, which can differentiate in vitro, did not differentiate in vivo. These results indicate that donor progenitor cells can differentiate in cloned animal.« less
-
Agro-economic impact of cattle cloning.
Faber, D C; Ferre, L B; Metzger, J; Robl, J M; Kasinathan, P
2004-01-01
The purpose of this paper is to review the economic and social implications of cloned cattle, their products, and their offspring as related to production agriculture. Cloning technology in cattle has several applications outside of traditional production agriculture. These applications can include bio-medical applications, such as the production of pharmaceuticals in the blood or milk of transgenic cattle. Cloning may also be useful in the production of research models. These models may or may not include genetic modifications. Uses in agriculture include many applications of the technology. These include making genetic copies of elite seed stock and prize winning show cattle. Other purposes may range from "insurance" to making copies of cattle that have sentimental value, similar to cloning of pets. Increased selection opportunities available with cloning may provide for improvement in genetic gain. The ultimate goal of cloning has often been envisioned as a system for producing quantity and uniformity of the perfect dairy cow. However, only if heritability were 100%, would clone mates have complete uniformity. Changes in the environment may have significant impact on the productivity and longevity of the resulting clones. Changes in consumer preferences and economic input costs may all change the definition of the perfect cow. The cost of producing such animals via cloning must be economically feasible to meet the intended applications. Present inefficiencies limit cloning opportunities to highly valued animals. Improvements are necessary to move the applications toward commercial application. Cloning has additional obstacles to conquer. Social and regulatory acceptance of cloning is paramount to its utilization in production agriculture. Regulatory acceptance will need to address the animal, its products, and its offspring. In summary, cloning is another tool in the animal biotechnology toolbox, which includes artificial insemination, sexing of semen, embryo sexing and in vitro fertilization. While it will not replace any of the above mentioned, its degree of utilization will depend on both improvement in efficiency as well as social and regulatory acceptance.
-
Wang, Zhongde
2011-01-01
Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.
-
Specific genetic modifications of domestic animals by gene targeting and animal cloning
Wang, Bin; Zhou, Jiangfeng
2003-01-01
The technology of gene targeting through homologous recombination has been extremely useful for elucidating gene functions in mice. The application of this technology was thought impossible in the large livestock species until the successful creation of the first mammalian clone "Dolly" the sheep. The combination of the technologies for gene targeting of somatic cells with those of animal cloning made it possible to introduce specific genetic mutations into domestic animals. In this review, the principles of gene targeting in somatic cells and the challenges of nuclear transfer using gene-targeted cells are discussed. The relevance of gene targeting in domestic animals for applications in bio-medicine and agriculture are also examined. PMID:14614774
-
Rødgaard, Tina; Skovgaard, Kerstin; Stagsted, Jan; Heegaard, Peter M H
2013-06-01
The objective of this study was to evaluate the usefulness of cloned pigs as porcine obesity models reflecting obesity-associated changes in innate immune factor gene expression profiles. Liver and adipose tissue expression of 43 innate immune genes as well as serum concentrations of six immune factors were analyzed in lean and diet-induced obese cloned domestic pigs and compared to normal domestic pigs (obese and lean). The number of genes affected by obesity was lower in cloned animals than in control animals. All genes affected by obesity in adipose tissues of clones were downregulated; both upregulation and downregulation were observed in the controls. Cloning resulted in a less differentiated adipose tissue expression pattern. Finally, the serum concentrations of two acute-phase proteins (APPs), haptoglobin (HP) and orosomucoid (ORM), were increased in obese clones as compared to obese controls as well as lean clones and controls. Generally, the variation in phenotype between individual pigs was not reduced in cloned siblings as compared to normal siblings. Therefore, we conclude that cloning limits both the number of genes responding to obesity as well as the degree of tissue-differentiated gene expression, concomitantly with an increase in APP serum concentrations only seen in cloned, obese pigs. This may suggest that the APP response seen in obese, cloned pigs is a consequence of the characteristic skewed gene response to obesity in cloned pigs, as described in this work. This should be taken into consideration when using cloned animals as models for innate responses to obesity.
-
Rødgaard, Tina; Skovgaard, Kerstin; Stagsted, Jan
2013-01-01
Abstract The objective of this study was to evaluate the usefulness of cloned pigs as porcine obesity models reflecting obesity-associated changes in innate immune factor gene expression profiles. Liver and adipose tissue expression of 43 innate immune genes as well as serum concentrations of six immune factors were analyzed in lean and diet-induced obese cloned domestic pigs and compared to normal domestic pigs (obese and lean). The number of genes affected by obesity was lower in cloned animals than in control animals. All genes affected by obesity in adipose tissues of clones were downregulated; both upregulation and downregulation were observed in the controls. Cloning resulted in a less differentiated adipose tissue expression pattern. Finally, the serum concentrations of two acute-phase proteins (APPs), haptoglobin (HP) and orosomucoid (ORM), were increased in obese clones as compared to obese controls as well as lean clones and controls. Generally, the variation in phenotype between individual pigs was not reduced in cloned siblings as compared to normal siblings. Therefore, we conclude that cloning limits both the number of genes responding to obesity as well as the degree of tissue-differentiated gene expression, concomitantly with an increase in APP serum concentrations only seen in cloned, obese pigs. This may suggest that the APP response seen in obese, cloned pigs is a consequence of the characteristic skewed gene response to obesity in cloned pigs, as described in this work. This should be taken into consideration when using cloned animals as models for innate responses to obesity. PMID:23668862
-
[Nuclear transfer and therapeutic cloning].
Xu, Xiao-Ming; Lei, An-Min; Hua, Jin-Lian; Dou, Zhong-Ying
2005-03-01
Nuclear transfer and therapeutic cloning have widespread and attractive prospects in animal agriculture and biomedical applications. We reviewed that the quality of oocytes and nuclear reprogramming of somatic donor cells were the main reasons of the common abnormalities in cloned animals and the low efficiency of cloning and showed the problems and outlets in therapeutic cloning, such as some basic problems in nuclear transfer affected clinical applications of therapeutic cloning. Study on isolation and culture of nuclear transfer embryonic stem (ntES) cells and specific differentiation of ntES cells into important functional cells should be emphasized and could enhance the efficiency. Adult stem cells could help to cure some great diseases, but could not replace therapeutic cloning. Ethics also impeded the development of therapeutic cloning. It is necessary to improve many techniques and reinforce the research of some basic theories, then somatic nuclear transfer and therapeutic cloning may apply to agriculture reproduction and benefit to human life better.
-
Factors influencing the commercialisation of cloning in the pork industry.
Pratt, S L; Sherrer, E S; Reeves, D E; Stice, S L
2006-01-01
Production of cloned pigs using somatic cell nuclear transfer (SCNT) is a repeatable and predictable procedure and multiple labs around the world have generated cloned pigs and genetically modified cloned pigs. Due to the integrated nature of the pork production industry, pork producers are the most likely to benefit and are in the best position to introduce cloning in to production systems. Cloning can be used to amplify superior genetics or be used in conjunction with genetic modifications to produce animals with superior economic traits. Though unproven, cloning could add value by reducing pig-to-pig variability in economically significant traits such as growth rate, feed efficiency, and carcass characteristics. However, cloning efficiencies using SCNT are low, but predictable. The inefficiencies are due to the intrusive nature of the procedure, the quality of oocytes and/or the somatic cells used in the procedure, the quality of the nuclear transfer embryos transferred into recipients, pregnancy rates of the recipients, and neonatal survival of the clones. Furthermore, in commercial animal agriculture, clones produced must be able to grow and thrive under normal management conditions, which include attainment of puberty and subsequent capability to reproduce. To integrate SCNT into the pork industry, inefficiencies at each step of the procedure must be overcome. In addition, it is likely that non-surgical embryo transfer will be required to deliver cloned embryos, and/or additional methods to generate high health clones will need to be developed. This review will focus on the state-of-the-art for SCNT in pigs and the steps required for practical implementation of pig cloning in animal agriculture.
-
Cloning: Past, Present, and the Exciting Future. Breakthroughs in Bioscience.
ERIC Educational Resources Information Center
Di Berardino, Marie A.
This document explores the history of cloning by focusing on Dolly the Sheep, one of the first large animal clonings. The disadvantages and advantages of transgenic clones are discussed as well as the future implications of cloning from the perspective of human health. (Contains 10 resources.) (YDS)
-
Cloned cows with short telomeres deliver healthy offspring with normal-length telomeres.
Miyashita, Norikazu; Kubo, Yasuaki; Yonai, Miharu; Kaneyama, Kanako; Saito, Norio; Sawai, Ken; Minamihashi, Akira; Suzuki, Toshiyuki; Kojima, Toshiyuki; Nagai, Takashi
2011-10-01
Dolly, the first mammal cloned from a somatic cell, had shorter telomeres than age-matched controls and died at an early age because of disease. To investigate longevity and lifetime performance in cloned animals, we produced cloned cows with short telomeres using oviductal epithelial cells as donor cells. At 5 years of age, despite the presence of short telomeres, all cloned cows delivered multiple healthy offspring following artificial insemination with conventionally processed spermatozoa from noncloned bulls, and their milk production was comparable to that of donor cows. Moreover, this study revealed that the offspring had normal-length telomeres in their leukocytes and major organs. Thus, cloned animals have normal functional germ lines, and therefore germ line function can completely restore telomere lengths in clone gametes by telomerase activity, resulting in healthy offspring with normal-length telomeres.
-
ERIC Educational Resources Information Center
Meghani, Zahra; de Melo-Martin, Inmaculada
2009-01-01
The U.S. Food and Drug Administration (FDA) announced recently that food products derived from some animal clones and their offspring are safe for human consumption. In response to criticism that it had failed to engage with ethical, social, and economic concerns raised by livestock cloning, the FDA argued that addressing normative issues prior to…
-
... have been cloned already, including two relatives of cattle called the guar and the banteng, mouflon sheep, ... are underway to clone agricultural animals, such as cattle and pigs, that are efficient producers of high- ...
-
Animal cloning by somatic cell nuclear transfer.
Smith, Lawrence C; Yoo, Jae-Gyu
2009-01-01
Animal cloning is becoming increasingly useful for its applications in biological inquiry and for its potential use in pharmaceutical, medical, and agricultural fields. Due to the complexity of the numerous steps required in reconstructing oocytes by nuclear transfer, detailed protocols are required to minimize the developmental damages inflicted during these manipulations and to standardize procedures across laboratories. Moreover, because oogenesis and early embryogenesis differ widely among mammalian species, it is essential that protocols be adapted according to each species concerned. Our objective here is to detail the protocols that have been most successful in producing laboratory and domestic animal clones.
-
Benefits and problems with cloning animals.
Smith, L C; Bordignon, V; Babkine, M; Fecteau, G; Keefer, C
2000-01-01
Animal cloning is becoming a useful technique for producing transgenic farm animals and is likely to be used to produce clones from valuable adults. Other applications will also undoubtedly be discovered in the near future, such as for preserving endangered breeds and species. Although cloning promises great advantages for commerce and research alike, its outcome is not always certain due to high pregnancy losses and high morbidity and mortality during the neonatal period. Research into the mechanisms involved in the reprogramming of the nucleus is being conducted throughout the world in an attempt to better understand the molecular and cellular mechanisms involved in correcting these problems. Although the cause of these anomalies remains mostly unknown, similar phenotypes have been observed in calves derived through in vitro fertilization, suggesting that culture conditions are involved in these phenomena. In the meantime, veterinarians and theriogenologists have an important role to play in improving the efficiency of cloning by finding treatments to assure normal gestation to term and to develop preventative and curative care for cloned neonates. Images Figure 1. PMID:11143925
-
Cloning cattle: the methods in the madness.
Oback, Björn; Wells, David N
2007-01-01
Somatic cell nuclear transfer (SCNT) is much more widely and efficiently practiced in cattle than in any other species, making this arguably the most important mammal cloned to date. While the initial objective behind cattle cloning was commercially driven--in particular to multiply genetically superior animals with desired phenotypic traits and to produce genetically modified animals-researchers have now started to use bovine SCNT as a tool to address diverse questions in developmental and cell biology. In this paper, we review current cattle cloning methodologies and their potential technical or biological pitfalls at any step of the procedure. In doing so, we focus on one methodological parameter, namely donor cell selection. We emphasize the impact of epigenetic and genetic differences between embryonic, germ, and somatic donor cell types on cloning efficiency. Lastly, we discuss adult phenotypes and fitness of cloned cattle and their offspring and illustrate some of the more imminent commercial cattle cloning applications.
-
[Product safety analysis of somatic cell cloned bovine].
Hua, Song; Lan, Jie; Song, Yongli; Lu, Chenglong; Zhang, Yong
2010-05-01
Somatic cell cloning (nuclear transfer) is a technique through which the nucleus (DNA) of a somatic cell is transferred into an enucleated oocyte for the generation of a new individual, genetically identical to the somatic cell donor. It could be applied for the enhancement of reproduction rate and the improvement of food products involving quality, yield and nutrition. In recent years, the United States, Japan and Europe as well as other countries announced that meat and milk products made from cloned cattle are safe for human consumption. Yet, cloned animals are faced with a wide range of health problems, with a high death rate and a high incidence of disease. The precise causal mechanisms for the low efficiency of cloning remain unclear. Is it safe that any products from cloned animals were allowed into the food supply? This review focuses on the security of meat, milk and products from cloned cattle based on the available data.
-
Somatic cell nuclear transfer: infinite reproduction of a unique diploid genome.
Kishigami, Satoshi; Wakayama, Sayaka; Hosoi, Yoshihiko; Iritani, Akira; Wakayama, Teruhiko
2008-06-10
In mammals, a diploid genome of an individual following fertilization of an egg and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual ending. Even as cultured cells from the individual, they cannot normally proliferate in perpetuity because of the "Hayflick limit". However, Dolly, the sheep cloned from an adult mammary gland cell, changes this scenario. Somatic cell nuclear transfer (SCNT) enables us to produce offspring without germ cells, that is, to "passage" a unique diploid genome. Animal cloning has also proven to be a powerful research tool for reprogramming in many mammals, notably mouse and cow. The mechanism underlying reprogramming, however, remains largely unknown and, animal cloning has been inefficient as a result. More momentously, in addition to abortion and fetal mortality, some cloned animals display possible premature aging phenotypes including early death and short telomere lengths. Under these inauspicious conditions, is it really possible for SCNT to preserve a diploid genome? Delightfully, in mouse and recently in primate, using SCNT we can produce nuclear transfer ES cells (ntES) more efficiently, which can preserve the eternal lifespan for the "passage" of a unique diploid genome. Further, new somatic cloning technique using histone-deacetylase inhibitors has been developed which can significantly increase the previous cloning rates two to six times. Here, we introduce SCNT and its value as a preservation tool for a diploid genome while reviewing aging of cloned animals on cellular and individual levels.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kishigami, Satoshi; Kinki University, 930 Nishimitani, Kinokawa 599-5993; Wakayama, Sayaka
In mammals, a diploid genome of an individual following fertilization of an egg and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual ending. Even as cultured cells from the individual, they cannot normally proliferate in perpetuity because of the 'Hayflick limit'. However, Dolly, the sheep cloned from an adult mammary gland cell, changes this scenario. Somatic cell nuclear transfer (SCNT) enables us to produce offspring without germ cells, that is, to 'passage' a unique diploid genome. Animal cloning has also proven to be a powerful research tool for reprogrammingmore » in many mammals, notably mouse and cow. The mechanism underlying reprogramming, however, remains largely unknown and, animal cloning has been inefficient as a result. More momentously, in addition to abortion and fetal mortality, some cloned animals display possible premature aging phenotypes including early death and short telomere lengths. Under these inauspicious conditions, is it really possible for SCNT to preserve a diploid genome? Delightfully, in mouse and recently in primate, using SCNT we can produce nuclear transfer ES cells (ntES) more efficiently, which can preserve the eternal lifespan for the 'passage' of a unique diploid genome. Further, new somatic cloning technique using histone-deacetylase inhibitors has been developed which can significantly increase the previous cloning rates two to six times. Here, we introduce SCNT and its value as a preservation tool for a diploid genome while reviewing aging of cloned animals on cellular and individual levels.« less
-
Cloning of aged animals: a medical model for tissue and organ regeneration.
Tian, X C; Kubota, C; Yang, X
2001-11-01
Cloning by nuclear transfer has great potential application in pharmaceutical protein production, xeno-transplantation, and perhaps most excitingly, therapeutic cloning. In therapeutic cloning a patient's own skin cells can be used to generate cloned embryos from which embryonic stem cells are isolated. Through targeted differentiation, embryonic stem cells can be directed to develop into the desired tissues/organs for replacement. The combination of homologous recombination of genes and nuclear transfer also offers the promise of correcting defective genes in humans. Demonstration of the successful cloning of aged animals is important for these future medical applications because degenerative diseases often afflict older adults. Our studies have demonstrated that skin fibroblast cells from aged adults, even after prolonged culture, provide nuclear donors equally as competent for cloning as cells from young adults or fetuses. These findings have paved the way for medically treating degenerative diseases of aged humans by tissue regeneration technologies made possible through cloning and homologous recombination.
-
Animal Cloning and Food Safety
... concluded that meat and milk from clones of cattle, swine (pigs), and goats, and the offspring of ... natural mating the composition of food products from cattle, swine, and goat clones, or the offspring of ...
-
Healy, David L; Weston, Gareth; Pera, Martin F; Rombauts, Luk; Trounson, Alan O
2002-05-01
This review summaries human cloning from a clinical perspective. Natural human clones, that is, monozygotic twins, are increasing in the general community. Iatrogenic human clones have been produced for decades in infertile couples given fertility treatment such as ovulation induction. A clear distinction must be made between therapeutic cloning using embryonic stem cells and reproductive cloning attempts. Unlike the early clinical years of in vitro fertilization, with cloning there is no animal model that is safe and dependable. Until there is such a model, 'Dolly'-style human cloning is medically unacceptable.
-
Pedersen, Rebecca; Andersen, Anders Daniel; Mølbak, Lars; Stagsted, Jan; Boye, Mette
2013-02-07
Obesity induced by a high-caloric diet has previously been associated with changes in the gut microbiota in mice and in humans. In this study, pigs were cloned to minimize genetic and biological variation among the animals with the aim of developing a controlled metabolomic model suitable for a diet-intervention study. Cloning of pigs may be an attractive way to reduce genetic influences when investigating the effect of diet and obesity on different physiological sites. The aim of this study was to assess and compare the changes in the composition of the gut microbiota of cloned vs. non-cloned pigs during development of obesity by a high-fat/high-caloric diet. Furthermore, we investigated the association between diet-induced obesity and the relative abundance of the phyla Firmicutes and Bacteroidetes in the fecal-microbiota. The fecal microbiota from obese cloned (n = 5) and non-cloned control pigs (n= 6) was investigated biweekly over a period of 136 days, by terminal restriction fragment length polymorphism (T-RFLP) and quantitative real time PCR (qPCR). A positive correlation was observed between body-weight at endpoint and percent body-fat in cloned (r=0.9, P<0.0001) and in non-cloned control pigs (r=0.9, P<0.0001). Shannon Weaver and principal component analysis (PCA) of the terminal restriction fragments (T-RFs) revealed no differences in the bacterial composition or variability of the fecal microbiota between the cloned pigs or between cloned and non-cloned control pigs. Body-weight correlated positively with the relative abundance of Firmicutes in both cloned (r=0.37; P<0.02) and non cloned-control pigs (r=0.45; P<0.006), and negatively with the abundance of Bacteroidetes in cloned pigs (r=-0.33, P<0.04), but not in the non-cloned control pigs. The cloned pigs did not have reduced inter-individual variation as compared to non-cloned pigs in regard to their gut microbiota in neither the obese nor the lean state. Diet-induced obesity was associated with an increase in the relative abundance of Firmicutes over time. Our results suggest that cloned pigs are not a more suitable animal model for gut microbiota-obesity related studies than non-cloned pigs. This study is the first to evaluate if cloned pigs provide a better animal model than conventional pigs in diet-intervention, obesity and gut microbiota research.
-
Learning, memory and exploratory similarities in genetically identical cloned dogs.
Shin, Chi Won; Kim, Geon A; Park, Won Jun; Park, Kwan Yong; Jeon, Jeong Min; Oh, Hyun Ju; Kim, Min Jung; Lee, Byeong Chun
2016-12-30
Somatic cell nuclear transfer allows generation of genetically identical animals using donor cells derived from animals with particular traits. To date, few studies have investigated whether or not these cloned dogs will show identical behavior patterns. To address this question, learning, memory and exploratory patterns were examined using six cloned dogs with identical nuclear genomes. The variance of total incorrect choice number in the Y-maze test among cloned dogs was significantly lower than that of the control dogs. There was also a significant decrease in variance in the level of exploratory activity in the open fields test compared to age-matched control dogs. These results indicate that cloned dogs show similar cognitive and exploratory patterns, suggesting that these behavioral phenotypes are related to the genotypes of the individuals.
-
Hoshino, Yoichiro; Hayashi, Noboru; Taniguchi, Shunji; Kobayashi, Naohiko; Sakai, Kenji; Otani, Tsuyoshi; Iritani, Akira; Saeki, Kazuhiro
2009-01-01
Frozen animal tissues without cryoprotectant have been thought to be inappropriate for use as a nuclear donor for somatic cell nuclear transfer (SCNT). We report the cloning of a bull using cells retrieved from testicles that had been taken from a dead animal and frozen without cryoprotectant in a -80 degrees C freezer for 10 years. We obtained live cells from defrosted pieces of the spermatic cords of frozen testicles. The cells proliferated actively in culture and were apparently normal. We transferred 16 SCNT embryos from these cells into 16 synchronized recipient animals. We obtained five pregnancies and four cloned calves developed to term. Our results indicate that complete genome sets are maintained in mammalian organs even after long-term frozen-storage without cryoprotectant, and that live clones can be produced from the recovered cells.
-
Hoshino, Yoichiro; Hayashi, Noboru; Taniguchi, Shunji; Kobayashi, Naohiko; Sakai, Kenji; Otani, Tsuyoshi; Iritani, Akira; Saeki, Kazuhiro
2009-01-01
Frozen animal tissues without cryoprotectant have been thought to be inappropriate for use as a nuclear donor for somatic cell nuclear transfer (SCNT). We report the cloning of a bull using cells retrieved from testicles that had been taken from a dead animal and frozen without cryoprotectant in a −80°C freezer for 10 years. We obtained live cells from defrosted pieces of the spermatic cords of frozen testicles. The cells proliferated actively in culture and were apparently normal. We transferred 16 SCNT embryos from these cells into 16 synchronized recipient animals. We obtained five pregnancies and four cloned calves developed to term. Our results indicate that complete genome sets are maintained in mammalian organs even after long-term frozen-storage without cryoprotectant, and that live clones can be produced from the recovered cells. PMID:19129919
-
Recent progress and problems in animal cloning.
Tsunoda, Y; Kato, Y
2002-01-01
It is remarkable that mammalian somatic cell nuclei can form whole individuals if they are transferred to enucleated oocytes. Advancements in nuclear transfer technology can now be applied for genetic improvement and increase of farm animals, rescue of endangered species, and assisted reproduction and tissue engineering in humans. Since July 1998, more than 200 calves have been produced by nuclear transfer of somatic cell nuclei in Japan, but half of them were stillborn or died within several months of parturition. Morphologic abnormalities have also been observed in cloned calves and embryonic stem cell-derived mice. In this review, we discuss the present situation and problems with animal cloning and the possibility for its application to human medicine.
-
Incidence of abnormal offspring from cloning and other assisted reproductive technologies.
Hill, Jonathan R
2014-02-01
In animals produced by assisted reproductive technologies, two abnormal phenotypes have been characterized. Large offspring syndrome (LOS) occurs in offspring derived from in vitro cultured embryos, and the abnormal clone phenotype includes placental and fetal changes. LOS is readily apparent in ruminants, where a large calf or lamb derived from in vitro embryo production or cloning may weigh up to twice the expected body weight. The incidence of LOS varies widely between species. When similar embryo culture conditions are applied to nonruminant species, LOS either is not as dramatic or may even be unapparent. Coculture with serum and somatic cells was identified in the 1990s as a risk factor for abnormal development of ruminant pregnancies. Animals cloned from somatic cells may display a combination of fetal and placental abnormalities that are manifested at different stages of pregnancy and postnatally. In highly interventional technologies, such as nuclear transfer (cloning), the incidence of abnormal offspring continues to be a limiting factor to broader application of the technique. This review details the breadth of phenotypes found in nonviable pregnancies, together with the phenotypes of animals that survive the transition to extrauterine life. The focus is on animals produced using in vitro embryo culture and nuclear transfer in comparison to naturally occurring phenotypes.
-
Claxton, John; Sachez, Elena; Matthiessen-Guyader, Line
2004-01-01
Cloned livestock have potential importance in the provision of improved medicine as well as in the development of livestock production. The public is, however, increasingly concerned about the social and ethical consequences of these advances in knowledge and techniques. There is unevenness throughout Europe in different Member States' attitudes to research into livestock cloning. Although there is EU legislation controlling the use of animals for research purposes, there is no legislation specifically governing cloning in livestock production. The main EU reference is the 9th Opinion of the European Group on Ethics, which states "Cloning of farm animals may prove to be of medical and agricultural as well as economic benefit. It is acceptable only when the aims and methods are ethically justified and when carried out under ethical conditions." The ethical justification includes the avoidance of suffering, the use of the 3Rs principle and a lack of better alternatives. The Commission addresses these issues in the 6th Framework Programme by promoting the integration of ethical, legal and social aspects in all proposals where they are relevant, by fostering ethical awareness and foresight in the proposals, by encouraging public dialogue, and by supporting specific actions to promote the debate. Research must respect fundamental ethical principles, including animal welfare requirements.
-
Regulation of animal biotechnology: research needs.
Rexroad, C E; Green, R D; Wall, R J
2007-09-01
Livestock that result from biotechnology have been a part of agricultural science for over 30 years but have not entered the market place as food or fiber. Two biotechnologies are at the forefront as challenges to the world's systems for regulating the market place: animal clones and transgenic animals. Both technologies have come before the Food and Drug Administration in the United States and it appears that action is imminent for clones. The FDA has asserted principles for evaluation of clones and asserts that "... remaining hazard(s) from cloning are likely to be subtle in nature." The science-based principles recognize that in some areas related to developmental biology and gene expression in clones, additional scientific information would be useful. The role of science then is to use the genomic tools that we have available to answer questions about epigenetic regulation of development and reprogramming of genes to the state found in germ cells. Transgenics pose additional challenges to regulators. If the transgenics are produced using cloning from modified cells then the additional scientific information needed will be related to the effects of insertion and expression of the transgenes. Other approaches such as retrovirally vectored transgenesis will elicit additional questions. These questions will be challenging because the science will have to be related to the expression and function of each gene or class of genes. For the promises of animal biotechnology to be fulfilled, scientists will have to resolve many questions for regulators and the public but tools to answer those questions are rapidly becoming available.
-
Genomics and Ethics: The Case of Cloned and/or Transgenic Animals
2003-01-01
The point of the present study is to illustrate and, if possible, promote the existing link between genomics and ethics, taking the example of cloned and transgenic animals. These ‘new animals’ raise theoretical and practical problems that concern applied ethics. We will explore more particularly an original strategy showing that it is possible, starting from philosophical questioning about the nature of identity, to use a genomic approach, based on amplification fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) detection, to provide useful tools to define more rigorously what cloned animals are, by testing their genetic and epigenetic identity. We expect from the future results of this combined approach to stimulate the creativity of the philosophical and ethical reflection about the impact of biotechnology on animals, and to increase scientific involvement in such issues. PMID:18629111
-
Analysis of an epigenetic argument against human reproductive cloning.
Nordgren, Anders
2006-08-01
Human reproductive cloning is a much disputed ethical issue. This technology is often condemned as being contrary to human dignity. However, there are also risk arguments. An ethical argument that is often put forward by scientists but seldom developed in more detail focuses on health risks in animal cloning. There is a high risk that animal clones exhibit abnormalities and these are increasingly believed to be due to errors in epigenetic reprogramming. The argument is that human reproductive cloning should not be carried out because human clones are also likely to exhibit abnormalities due to inappropriate epigenetic reprogramming. Different versions of this epigenetic argument are analysed, a categorical version and a non-categorical. The non-categorical version is suggested to be more well-considered. With regard to policy making on human reproductive cloning, the categorical version can be used to prescribe a permanent ban, while the non-categorical version can be used to prescribe a temporary ban. The implications of the precautionary principle--as interpreted in the European Union--are investigated. The conclusion is that it seems possible to support a temporary ban by reference to this principle.
-
Revel, M
2000-07-01
Reproduction by cloning has been achieved by transfer into enucleated oocytes of nuclei from embryonic cells and more recently from cells of adult animals. The efficiency at which embryos produced by such nuclear transfers will develop into healthy newborns is very low but has succeeded in producing some cloned bovines, ovines and mice. Since the first report of sheep cloning from an adult cell in 1997, the potential applications of reproductive cloning in human medicine have been envisaged amidst a flurry of moral debates. Although the technology is still far from being ready for any human use, it has been condemned up front. It has also led to irrational fantasies and fears, based mainly on the misconception that genetic identity means identical twin personalities. Scientific research is ongoing to refine the cloning technology for applications in the production of genetically homogeneous farm animals with useful nutritional or therapeutic genetic traits. A new area of research is non-reproductive therapeutic cloning for the purpose of producing autologous embryonic cells and tissues for transplantation.
-
Production of healthy cloned mice from bodies frozen at -20 degrees C for 16 years.
Wakayama, Sayaka; Ohta, Hiroshi; Hikichi, Takafusa; Mizutani, Eiji; Iwaki, Takamasa; Kanagawa, Osami; Wakayama, Teruhiko
2008-11-11
Cloning animals by nuclear transfer provides an opportunity to preserve endangered mammalian species. However, it has been suggested that the "resurrection" of frozen extinct species (such as the woolly mammoth) is impracticable, as no live cells are available, and the genomic material that remains is inevitably degraded. Here we report production of cloned mice from bodies kept frozen at -20 degrees C for up to 16 years without any cryoprotection. As all of the cells were ruptured after thawing, we used a modified cloning method and examined nuclei from several organs for use in nuclear transfer attempts. Using brain nuclei as nuclear donors, we established embryonic stem cell lines from the cloned embryos. Healthy cloned mice were then produced from these nuclear transferred embryonic stem cells by serial nuclear transfer. Thus, nuclear transfer techniques could be used to "resurrect" animals or maintain valuable genomic stocks from tissues frozen for prolonged periods without any cryopreservation.
-
Production of healthy cloned mice from bodies frozen at −20°C for 16 years
Wakayama, Sayaka; Ohta, Hiroshi; Hikichi, Takafusa; Mizutani, Eiji; Iwaki, Takamasa; Kanagawa, Osami; Wakayama, Teruhiko
2008-01-01
Cloning animals by nuclear transfer provides an opportunity to preserve endangered mammalian species. However, it has been suggested that the “resurrection” of frozen extinct species (such as the woolly mammoth) is impracticable, as no live cells are available, and the genomic material that remains is inevitably degraded. Here we report production of cloned mice from bodies kept frozen at −20 °C for up to 16 years without any cryoprotection. As all of the cells were ruptured after thawing, we used a modified cloning method and examined nuclei from several organs for use in nuclear transfer attempts. Using brain nuclei as nuclear donors, we established embryonic stem cell lines from the cloned embryos. Healthy cloned mice were then produced from these nuclear transferred embryonic stem cells by serial nuclear transfer. Thus, nuclear transfer techniques could be used to “resurrect” animals or maintain valuable genomic stocks from tissues frozen for prolonged periods without any cryopreservation. PMID:18981419
-
Six cloned calves produced from adult fibroblast cells after long-term culture
Kubota, Chikara; Yamakuchi, Hiroshi; Todoroki, Junichi; Mizoshita, Kazunori; Tabara, Norio; Barber, Michele; Yang, Xiangzhong
2000-01-01
Cloning whole animals with somatic cells as parents offers the possibility of targeted genetic manipulations in vitro such as “gene knock-out” by homologous recombination. However, such manipulation requires prolonged culture of nuclear donor cells. Previous successes in cloning have been limited to the use of cells collected either fresh or after short-term culture. Therefore, demonstration of genetic totipotency of cells after prolonged culture is pivotal to combining site-specific genetic manipulations and cloning. Here we report birth of six clones of an aged (17-year-old) Japanese Black Beef bull using ear skin fibroblast cells as nuclear donor cells after up to 3 months of in vitro culture (10–15 passages). We observed higher developmental rates for embryos derived from later passages (10 and 15) as compared with those embryos from an early passage (passage 5). The four surviving clones are now 10–12 months of age and appear normal, similar to their naturally reproduced peers. These data show that fibroblasts of aged animals remain competent for cloning, and prolonged culture does not affect the cloning competence of adult somatic donor cells. PMID:10655472
-
Transgenic and cloned animals in the food chain--are we prepared to tackle it?
Jagadeesan, Premanandh; Bin Salem, Samara
2015-11-01
Transgenic and cloned animal production for various purposes has been increasing rapidly in recent times. While the actual impact of these animals in the food chain is unknown, the significance of tracking and monitoring measures to curb accidental and or deliberate release has been discussed. Religious perspectives from different faiths and traditions have been presented. Although the concept of substantial equivalence satisfies the technical and nutritional requirements of these products when assessed against comparators, public opinion and religious concerns should also be considered by the regulators while developing policy regulations. In conclusion, measures to prevent real or perceived risks of transgenic and cloned animals in food production require global coordinated action. It is worthwhile to consider establishing effective tracking systems and analytical procedures as this will be a valuable tool if a global consensus is not reached on policy regulation. © 2015 Society of Chemical Industry.
-
Selokar, Naresh L; Sharma, Papori; Krishna, Ananth; Kumar, Deepak; Kumar, Dharmendra; Saini, Monika; Sharma, Arpna; Vijayalakshmy, Kennady; Yadav, Prem Singh
2018-06-01
Biobanks of cryopreserved gametes and embryos of domestic animals have been utilized to spread desired genotypes and to conserve the animal germplasm of endangered breeds. In principle, somatic cells can be used for the same purposes, and for reviving of animals, the somatic cells must be suitable for animal cloning techniques, such as somatic cell nuclear transfer. In the present study, we derived and cryopreserved somatic cells from three breeds of riverine and swamp-like type buffaloes and established a somatic cell bank. In total, 350 cryovials of 14 different individual animals (25 cryovials per animal) were cryopreserved and informative data such as breed value, origin, and others were documented. Immunostaining of the established cells against vimentin and cytokeratin suggested a commitment to the fibroblast lineage. In addition, microsatellite analysis was performed and documented for unambiguous parentage verification of clones in the future. Subsequently, the cryopreserved cells were tested for their suitability as nuclear donors (n = 7) using handmade cloning, and the reconstructed embryos were cultured in vitro. The cleavage rates (95.99% ± 2.17% vs. 82.18% ± 2.50%) and blastocyst rates (37.73% ± 1.54% vs. 24.31% ± 1.78%) were higher (p < 0.05) for riverine buffalo cells than that of swamp-like buffalo cells, whereas the total cell numbers of blastocysts (258.16 ± 36.25 vs. 198.16 ± 36.25, respectively) were similar. In conclusion, we demonstrated the feasibility of biobanking of buffalo somatic cells, and that the cryopreserved cells can be used to produce cloned embryos. This study encourages the development of somatic cell biobanks of domestic livestock, including endangered breeds of buffalo, to preserve valuable genotypes for future revitalization by animal cloning techniques.
-
Mapping, fine mapping, and molecular dissection of quantitative trait Loci in domestic animals.
Georges, Michel
2007-01-01
Artificial selection has created myriad breeds of domestic animals, each characterized by unique phenotypes pertaining to behavior, morphology, physiology, and disease. Most domestic animal populations share features with isolated founder populations, making them well suited for positional cloning. Genome sequences are now available for most domestic species, and with them a panoply of tools including high-density single-nucleotide polymorphism panels. As a result, domestic animal populations are becoming invaluable resources for studying the molecular architecture of complex traits and of adaptation. Here we review recent progress and issues in the positional identification of genes underlying complex traits in domestic animals. As many phenotypes studied in animals are quantitative, we focus on mapping, fine mapping, and cloning of quantitative trait loci.
-
Oback, Björn
2008-07-01
Despite more than a decade of research efforts, farm animal cloning by somatic cell nuclear transfer (SCNT) is still frustratingly inefficient. Inefficiency manifests itself at different levels, which are currently not well integrated. At the molecular level, it leads to widespread genetic, epigenetic and transcriptional aberrations in cloned embryos. At the organismal level, these genome-wide abnormalities compromise development of cloned foetuses and offspring. Specific molecular defects need to be causally linked to specific cloned phenotypes, in order to design specific treatments to correct them. Cloning efficiency depends on the ability of the nuclear donor cell to be fully reprogrammed into an embryonic state and the ability of the enucleated recipient cell to carry out the reprogramming reactions. It has been postulated that reprogrammability of the somatic donor cell epigenome is influenced by its differentiation status. However, direct comparisons between cells of divergent differentiation status within several somatic lineages have found no conclusive evidence for this. Choosing somatic stem cells as donors has not improved cloning efficiency, indicating that donor cell type may be less critical for cloning success. Different recipient cells, on the other hand, vary in their reprogramming ability. In bovine, using zygotes instead of oocytes has increased cloning success. Other improvements in livestock cloning efficiency include better coordinating donor cell type with cell cycle stage and aggregating cloned embryos. In the future, it will be important to demonstrate if these small increases at every step are cumulative, adding up to an integrated cloning protocol with greatly improved efficiency.
-
Federal Register 2010, 2011, 2012, 2013, 2014
2011-06-23
.... (n/k/a Bancorp Energy, Inc.): Order of Suspension of Trading June 21, 2011. It appears to the... securities of Animal Cloning Sciences, Inc. (n/k/a Bancorp Energy, Inc.) because it has not filed any... above-listed company. Therefore, it is ordered, pursuant to Section 12(k) of the Securities Exchange Act...
-
... to have health problems all their lives. Myth: Cow clones make human pharmaceuticals in their milk. Myth: ... actual animal. For example, if they’re Holstein cows, the pattern of their spots, or the shape ...
-
[Telomere lengthening by trichostatin A treatment in cloned pigs].
Xie, Bing-Teng; Ji, Guang-Zhen; Kong, Qing-Ran; Mao, Jian; Shi, Yong-Qian; Liu, Shi-Chao; Wu, Mei-Ling; Wang, Juan; Liu, Lin; Liu, Zhong-Hua
2012-12-01
Telomeres are repeated GC rich sequences at the end of chromosomes, and shorten with each cell division due to DNA end replication problem. Previously, reprogrammed somatic cells of cloned animals display variable telomere elongation. However, it was reported that the cloned animals including Dolly do not reset telomeres and show premature aging. In this study, we investigated telomere function in cloned or transgenic cloned pigs, including the cloned Northeast Min pigs, eGFP, Mx, and PGC1α transgenic cloned pigs, and found that the telomere lengths of cloned pigs were significantly shorter than the nuclear donor adult fibroblasts and age-matched noncloned pigs (P<0.05), indicating that nuclear reprogramming did not restore cellular age of donor cells after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA), an inhibitor of histone deacetylase, has proven to enhance the efficiency of nuclear reprogramming in several species. In order to test whether TSA also can effectively enhance reprogramming of telomeres, TSA (40 nmol/L) was used to treat porcine cloned embryos at 1-cell stage for 24 h. Consistent with previous reports, the developmental rate of SCNT embryos to the blastocyst stage was significantly increased compared with those of the control group (16.35% vs. 27.09%, 21.60% vs. 34.90%, P<0.05). Notably, the telomere length of cloned porcine blastocysts was also significantly elongated (P<0.05). Although TSA did not improve the cloning efficiency (1.3% vs. 1.7%, TSA vs. control), the telomere lengths of cloned pig-lets were significantly longer compared with those of the control group and the donor fibroblasts (P<0.05). In conclusion, telomeres have not been effectively restored by SCNT in pigs but TSA can effectively lengthen the telomere lengths of cloned pigs.
-
Bressan, Fabiana Fernandes; Dos Santos Miranda, Moyses; Perecin, Felipe; De Bem, Tiago Henrique; Pereira, Flavia Thomaz Verechia; Russo-Carbolante, Elisa Maria; Alves, Daiani; Strauss, Bryan; Bajgelman, Marcio; Krieger, José Eduardo; Binelli, Mario; Meirelles, Flavio Vieira
2011-02-01
Animal cloning by nuclear transfer (NT) has made the production of transgenic animals using genetically modified donor cells possible and ensures the presence of the gene construct in the offspring. The identification of transgene insertion sites in donor cells before cloning may avoid the production of animals that carry undesirable characteristics due to positional effects. This article compares blastocyst development and competence to establish pregnancies of bovine cloned embryos reconstructed with lentivirus-mediated transgenic fibroblasts containing either random integration of a transgene (random integration group) or nuclear transfer derived transgenic fibroblasts with known transgene insertion sites submitted to recloning (recloned group). In the random integration group, eGFP-expressing bovine fetal fibroblasts were selected by fluorescence activated cell sorting (FACS) and used as nuclei donor cells for NT. In the recloned group, a fibroblast cell line derived from a transgenic cloned fetus was characterized regarding transgene insertion and submitted to recloning. The recloned group had higher blastocyst production (25.38 vs. 14.42%) and higher percentage of 30-day pregnancies (14.29 vs. 2.56%) when compared to the random integration group. Relative eGFP expression analysis in fibroblasts derived from each cloned embryo revealed more homogeneous expression in the recloned group. In conclusion, the use of cell lines recovered from transgenic fetuses after identification of the transgene integration site allowed for the production of cells and fetuses with stable transgene expression, and recloning may improve transgenic animal yields.
-
Somatic Cell Nuclear Transfer in the Mouse
NASA Astrophysics Data System (ADS)
Kishigami, Satoshi; Wakayama, Teruhiko
Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.
-
Characterization of Streptococcus pyogenes from Animal Clinical Specimens, Spain
Vela, Ana Isabel; Villalón, Pilar; Sáez-Nieto, Juan Antonio; Chacón, Gema; Domínguez, Lucas
2017-01-01
Streptococcus pyogenes appears to be almost exclusively restricted to humans, with few reports on isolation from animals. We provide a detailed characterization (emm typing, pulsed-field gel electrophoresis [PFGE], and multilocus sequence typing [MLST]) of 15 S. pyogenes isolates from animals associated with different clinical backgrounds. We also investigated erythromycin resistance mechanisms and phenotypes and virulence genes. We observed 2 emm types: emm12 (11 isolates) and emm77 (4 isolates). Similarly, we observed 2 genetic linages, sequence type (ST) 26 and ST63. Most isolates exhibited the M macrolide resistance phenotype and the mefA/ermB genotype. Isolates were grouped into 2 clones on the basis of emm-MLST-PFGE-virulence gene profile combinations: clone 1, characterized by the combined genotype emm12-ST36-pulsotype A-speG; and clone 2, characterized by the genotype emm77-ST63-pulsotype B-speC. Our results do not show conclusively that animals may represent a new reservoir of S. pyogenes but indicate the ability of human-derived S. pyogenes isolates to colonize and infect animals. PMID:29148379
-
Wohlwend, Nadia; Francey, Thierry
2015-01-01
Characterization of third-generation-cephalosporin-resistant Klebsiella pneumoniae isolates originating mainly from one human hospital (n = 22) and one companion animal hospital (n = 25) in Bern (Switzerland) revealed the absence of epidemiological links between human and animal isolates. Human infections were not associated with the spread of any specific clone, while the majority of animal infections were due to K. pneumoniae sequence type 11 isolates producing plasmidic DHA AmpC. This clonal dissemination within the veterinary hospital emphasizes the need for effective infection control practices. PMID:25733505
-
Mizutani, Eiji; Wakayama, Sayaka; Wakayama, Teruhiko
2015-01-01
The successful production of cloned animals by somatic cell nuclear transfer (SCNT) is a promising technology with many potential applications in basic research, medicine, and agriculture. However, the low efficiency and the difficulty of cloning are major obstacles to the widespread use of this technology. Since the first mammal cloned from an adult donor cell was born, many attempts have been made to improve animal cloning techniques, and some approaches have successfully improved its efficiency. Nuclear transfer itself is still difficult because it requires an accomplished operator with a practiced technique. Thus, it is very important to find simple and reproducible methods for improving the success rate of SCNT. In this chapter, we will review our recent protocols, which seem to be the simplest and most reliable method to date to improve development of SCNT embryos.
-
"Mouse Clone Model" for evaluating the immunogenicity and tumorigenicity of pluripotent stem cells.
Zhang, Gang; Zhang, Yi
2015-12-18
To investigate the immune-rejection and tumor-formation potentials of induced pluripotent stem cells and other stem cells, we devised a model-designated the "Mouse Clone Model"-which combined the theory of somatic animal cloning, tetraploid complementation, and induced pluripotent stem cells to demonstrate the applicability of stem cells for transplantation therapy.
-
Rescuing valuable genomes by animal cloning: a case for natural disease resistance in cattle.
Westhusin, M E; Shin, T; Templeton, J W; Burghardt, R C; Adams, L G
2007-01-01
Tissue banking and animal cloning represent a powerful tool for conserving and regenerating valuable animal genomes. Here we report an example involving cattle and the rescue of a genome affording natural disease resistance. During the course of a 2-decade study involving the phenotypic and genotypic analysis for the functional and genetic basis of natural disease resistance against bovine brucellosis, a foundation sire was identified and confirmed to be genetically resistant to Brucella abortus. This unique animal was utilized extensively in numerous animal breeding studies to further characterize the genetic basis for natural disease resistance. The bull died in 1996 of natural causes, and no semen was available for AI, resulting in the loss of this valuable genome. Fibroblast cell lines had been established in 1985, cryopreserved, and stored in liquid nitrogen for future genetic analysis. Therefore, we decided to utilize these cells for somatic cell nuclear transfer to attempt the production of a cloned bull and salvage this valuable genotype. Embryos were produced by somatic cell nuclear transfer and transferred to 20 recipient cows, 10 of which became pregnant as determined by ultrasound at d 40 of gestation. One calf survived to term. At present, the cloned bull is 4.5 yr old and appears completely normal as determined by physical examination and blood chemistry. Furthermore, in vitro assays performed to date indicate this bull is naturally resistant to B. abortus, Mycobacterium bovis, and Salmonella typhimurium, as was the original genetic donor.
-
KIM, Eun Young; SONG, Dong Hwan; PARK, Min Jee; PARK, Hyo Young; LEE, Seung Eun; CHOI, Hyun Yong; MOON, Jeremiah Jiman; KIM, Young Hoon; MUN, Seong Ho; OH, Chang Eon; KO, Moon Suck; LEE, Dong Sun; RIU, Key Zung; PARK, Se Pill
2013-01-01
Abstract To preserve Jeju black cattle (JBC; endangered native Korean cattle), a pair of cattle, namely a post-death cloned JBC bull and cow, were produced by somatic cell nuclear transfer (SCNT) in a previous study. In the present study, we examined the in vitro fertilization and reproductive potentials of these post-death cloned animals. Sperm motility, in vitro fertilization and developmental capacity were examined in a post-death cloned bull (Heuk Oll Dolee) and an extinct nuclear donor bull (BK94-13). We assessed reproductive ability in another post-death cloned cow (Heuk Woo Sunee) using cloned sperm for artificial insemination (AI). There were no differences in sperm motility or developmental potential of in vitro fertilized embryos between the post-death cloned bull and its extinct nuclear donor bull; however, the embryo development ratio was slightly higher in the cloned sperm group than in the nuclear donor sperm group. After one attempt at AI, the post-death cloned JBC cow became pregnant, and gestation proceeded normally until day 287. From this post-death cloned sire and dam, a JBC male calf (Heuk Woo Dolee) was delivered naturally (weight, 25 kg). The genetic paternity/maternity of the cloned JBC bull and cow with regard to their offspring was confirmed using International Society for Animal Genetics standard microsatellite markers. Presently, Heuk Woo Dolee is 5 months of age and growing normally. In addition, there were no significant differences in blood chemistry among the post-death cloned JBC bull, the cow, their offspring and cattle bred by AI. This is the first report showing that a pair of cattle, namely, a post-death cloned JBC bull and cow, had normal fertility. Therefore, SCNT can be used effectively to increase the population of endangered JBC. PMID:23955237
-
Kim, Eun Young; Song, Dong Hwan; Park, Min Jee; Park, Hyo Young; Lee, Seung Eun; Choi, Hyun Yong; Moon, Jeremiah Jiman; Kim, Young Hoon; Mun, Seong Ho; Oh, Chang Eon; Ko, Moon Suck; Lee, Dong Sun; Riu, Key Zung; Park, Se Pill
2013-12-17
To preserve Jeju black cattle (JBC; endangered native Korean cattle), a pair of cattle, namely a post-death cloned JBC bull and cow, were produced by somatic cell nuclear transfer (SCNT) in a previous study. In the present study, we examined the in vitro fertilization and reproductive potentials of these post-death cloned animals. Sperm motility, in vitro fertilization and developmental capacity were examined in a post-death cloned bull (Heuk Oll Dolee) and an extinct nuclear donor bull (BK94-13). We assessed reproductive ability in another post-death cloned cow (Heuk Woo Sunee) using cloned sperm for artificial insemination (AI). There were no differences in sperm motility or developmental potential of in vitro fertilized embryos between the post-death cloned bull and its extinct nuclear donor bull; however, the embryo development ratio was slightly higher in the cloned sperm group than in the nuclear donor sperm group. After one attempt at AI, the post-death cloned JBC cow became pregnant, and gestation proceeded normally until day 287. From this post-death cloned sire and dam, a JBC male calf (Heuk Woo Dolee) was delivered naturally (weight, 25 kg). The genetic paternity/maternity of the cloned JBC bull and cow with regard to their offspring was confirmed using International Society for Animal Genetics standard microsatellite markers. Presently, Heuk Woo Dolee is 5 months of age and growing normally. In addition, there were no significant differences in blood chemistry among the post-death cloned JBC bull, the cow, their offspring and cattle bred by AI. This is the first report showing that a pair of cattle, namely, a post-death cloned JBC bull and cow, had normal fertility. Therefore, SCNT can be used effectively to increase the population of endangered JBC.
-
Lessons learned from cloning dogs.
Kim, M J; Oh, H J; Kim, G A; Park, J E; Park, E J; Jang, G; Ra, J C; Kang, S K; Lee, B C
2012-08-01
The aim of this article is to review dog cloning research and to suggest its applications based on a discussion about the normality of cloned dogs. Somatic cell nuclear transfer was successfully used for production of viable cloned puppies despite limited understanding of in vitro dog embryo production. Cloned dogs have similar growth characteristics to those born from natural fertilization, with no evidence of serious adverse effects. The offspring of cloned dogs also have similar growth performance and health to those of naturally bred puppies. Therefore, cloning in domestic dogs can be applied as an assisted reproductive technique to conserve endangered species, to treat sterile canids or aged dogs, to improve reproductive performance of valuable individuals and to generate disease model animals. © 2012 Blackwell Verlag GmbH.
-
Mouse cloning using a drop of peripheral blood.
Kamimura, Satoshi; Inoue, Kimiko; Ogonuki, Narumi; Hirose, Michiko; Oikawa, Mami; Yo, Masahiro; Ohara, Osamu; Miyoshi, Hiroyuki; Ogura, Atsuo
2013-08-01
Somatic cell nuclear transfer (SCNT) is a unique technology that produces cloned animals from single cells. It is desirable from a practical viewpoint that donor cells can be collected noninvasively and used readily for nuclear transfer. The present study was undertaken to determine whether peripheral blood cells freshly collected from living mice could be used for SCNT. We collected a drop of peripheral blood (15-45 μl) from the tail of a donor. A nucleated cell (leukocyte) suspension was prepared by lysing the red blood cells. Following SCNT using randomly selected leukocyte nuclei, cloned offspring were born at a 2.8% birth rate. Fluorescence-activated cell sorting revealed that granulocytes/monocytes and lymphocytes could be roughly distinguished by their sizes, the former being significantly larger. We then cloned putative granulocytes/monocytes and lymphocytes separately and obtained 2.1% and 1.7% birth rates, respectively (P > 0.05). Because the use of lymphocyte nuclei inevitably results in the birth of offspring with DNA rearrangements, we applied granulocyte/monocyte cloning to two genetically modified strains and two recombinant inbred strains. Normal-looking offspring were obtained from all four strains tested. The present study clearly indicated that genetic copies of mice could be produced using a drop of peripheral blood from living donors. This strategy will be applied to the rescue of infertile founder animals or a "last-of-line" animal possessing invaluable genetic resources.
-
Progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer
Li, Ziyi; Engelhardt, John F
2003-01-01
Mammalian cloning by nuclear transfer from somatic cells has created new opportunities to generate animal models of genetic diseases in species other than mice. Although genetic mouse models play a critical role in basic and applied research for numerous diseases, often mouse models do not adequately reproduce the human disease phenotype. Cystic fibrosis (CF) is one such disease. Targeted ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in mice does not adequately replicate spontaneous bacterial infections observed in the human CF lung. Hence, several laboratories are pursuing alternative animal models of CF in larger species such as the pig, sheep, rabbits, and ferrets. Our laboratory has focused on developing the ferret as a CF animal model. Over the past few years, we have investigated several experimental parameters required for gene targeting and nuclear transfer (NT) cloning in the ferret using somatic cells. In this review, we will discuss our progress and the hurdles to NT cloning and gene-targeting that accompany efforts to generate animal models of genetic diseases in species such as the ferret. PMID:14613541
-
Sung, Li-Ying; Gao, Shaorong; Shen, Hongmei; Yu, Hui; Song, Yifang; Smith, Sadie L; Chang, Ching-Chien; Inoue, Kimiko; Kuo, Lynn; Lian, Jin; Li, Ao; Tian, X Cindy; Tuck, David P; Weissman, Sherman M; Yang, Xiangzhong; Cheng, Tao
2006-11-01
Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.
-
Ethical issues regarding human cloning: a nursing perspective.
Dinç, Leyla
2003-05-01
Advances in cloning technology and successful cloning experiments in animals raised concerns about the possibility of human cloning in recent years. Despite many objections, this is not only a possibility but also a reality. Human cloning is a scientific revolution. However, it also introduces the potential for physical and psychosocial harm to human beings. From this point of view, it raises profound ethical, social and health related concerns. Human cloning would have an impact on the practice of nursing because it could result in the creation of new physiological and psychosocial conditions that would require nursing care. The nursing profession must therefore evaluate the ethics of human cloning, in particular the potential role of nurses. This article reviews the ethical considerations of reproductive human cloning, discusses the main reasons for concern, and reflects a nursing perspective regarding this issue.
-
Production of cloned mice from somatic cells, ES cells, and frozen bodies.
Wakayama, Sayaka; Mizutani, Eiji; Wakayama, Teruhiko
2010-01-01
Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, therefore, the nuclear transfer (NT) method has been thought of as a "black box approach" and inadequate to determine the detail of how genomic reprogramming occurs. However, only the NT approach can reveal dynamic and global modifications in the epigenome without using genetic modification, as well as can create live animals. At present, this is the only technique available for the preservation and propagation of valuable genetic resources from mutant mice that are infertile or too old, or recovered from carcasses, without the use of germ cells. This chapter describes a basic protocol for mouse cloning and embryonic stem (ES) cell establishment from cloned embryo using a piezo-actuated micromanipulator. This technique will greatly help not only in mouse cloning but also in other forms of micromanipulation such as intracytoplasmic sperm injection (ICSI) into oocytes or ES cell injection into blastocysts. In addition, we describe a new, more efficient mouse cloning protocol using histone deacetylase inhibitor (HDACi), which increases the success rates of cloned mice or establish rate of ES cells to fivefold. Copyright (c) 2010 Elsevier Inc. All rights reserved.
-
Cloning animals by somatic cell nuclear transfer – biological factors
Tian, X Cindy; Kubota, Chikara; Enright, Brian; Yang, Xiangzhong
2003-01-01
Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other specie, this review will be focused on somatic cell cloning of cattle. PMID:14614770
-
Cloning animals by somatic cell nuclear transfer--biological factors.
Tian, X Cindy; Kubota, Chikara; Enright, Brian; Yang, Xiangzhong
2003-11-13
Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other species, this review will be focused on somatic cell cloning of cattle.
-
Terashita, Yukari; Wakayama, Sayaka; Yamagata, Kazuo; Li, Chong; Sato, Eimei; Wakayama, Teruhiko
2012-06-01
Although animal cloning is becoming more practicable, there are many abnormalities in cloned embryos, and the success rate of producing live animals by cloning has been low. Here, we focused on the procedure for preventing pseudo-second polar body extrusion from somatic cell nuclear transfer (SCNT)-derived oocytes. Typically, reconstructed oocytes are treated with cytochalasin B (CB), but here latrunculin A (LatA) was used instead of CB to prevent pseudo-second polar body extrusion by inhibiting actin polymerization. CB caps F-actin, LatA binds G-actin, and both drugs prevent their polymerization. When the localization of F-actin was examined using phalloidin staining, it was abnormally scattered in the cytoplasm of CB-treated 1-cell embryos, but this was not detected in LatA-treated or in vitro fertilization-derived control embryos. The spindle was larger in CB-treated oocytes than in LatA-treated or untreated control oocytes. LatA treatment also doubled the rate of full-term development after embryo transfer. These results suggest that cloning efficiency in mice can be improved by optimizing each step of the SCNT procedure. Moreover, by using LatA, we could simplify the procedure with a higher birth rate of cloned mice compared with our original method.
-
Uhlemann, Anne-Catrin; Porcella, Stephen F.; Trivedi, Sheetal; Sullivan, Sean B.; Hafer, Cory; Kennedy, Adam D.; Barbian, Kent D.; McCarthy, Alex J.; Street, Craig; Hirschberg, David L.; Lipkin, W. Ian; Lindsay, Jodi A.; DeLeo, Frank R.; Lowy, Franklin D.
2012-01-01
ABSTRACT A methicillin-resistant Staphylococcus aureus (MRSA) clone known as ST398 has emerged as a major cause of acute infections in individuals who have close contact with livestock. More recently, the emergence of an animal-independent ST398 methicillin-sensitive S. aureus (MSSA) clone has been documented in several countries. However, the limited surveillance of MSSA has precluded an accurate assessment of the global spread of ST398 and its clinical relevance. Here we provide evidence that ST398 is a frequent source of MSSA infections in northern Manhattan and is readily transmitted between individuals in households. This contrasts with the limited transmissibility of livestock-associated ST398 (LA-ST398) MRSA strains between humans. Our whole-genome sequence analysis revealed that the chromosome of the human-associated ST398 MSSA clone is smaller than that of the LA-ST398 MRSA reference strain S0385, due mainly to fewer mobile genetic elements (MGEs). In contrast, human ST398 MSSA isolates harbored the prophage φ3 and the human-specific immune evasion cluster (IEC) genes chp and scn. While most of the core genome was conserved between the human ST398 MSSA clone and S0385, these strains differed substantially in their repertoire and composition of intact adhesion genes. These genetic changes were associated with significantly enhanced adhesion of human ST398 MSSA isolates to human skin keratinocytes and keratin. We propose that the human ST398 MSSA clone can spread independent of animal contact using an optimized repertoire of MGEs and adhesion molecules adapted to transmission among humans. PMID:22375071
-
García-Sancho, Miguel
2015-09-01
This paper addresses the 1996 cloning of Dolly the sheep, locating it within a long-standing tradition of animal breeding research in Edinburgh. Far from being an end in itself, the cell-nuclear transfer experiment from which Dolly was born should be seen as a step in an investigative pathway that sought the production of medically relevant transgenic animals. By historicising Dolly, I illustrate how the birth of this sheep captures a dramatic redefinition of the life sciences, when in the 1970s and 1980s the rise of neo-liberal governments and the emergence of the biotechnology market pushed research institutions to show tangible applications of their work. Through this broader interpretative framework, the Dolly story emerges as a case study of the deep transformations of agricultural experimentation during the last third of the twentieth century. The reorganisation of laboratory practice, human resources and institutional settings required by the production of transgenic animals had unanticipated consequences. One of these unanticipated effects was that the boundaries between animal and human health became blurred. As a result of this, new professional spaces emerged and the identity of Dolly the sheep was reconfigured, from an instrument for livestock improvement in the farm to a more universal symbol of the new cloning age.
-
Propagation of senescent mice using nuclear transfer embryonic stem cell lines.
Mizutani, Eiji; Ono, Tetsuo; Li, Chong; Maki-Suetsugu, Rinako; Wakayama, Teruhiko
2008-09-01
Senescent mice are often infertile, and the cloning success rate decreases with age, making it almost impossible to produce cloned progeny directly from such animals. In this study, we tried to produce offspring from such "unclonable" senescent mice using nuclear transfer techniques. Donor fibroblasts were obtained from the tail tips of mice aged up to 2 years and 9 months. Although most attempts failed to produce cloned mice by direct somatic cell nuclear transfer, we managed to establish nuclear transfer embryonic stem (ntES) cell lines from all aged mice with an establishment rate of 10-25%, irrespective of sex or strain. Finally, cloned mice were obtained from these ntES cells by a second round of nuclear transfer. In addition, healthy offspring was obtained from all aged donors via germline transmission of ntES cells in chimeric mice. This technique is thus applicable to the propagation of a variety of animals, irrespective of age or fertile potential.
-
Hyper-reactive cloned mice generated by direct nuclear transfer of antigen-specific CD4+ T cells.
Kaminuma, Osamu; Katayama, Kazufumi; Inoue, Kimiko; Saeki, Mayumi; Nishimura, Tomoe; Kitamura, Noriko; Shimo, Yusuke; Tofukuji, Soichi; Ishida, Satoru; Ogonuki, Narumi; Kamimura, Satoshi; Oikawa, Mami; Katoh, Shigeki; Mori, Akio; Shichijo, Michitaka; Hiroi, Takachika; Ogura, Atsuo
2017-06-01
T-cell receptor (TCR)-transgenic mice have been employed for evaluating antigen-response mechanisms, but their non-endogenous TCR might induce immune response differently than the physiologically expressed TCR Nuclear transfer cloning produces animals that retain the donor genotype in all tissues including germline and immune systems. Taking advantage of this feature, we generated cloned mice that carry endogenously rearranged TCR genes from antigen-specific CD4 + T cells. We show that T cells of the cloned mice display distinct developmental pattern and antigen reactivity because of their endogenously pre-rearranged TCRα (rTα) and TCRβ (rTβ) alleles. These alleles were transmitted to the offspring, allowing us to establish a set of mouse lines that show chronic-type allergic phenotypes, that is, bronchial and nasal inflammation, upon local administrations of the corresponding antigens. Intriguingly, the existence of either rTα or rTβ is sufficient to induce in vivo hypersensitivity. These cloned mice expressing intrinsic promoter-regulated antigen-specific TCR are a unique animal model with allergic predisposition for investigating CD4 + T-cell-mediated pathogenesis and cellular commitment in immune diseases. © 2017 The Authors.
-
Cloning of Buffalo, a Highly Valued Livestock Species of South and Southeast Asia: Any Achievements?
Selokar, Naresh L; Saini, Monika; Palta, Prabhat; Chauhan, Manmohan S; Manik, Radhey S; Singla, Suresh K
2018-04-01
Buffalo (Bubalus bubalis) is a major source of milk, meat, and draught power in many developing countries in Asia. Animal cloning holds a lot of potential for fast multiplication of elite buffaloes and conservation of their valuable germplasm. Although the progress of buffalo cloning has been slow in comparison to cattle or pig, several breakthroughs were reported in buffalo cloning such as the production of cloned calves from somatic cells isolated from over one-decade old frozen-thawed semen or from urine-derived cells. Since the initiation of buffalo cloning, several approaches have been tried to refine nuclear transfer protocols. This has resulted in increasing the blastocyst production rate and improving their quality leading to an increase in live birth rate. In this review, we discuss current developments in buffalo cloning, its challenges, and the future roadmap.
-
Tang, Yizhi; Meinersmann, Richard J.; Sahin, Orhan; Wu, Zuowei; Dai, Lei; Carlson, James; Plumblee Lawrence, Jodie; Genzlinger, Linda; LeJeune, Jeffrey T.
2017-01-01
ABSTRACT Campylobacter jejuni clone SA is the major cause of sheep abortion and contributes significantly to foodborne illnesses in the United States. Clone SA is hypervirulent because of its distinct ability to produce systemic infection and its predominant role in clinical sheep abortion. Despite the importance of clone SA, little is known about its distribution and epidemiological features in cattle. Here we describe a prospective study on C. jejuni clone SA prevalence in 35 feedlots in 5 different states in the United States and a retrospective analysis of clone SA in C. jejuni isolates collected by National Animal Health Monitoring System (NAHMS) dairy studies in 2002, 2007, and 2014. In feedlot cattle feces, the overall prevalence of Campylobacter organisms was 72.2%, 82.1% of which were C. jejuni. Clone SA accounted for 5.8% of the total C. jejuni isolates, but its prevalence varied by feedlot and state. Interestingly, starlings on the feedlots harbored C. jejuni in feces, including clone SA, suggesting that these birds may play a role in the transmission of Campylobacter. In dairy cattle, the overall prevalence of clone SA was 7.2%, but a significant decrease in the prevalence was observed from 2002 to 2014. Whole-genome sequence analysis of the dairy clone SA isolates revealed that it was genetically stable over the years and most of the isolates carried the tetracycline resistance gene tet(O) in the chromosome. These findings indicate that clone SA is widely distributed in both beef and dairy cattle and provide new insights into the molecular epidemiology of clone SA in ruminants. IMPORTANCE C. jejuni clone SA is a major cause of small-ruminant abortion and an emerging threat to food safety because of its association with foodborne outbreaks. Cattle appear to serve as a major reservoir for this pathogenic organism, but there is a major gap in our knowledge about the epidemiology of clone SA in beef and dairy cattle. By taking advantage of surveillance studies conducted on a national scale, we found a wide but variable distribution of clone SA in feedlot cattle and dairy cows in the United States. Additionally, the work revealed important genomic features of clone SA isolates from cattle. These findings provide critically needed information for the development of preharvest interventions to control the transmission of this zoonotic pathogen. Control of C. jejuni clone SA will benefit both animal health and public health, as it is a zoonotic pathogen causing disease in both ruminants and humans. PMID:28970227
-
Tang, Yizhi; Meinersmann, Richard J; Sahin, Orhan; Wu, Zuowei; Dai, Lei; Carlson, James; Plumblee, Jodie; Genzlinger, Linda; LeJeune, Jeffrey T; Zhang, Qijing
2017-09-29
Campylobacter jejuni clone SA is the major cause of sheep abortion and contributes significantly to foodborne illnesses in the United States. Clone SA is hypervirulent because of its distinct ability to produce systemic infection and its predominant role in clinical sheep abortion. Despite the importance of clone SA, little is known about its distribution and epidemiological features in cattle. Here, we describe a prospective study on C. jejuni clone SA prevalence in 35 feedlots in 5 different states in the U.S. and a retrospective analysis of clone SA in C. jejuni isolates collected by National Animal Health Monitoring System (NAHMS) Dairy Studies 2002, 2007 and 2014. In feedlot cattle feces, the overall prevalence of Campylobacter was 72.2%, 82.1% of which were C. jejuni Clone SA accounted for 5.8% of the total C. jejuni isolates, but its prevalence varied by feedlot and state. Interestingly, starlings on the feedlots harbored C. jejuni in feces including clone SA, suggesting it may play a role in the transmission of Campylobacter In dairy cattle, the overall prevalence of clone SA was 7.2%, but a significant decrease in the prevalence was observed from 2002 to 2014. Whole genome sequence analysis of the dairy clone SA isolates revealed that it was genetically stable over the years and most of the isolates carried the tetracycline resistance gene tet(O ) in the chromosome. These findings indicate clone SA is widely distributed in both beef and dairy cattle, and provide new insights into the molecular epidemiology of clone SA in ruminants. Importance C. jejuni clone SA is a major cause of small ruminant abortion and an emerging threat to food safety because of its association with foodborne outbreaks. Cattle appears to serve as a major reservoir for this pathogenic organism, but there is a major gap in our knowledge about the epidemiology of clone SA in beef and dairy cattle. By taking advantage of surveillance studies conducted on a national scale, this manuscript describes wide but variable distribution of clone SA in feedlot cattle and dairy cows in the United States. Additionally, the work revealed important genomic features of clone SA isolates from cattle. These findings provide critically needed information for the development of pre-harvest interventions to control the transmission of this zoonotic pathogen. Control of C. jejuni clone SA will benefit both animal health and public health as it is a zoonotic pathogen causing disease in both ruminants and humans. Copyright © 2017 Tang et al.
-
Cloning and characterization of new bioluminescent proteins
NASA Astrophysics Data System (ADS)
Szent-Gyorgyi, Christopher; Ballou, Byron T.; Dagnal, Erich; Bryan, Bruce
1999-07-01
Over the past two years Prolume has undertaken a comprehensive program to clone luciferases and associated 'green fluorescent proteins' (GFPs) from marine animals that use coelenterazine as the luciferin. To data we have cloned several bioluminescent proteins, including two novel copepod luciferases and two anthozoan GFPs. These four proteins have sequences that differ greatly form previously cloned analogous proteins; the sequence diversity apparently is due to independent evolutionary origins and unusual evolutionary constraints. Thus coelenterazine-based bioluminescent systems may also manifest a variety of useful properties. We discuss form this taxonomic perspective the initial biochemical and spectral characterization of our cloned proteins. Emphasis is placed on the anthozoan luciferase-GFP systems, whose efficient resonance energy transfer has elicited much current interest.
-
Wakayama, Sayaka; Wakayama, Teruhiko
2010-01-01
Nuclear transfer-derived ES (ntES) cell lines can be established from somatic cell nuclei with a relatively high success rate. Although ntES cells have been shown to be equivalent to ES cells, there are ethical objections concerning human cells, such as the use of fresh oocyte donation from young healthy woman. In contrast, the use of induced pluripotent stem (iPS) cells for cloning poses few ethical problems and is a relatively easy technique compared with nuclear transfer. Therefore, although there are several reports proposing the use of ntES cells as a model of regenerative medicine, the use of these cells in preliminary medical research is waning. However, in theory, 5 to 10 donor cells can establish one ntES cell line and, once established, these cells will propagate indefinitely. These cells can be used to generate cloned animals from ntES cell lines using a second round of NT. Even in infertile and "unclonable" strains of mice, we can generate offspring from somatic cells by combining cloning with ntES technology. Moreover, cloned offspring can be generated potentially even from the nuclei of dead bodies or freeze-dried cells via ntES cells, such as from an extinct frozen animal. Currently, only the ntES technology is available for this purpose, because all other techniques, including iPS cell derivation, require significant numbers of living donor cells. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.
-
DNA methylation profiles of donor nuclei cells and tissues of cloned bovine fetuses.
Kremenskoy, Maksym; Kremenska, Yuliya; Suzuki, Masako; Imai, Kei; Takahashi, Seiya; Hashizume, Kazuyoshi; Yagi, Shintaro; Shiota, Kunio
2006-04-01
Methylation of DNA in CpG islands plays an important role during fetal development and differentiation because CpG islands are preferentially located in upstream regions of mammalian genomic DNA, including the transcription start site of housekeeping genes and are also associated with tissue-specific genes. Somatic nuclear transfer (NT) technology has been used to generate live clones in numerous mammalian species, but only a low percentage of nuclear transferred animals develop to term. Abnormal epigenetic changes in the CpG islands of donor nuclei after nuclear transfer could contribute to a high rate of abortion during early gestation and increase perinatal death. These changes have yet to be explored. Thus, we investigated the genome-wide DNA methylation profiles of CpG islands in nuclei donor cells and NT animals. Using Restriction Landmark Genomic Scanning (RLGS), we showed, for the first time, the epigenetic profile formation of tissues from NT bovine fetuses produced from cumulus cells. From approximately 2600 unmethylated NotI sites visualized on the RLGS profile, at least 35 NotI sites showed different methylation statuses. Moreover, we proved that fetal and placental tissues from artificially inseminated and cloned cattle have tissue-specific differences in the genome-wide methylation profiles of the CpG islands. We also found that possible abnormalities occurred in the fetal brain and placental tissues of cloned animals.
-
Federal Register 2010, 2011, 2012, 2013, 2014
2010-01-19
... Engineered Eucalyptus Hybrid AGENCY: Animal and Plant Health Inspection Service, USDA. ACTION: Notice... for a proposed controlled field release of a genetically engineered clone of a Eucalyptus hybrid. This... proposed controlled field release of a genetically engineered clone of a Eucalyptus hybrid. \\1\\ To view the...
-
Muldoon, L. L.; Neuwelt, E. A.; Pagel, M. A.; Weiss, D. L.
1994-01-01
The Korat cat provides an animal model for type II GM2-gangliosidosis (Sandhoff disease) that may be suitable for tests of gene replacement therapy with the HEXB gene encoding the beta subunit of the beta-hexosaminidases. In the present report, we examined the brain and liver pathology of a typical Sandhoff-affected cat. We characterized the feline HEXB complementary DNA (cDNA) and determined the molecular defect in this feline model. cDNA libraries were produced from one normal and one affected animal, and cDNA clones homologous to human HEXB were sequenced. In the affected cDNA clone, the deletion of a cytosine residue at position +39 of the putative coding region results in a frame shift and a stop codon at base +191. This disease-related deletion was consistently detected by sequencing of cloned polymerase chain reaction amplified reverse transcribed messenger RNA from one more normal Korat and two additional affected animals. The defect was further demonstrated using single-strand conformational polymorphism analysis of the polymerase chain reaction products. In addition, alternative splicing of both normal and affected messenger RNAs was demonstrated. These results should facilitate the use of this animal model to assess gene therapy. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:8178934
-
Muldoon, L L; Neuwelt, E A; Pagel, M A; Weiss, D L
1994-05-01
The Korat cat provides an animal model for type II GM2-gangliosidosis (Sandhoff disease) that may be suitable for tests of gene replacement therapy with the HEXB gene encoding the beta subunit of the beta-hexosaminidases. In the present report, we examined the brain and liver pathology of a typical Sandhoff-affected cat. We characterized the feline HEXB complementary DNA (cDNA) and determined the molecular defect in this feline model. cDNA libraries were produced from one normal and one affected animal, and cDNA clones homologous to human HEXB were sequenced. In the affected cDNA clone, the deletion of a cytosine residue at position +39 of the putative coding region results in a frame shift and a stop codon at base +191. This disease-related deletion was consistently detected by sequencing of cloned polymerase chain reaction amplified reverse transcribed messenger RNA from one more normal Korat and two additional affected animals. The defect was further demonstrated using single-strand conformational polymorphism analysis of the polymerase chain reaction products. In addition, alternative splicing of both normal and affected messenger RNAs was demonstrated. These results should facilitate the use of this animal model to assess gene therapy.
-
Advancing pig cloning technologies towards application in regenerative medicine.
Nagashima, H; Matsunari, H; Nakano, K; Watanabe, M; Umeyama, K; Nagaya, M
2012-08-01
Regenerative medicine is expected to make a significant contribution by development of novel therapeutic treatments for intractable diseases and for improving the quality of life of patients. Many advances in regenerative medicine, including basic and translational research, have been developed and tested in experimental animals; pigs have played an important role in various aspects of this work. The value of pigs as a model species is being enhanced by the generation of specially designed animals through cloning and genetic modifications, enabling more sophisticated research to be performed and thus accelerating the clinical application of regenerative medicine. This article reviews the significant aspects of the creation and application of cloned and genetically modified pigs in regenerative medicine research and considers the possible future directions of the technology. We also discuss the importance of reproductive biology as an interface between basic science and clinical medicine. © 2012 Blackwell Verlag GmbH.
-
[Scientific ethics of human cloning].
Valenzuela, Carlos Y
2005-01-01
True cloning is fission, budding or other types of asexual reproduction. In humans it occurs in monozygote twinning. This type of cloning is ethically and religiously good. Human cloning can be performed by twinning (TWClo) or nuclear transfer (NTClo). Both methods need a zygote or a nuclear transferred cell, obtained in vitro (IVTec). They are under the IVTec ethics. IVTecs use humans (zygotes, embryos) as drugs or things; increase the risk of malformations; increase development and size of abnormalities and may cause long-term changes. Cloning for preserving extinct (or almost extinct) animals or humans when sexual reproduction is not possible is ethically valid. The previous selection of a phenotype in human cloning violates some ethical principles. NTClo for reproductive or therapeutic purposes is dangerous since it increases the risk for nucleotide or chromosome mutations, de-programming or re-programming errors, aging or malignancy of the embryo cells thus obtained.
-
[TSA improve transgenic porcine cloned embryo development and transgene expression].
Kong, Qing-Ran; Zhu, Jiang; Huang, Bo; Huan, Yan-Jun; Wang, Feng; Shi, Yong-Qian; Liu, Zhong-Feng; Wu, Mei-Ling; Liu, Zhong-Hua
2011-07-01
Uncompleted epigenetic reprogramming is attributed to the low efficiency of producing transgenic cloned animals. Histone modification associated with epigenetics can directly influence the embryo development and transgene expression. Trichostatin A (TSA), as an inhibitor of histone deacetylase, can change the status of histone acetylation, improve somatic cell reprogramming, and enhance cloning efficiency. TSA prevents the chromatin structure from being condensed, so that transcription factor could binds to DNA sequence easily and enhance transgene expression. Our study established the optimal TSA treatment on porcine donor cells and cloned embryos, 250 nmol/L, 24 h and 40 nmol/L, 24 h, respectively. Furthermore, we found that both the cloned embryo and the donor cell treated by TSA resulted in the highest development efficiency. Meanwhile, TSA can improve transgene expression in donor cell and cloned embryo. In summary, TSA can significantly improve porcine reconstructed embryo development and transgene expression.
-
Preservation and Reproduction of Microminipigs by Cloning Technology.
Enya, Satoko; Kawarasaki, Tatsuo; Otake, Masayoshi; Kangawa, Akihisa; Uenishi, Hirohide; Mikawa, Satoshi; Nishimura, Takashi; Kuwahawa, Yasushi; Shibata, Masatoshi
Microminipigs have been maintained in small populations of closed colonies, involving risks of inbreeding depression and genetic drift. In order to avoid these risks, we assessed the applicability of cloning technology. Male and female clones were produced from a stock of cryopreserved somatic cells, obtaining offspring by means of natural mating. Phenotypic and genotypic characteristics of original microminipigs, clones and their offspring were analyzed and recorded. Clones presented characteristics similar to those of the cell-stock data. Although the body weight of clones tended to be heavier than that of the cell-stock data, body weights of their offspring were similar to those of previous reports. Thus, cloned microminipigs have the potential to be a valuable genetic resource for reproduction and breeding. Our proposed methodology might be useful to provide a large number of animals with adequate quality from a limited population with sufficient genetic diversity. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
-
Molecular Characteristics of Staphylococcus aureus Causing Bovine Mastitis between 2014 and 2015.
Li, Tianming; Lu, Huiying; Wang, Xing; Gao, Qianqian; Dai, Yingxin; Shang, Jun; Li, Min
2017-01-01
Staphylococcus aureus is highly pathogenic and can cause diseases in both humans and domestic animals. In animal species, including ruminants, S. aureus may cause severe or sub-clinical mastitis. This study aimed to investigate the molecular profile, antimicrobial resistance, and genotype/phenotype correlation of 212 S. aureus isolates recovered from cases of bovine mastitis from 2014 to 2015 in the Shanghai and Zhejiang areas of China. Nineteen sequence types (STs) were determined by multi-locus sequence typing, while the dominant ST was ST97, followed by ST520, ST188, ST398, ST7, and ST9. Within 14 methicillin-resistant S. aureus (MRSA) isolates and 198 methicillin-susceptible S. aureus (MSSA) isolates, ST97 was the predominant MSSA clone and ST9-MRSA-SCCmecXII-spa t899 was the most common MRSA clone. The MRSA strains showed much higher rates of resistance to multiple antibiotics than did MSSA strains. Compared with other MSSA strains, MSSA ST398 was more resistant to clindamycin, erythromycin, and ciprofloxacin. No isolates were resistant to vancomycin, teicoplanin, or linezolid. The molecular profiles of the virulence genes varied in different strains. ST520 strains carried seg-sei-sem-sen-seo genes, and ST9 and ST97 harbored sdrD-sdrE genes. Virulence phenotype analysis showed diversity in different clones. Biofilm formation ability was significantly enhanced in ST188 and ST7, and red blood cell lysis capacity was relatively strong in all S. aureus strains of animal origin except ST7. Our results indicate that MSSA was the predominant S. aureus strain causing bovine mastitis in eastern regions of China. However, the presence of multidrug resistant and toxigenic MRSA clone ST9 suggests that comprehensive surveillance of S. aureus infection should be implemented in the management of animal husbandry products.
-
Social Media: Menagerie of Metrics
2010-01-27
intelligence, an evolutionary algorithm (EA) is a subset of evolutionary computation, a generic population-based metaheuristic optimization algorithm . An EA...Cloning - 22 Animals were cloned to date; genetic algorithms can help prediction (e.g. “elitism” - attempts to ensure selection by including performers...28, 2010 Evolutionary Algorithm • Evolutionary algorithm From Wikipedia, the free encyclopedia Artificial intelligence portal In artificial
-
Njenga, M. Kariuki; Njagi, Leonard; Thumbi, S. Mwangi; Kahariri, Samuel; Githinji, Jane; Omondi, Eunice; Baden, Amy; Murithi, Mbabu; Paweska, Janusz; Ithondeka, Peter M.; Ngeiywa, Kisa J.; Dungu, Baptiste; Donadeu, Meritxell; Munyua, Peninah M.
2015-01-01
Background Although livestock vaccination is effective in preventing Rift Valley fever (RVF) epidemics, there are concerns about safety and effectiveness of the only commercially available RVF Smithburn vaccine. We conducted a randomized controlled field trial to evaluate the immunogenicity and safety of the new RVF Clone 13 vaccine, recently registered in South Africa. Methods In a blinded randomized controlled field trial, 404 animals (85 cattle, 168 sheep, and 151 goats) in three farms in Kenya were divided into three groups. Group A included males and non-pregnant females that were randomized and assigned to two groups; one vaccinated with RVF Clone 13 and the other given placebo. Groups B included animals in 1st half of pregnancy, and group C animals in 2nd half of pregnancy, which were also randomized and either vaccinated and given placebo. Animals were monitored for one year and virus antibodies titers assessed on days 14, 28, 56, 183 and 365. Results In vaccinated goats (N = 72), 72% developed anti-RVF virus IgM antibodies and 97% neutralizing IgG antibodies. In vaccinated sheep (N = 77), 84% developed IgM and 91% neutralizing IgG antibodies. Vaccinated cattle (N = 42) did not develop IgM antibodies but 67% developed neutralizing IgG antibodies. At day 14 post-vaccination, the odds of being seropositive for IgG in the vaccine group was 3.6 (95% CI, 1.5 – 9.2) in cattle, 90.0 (95% CI, 25.1 – 579.2) in goats, and 40.0 (95% CI, 16.5 – 110.5) in sheep. Abortion was observed in one vaccinated goat but histopathologic analysis did not indicate RVF virus infection. There was no evidence of teratogenicity in vaccinated or placebo animals. Conclusions The results suggest RVF Clone 13 vaccine is safe to use and has high (>90%) immunogenicity in sheep and goats but moderate (> 65%) immunogenicity in cattle. PMID:25756501
-
Vanin, E F; Kaloss, M; Broscius, C; Nienhuis, A W
1994-01-01
Rapidly progressive T-cell lymphomas were observed in 3 of 10 rhesus monkeys several months after autologous transplantation of enriched bone marrow stem cells that had been transduced with a retroviral vector preparation containing replication-competent virus (R. E. Donahue, S. W. Kessler, D. Bodice, K. McDonagh, C. Dunbar, S. Goodman, B. Agricola, E. Byrne, M. Raffeld, R. Moen, J. Bacher, K. M. Zsebo, and A. W. Nienhuis, J. Exp. Med. 176:1124-1135, 1992). The animals with lymphoma appeared to be tolerant to retroviral antigens in that their sera lacked antibodies reactive with viral proteins and contained 10(4) to 10(5) infectious virus particles per ml. By molecular cloning and DNA sequencing, we have now demonstrated that the serum from one of the monkeys contained a replication-competent retrovirus that arose by recombination between vector and packaging encoding sequences (vector/helper [V/H] recombinant) in the producer clone used for transduction of bone marrow stem cells. Southern blot analysis demonstrated 14 or 25 copies of this genome per cell where present in two animals. The genome of a second replication-competent virus was also recovered by molecular cloning; it arose by recombination involving the genome of the V/H recombinant and endogenous murine retroviral genomes in the producer clone. Twelve copies of this amphotropic virus/mink cell focus-forming virus genome were present in tumor DNA of one animal, but it was not found in tumor DNA of the other two animals with lymphoma. Southern blot analysis of DNA from various tissues demonstrated common insertion site bands in several samples of tumor DNA from one animal, suggesting clonal origin of the lymphoma. Our data are most consistent with a pathogenic mechanism in which chronic productive retroviral infection allowed insertional mutagenesis of critical growth control genes, leading to cell transformation and clonal tumor evolution. Images PMID:8207799
-
Cloning of a Gene Whose Expression is Increased in Scrapie and in Senile Plaques in Human Brain
NASA Astrophysics Data System (ADS)
Wietgrefe, S.; Zupancic, M.; Haase, A.; Chesebro, B.; Race, R.; Frey, W.; Rustan, T.; Friedman, R. L.
1985-12-01
A complementary DNA library was constructed from messenger RNA's extracted from the brains of mice infected with the scrapie agent. The library was differentially screened with the objectives of finding clones that might be used as markers of infection and finding clones of genes whose increased expression might be correlated with the pathological changes common to scrapie and Alzheimer's disease. A gene was identified whose expression is increased in scrapie. The complementary DNA corresponding to this gene hybridized preferentially and focally to cells in the brains of scrapie-infected animals. The cloned DNA also hybridized to the neuritic plaques found with increased frequency in brains of patients with Alzheimer's disease.
-
Fang, Ying; Rowland, Raymond R R; Roof, Michael; Lunney, Joan K; Christopher-Hennings, Jane; Nelson, Eric A
2006-12-01
The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States.
-
Fang, Ying; Rowland, Raymond R. R.; Roof, Michael; Lunney, Joan K.; Christopher-Hennings, Jane; Nelson, Eric A.
2006-01-01
The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States. PMID:16971421
-
Monzani, P S; Sangalli, J R; De Bem, T H C; Bressan, F F; Fantinato-Neto, P; Pimentel, J R V; Birgel-Junior, E H; Fontes, A M; Covas, D T; Meirelles, F V
2013-02-28
Recombinant coagulation factor IX must be produced in mammalian cells because FIX synthesis involves translational modifications. Human cell culture-based expression of human coagulation factor IX (hFIX) is expensive, and large-scale production capacity is limited. Transgenic animals may greatly increase the yield of therapeutic proteins and reduce costs. In this study, we used a lentiviral system to obtain transgenic cells and somatic cell nuclear transfer (SCNT) to produce transgenic animals. Lentiviral vectors carrying hFIX driven by 3 bovine β-casein promoters were constructed. Bovine epithelial mammary cells were transduced by lentivirus, selected with blasticidin, plated on extracellular matrix, and induced by lactogenic hormones; promoter activity was evaluated by quantitative PCR. Transcriptional activity of the 5.335-kb promoter was 6-fold higher than the 3.392- and 4.279-kb promoters, which did not significantly differ. Transgenic bovine fibroblasts were transduced with lentivirus carrying the 5.335-kb promoter and used as donor cells for SCNT. Cloned transgenic embryo production yielded development rates of 28.4%, similar to previous reports on cloned non-transgenic embryos. The embryos were transferred to recipient cows (N = 21) and 2 births of cloned transgenic cattle were obtained. These results suggest combination of the lentiviral system and cloning may be a good strategy for production of transgenic cattle.
-
Cen, Junyu; Liu, Xueqin; Li, Jinnian; Zhang, Ming; Wang, Wei
2013-01-01
Vibrio mimicus (V. mimicus) is the causative agent of ascites disease in aquatic animals. Outer membrane protein U (OmpU) is an important antigen of V. mimicus, but its protective epitopes are still unclear. A random 12-mer phage-displayed peptide library was used to screen and identify immunodominant mimotopes of the OmpU protein in V. mimicus by panning against purified OmpU-specific polyclonal antibody. Then the immunogenicity and immunoprotection in fish of these mimotopes was evaluated. Nine positive phage clones presented seven different 12- peptide sequences and more than 50% of them carried a consensus core motif of DSSK-P. These positive clones reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by OmpU protein. Intraperitoneal injection of seven positive phage clones into fish induced a specific antibody response to OmpU protein. The fish immunized respectively with the positive phage clones C17, C24, C60 and C66 obtained 100% immunoprotective effect against experimental V. mimicus challenge. Taken together, these mimotopes presented by clone C17, C24, C60 and C66 were immunodominant mimotopes of the OmpU protein and exhibited a more appropriate candidate as epitope-based vaccine against V. mimicus infection in aquatic animals. Copyright © 2012 Elsevier Ltd. All rights reserved.
-
Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer.
Lanza, R P; Cibelli, J B; Diaz, F; Moraes, C T; Farin, P W; Farin, C E; Hammer, C J; West, M D; Damiani, P
2000-01-01
Approximately 100 species become extinct a day. Despite increasing interest in using cloning to rescue endangered species, successful interspecies nuclear transfer has not been previously described, and only a few reports of in vitro embryo formation exist. Here we show that interspecies nuclear transfer can be used to clone an endangered species with normal karyotypic and phenotypic development through implantation and the late stages of fetal growth. Somatic cells from a gaur bull (Bos gaurus), a large wild ox on the verge of extinction, (Species Survival Plan < 100 animals) were electrofused with enucleated oocytes from domestic cows. Twelve percent of the reconstructed oocytes developed to the blastocyst stage, and 18% of these embryos developed to the fetal stage when transferred to surrogate mothers. Three of the fetuses were electively removed at days 46 to 54 of gestation, and two continued gestation longer than 180 (ongoing) and 200 days, respectively. Microsatellite marker and cytogenetic analyses confirmed that the nuclear genome of the cloned animals was gaurus in origin. The gaur nuclei were shown to direct normal fetal development, with differentiation into complex tissue and organs, even though the mitochondrial DNA (mtDNA) within all the tissue types evaluated was derived exclusively from the recipient bovine oocytes. These results suggest that somatic cell cloning methods could be used to restore endangered, or even extinct, species and populations.
-
Delivery of cloned offspring: experience in Zebu cattle (Bos indicus).
Meirelles, Flávio V; Birgel, Eduardo H; Perecin, Felipe; Bertolini, Marcelo; Traldi, Anneliese S; Pimentel, José Rodrigo V; Komninou, Eliza R; Sangalli, Juliano R; Neto, Paulo Fantinato; Nunes, Mariana Tikuma; Pogliani, Fábio Celidonio; Meirelles, Flávia D P; Kubrusly, Flávia S; Vannucchi, Camila I; Silva, Liege C G
2010-01-01
The production of a healthy cloned calf is dependent on a multitude of successful steps, including reprogramming mediated by the oocyte, the development of a functional placenta, adequate maternal-fetal interaction, the establishment of a physiological metabolic setting and the formation of a complete set of well-differentiated cells that will eventually result in well-characterised and fully competent tissues and organs. Although the efficiency of nuclear transfer has improved significantly since the first report of a somatic cell nuclear transfer-derived animal, there are many descriptions of anomalies concerning cloned calves leading to high perinatal morbidity and mortality. The present article discusses some our experience regarding perinatal and neonatal procedures for cloned Zebu cattle (B. indicus) that has led to improved survival rates in Nellore cloned calves following the application of such 'labour-intensive technology'.
-
Solomon, Louis M; Noll, Rebekka C; Mordkoff, David S; Murphy, Patrick; Rolerson, Marcy
2009-09-01
To meet its public mandate, the US Food and Drug Administration (FDA) collected studies on the potential health hazards of eating or drinking cloned food products. Based on an earlier National Academy of Sciences study that, on closer analysis, was not nearly as sanguine, the FDA's report found no evidence of a health risk from the public's ingestion of cloned food products. This article analyzes the risks the FDA considered, and concludes that there is a disconnect between the risks the FDA assessed in these studies and the risks that might arise from cloned food products. The FDA should consider instituting effective tracking mechanisms and other diagnostics that would permit scientists and the public to answer the question of health risks posed by cloned food products.
-
Huang, Xiao Dan; Tan, Hui Yin; Long, Ruijun; Liang, Juan Boo; Wright, André-Denis G
2012-10-19
Methane emissions by methanogen from livestock ruminants have significantly contributed to the agricultural greenhouse gas effect. It is worthwhile to compare methanogen from "energy-saving" animal (yak) and normal animal (cattle) in order to investigate the link between methanogen structure and low methane production. Diversity of methanogens from the yak and cattle rumen was investigated by analysis of 16S rRNA gene sequences from rumen digesta samples from four yaks (209 clones) and four cattle (205 clones) from the Qinghai-Tibetan Plateau area (QTP). Overall, a total of 414 clones (i.e. sequences) were examined and assigned to 95 operational taxonomic units (OTUs) using MOTHUR, based upon a 98% species-level identity criterion. Forty-six OTUs were unique to the yak clone library and 34 OTUs were unique to the cattle clone library, while 15 OTUs were found in both libraries. Of the 95 OTUs, 93 putative new species were identified. Sequences belonging to the Thermoplasmatales-affiliated Linage C (TALC) were found to dominate in both libraries, accounting for 80.9% and 62.9% of the sequences from the yak and cattle clone libraries, respectively. Sequences belonging to the Methanobacteriales represented the second largest clade in both libraries. However, Methanobrevibacter wolinii (QTPC 110) was only found in the cattle library. The number of clones from the order Methanomicrobiales was greater in cattle than in the yak clone library. Although the Shannon index value indicated similar diversity between the two libraries, the Libshuff analysis indicated that the methanogen community structure of the yak was significantly different than those from cattle. This study revealed for the first time the molecular diversity of methanogen community in yaks and cattle in Qinghai-Tibetan Plateau area in China. From the analysis, we conclude that yaks have a unique rumen microbial ecosystem that is significantly different from that of cattle, this may also help to explain why yak produce less methane than cattle.
-
2012-01-01
Background Methane emissions by methanogen from livestock ruminants have significantly contributed to the agricultural greenhouse gas effect. It is worthwhile to compare methanogen from “energy-saving” animal (yak) and normal animal (cattle) in order to investigate the link between methanogen structure and low methane production. Results Diversity of methanogens from the yak and cattle rumen was investigated by analysis of 16S rRNA gene sequences from rumen digesta samples from four yaks (209 clones) and four cattle (205 clones) from the Qinghai-Tibetan Plateau area (QTP). Overall, a total of 414 clones (i.e. sequences) were examined and assigned to 95 operational taxonomic units (OTUs) using MOTHUR, based upon a 98% species-level identity criterion. Forty-six OTUs were unique to the yak clone library and 34 OTUs were unique to the cattle clone library, while 15 OTUs were found in both libraries. Of the 95 OTUs, 93 putative new species were identified. Sequences belonging to the Thermoplasmatales-affiliated Linage C (TALC) were found to dominate in both libraries, accounting for 80.9% and 62.9% of the sequences from the yak and cattle clone libraries, respectively. Sequences belonging to the Methanobacteriales represented the second largest clade in both libraries. However, Methanobrevibacter wolinii (QTPC 110) was only found in the cattle library. The number of clones from the order Methanomicrobiales was greater in cattle than in the yak clone library. Although the Shannon index value indicated similar diversity between the two libraries, the Libshuff analysis indicated that the methanogen community structure of the yak was significantly different than those from cattle. Conclusion This study revealed for the first time the molecular diversity of methanogen community in yaks and cattle in Qinghai-Tibetan Plateau area in China. From the analysis, we conclude that yaks have a unique rumen microbial ecosystem that is significantly different from that of cattle, this may also help to explain why yak produce less methane than cattle. PMID:23078429
-
Nutritional value of milk and meat products derived from cloning.
Tomé, Daniel; Dubarry, Michel; Fromentin, Gilles
2004-01-01
The development and use of milk and meat products derived from cloning depends on their safety and on the nutritional advantages they can confer to the products as perceived by consumers. The development of such products thus implies (i) to demonstrate their safety and security, (ii) to show that their nutritional value is equivalent to the traditional products, and (iii) to identify the conditions under which cloning could allow additional nutritional and health benefit in comparison to traditional products for the consumers. Both milk and meat products are a source of high quality protein as determined from their protein content and essential amino acid profile. Milk is a source of calcium, phosphorus, zinc, magnesium and vitamin B2 and B12. Meat is a source of iron, zinc and vitamin B12. An important issue regarding the nutritional quality of meat and milk is the level and quality of fat which usually present a high content in saturated fat and some modification of the fat fraction could improve the nutritional quality of the products. The role of the dietary proteins as potential allergens has to be taken into account and an important aspect regarding this question is to evaluate whether the cloning does not produce the appearance of novel allergenic structures. The presence of bio-activities associated to specific components of milk (lactoferrin, immunoglobulins, growth factors, anti-microbial components) also represents a promising development. Preliminary results obtained in rats fed cow's milk or meat-based diets prepared from control animals or from animals derived from cloning did not show any difference between control and cloning-derived products.
-
Cloned ferrets produced by somatic cell nuclear transfer.
Li, Ziyi; Sun, Xingshen; Chen, Juan; Liu, Xiaoming; Wisely, Samantha M; Zhou, Qi; Renard, Jean-Paul; Leno, Gregory H; Engelhardt, John F
2006-05-15
Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (approximately 3-4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink.
-
USDA-ARS?s Scientific Manuscript database
Somatic cell nuclear transfer (SCNT) in animals has been around for nearly 20 years and has been successfully used for cloning of various livestock species. In this study, goat oocytes were collected from large follicles (>3mm) and small follicles (<3mm) to compare the success rate when used in goat...
-
Aging and longevity in the simplest animals and the quest for immortality
Petralia, Ronald S.; Mattson, Mark P.; Yao, Pamela J.
2014-01-01
Here we review the examples of great longevity and potential immortality in the earliest animal types and contrast and compare these to humans and other higher animals. We start by discussing aging in single-celled organisms such as yeast and ciliates, and the idea of the immortal cell clone. Then we describe how these cell clones could become organized into colonies of different cell types that lead to multicellular animal life. We survey aging and longevity in all of the basal metazoan groups including ctenophores (comb jellies), sponges, placozoans, cnidarians (hydras, jellyfish, corals and sea anemones) and myxozoans. Then we move to the simplest bilaterian animals (with a head, three body cell layers, and bilateral symmetry), the two phyla of flatworms. A key determinant of longevity and immortality in most of these simple animals is the large numbers of pluripotent stem cells that underlie the remarkable abilities of these animals to regenerate and rejuvenate themselves. Finally, we discuss briefly the evolution of the higher bilaterians and how longevity was reduced and immortality lost due to attainment of greater body complexity and cell cycle strategies that protect these complex organisms from developing tumors. We also briefly consider how the evolution of multiple aging-related mechanisms/pathwayshinders our ability to understand and modify the aging process in higher organisms. PMID:24910306
-
Ostertag, Eric M.; Kacir, Stephen; Thiboutot, Michelle; Gulendran, Gayathri; Zheng, X. Long; Cines, Douglas B.; Siegel, Don L.
2016-01-01
BACKGROUND Acquired thrombotic thrombocytopenia purpura (TTP) is a life-threatening illness caused by autoantibodies that decrease the activity of ADAMTS13, the von Willebrand Factor cleaving protease. Despite efficacy of plasma exchange, mortality remains high and relapse is common. Improved therapies may come from understanding the diversity of pathogenic autoantibodies on a molecular/genetic level. Cloning comprehensive repertoires of patient autoantibodies can provide the necessary tools for studying immunobiology of disease and developing animal models. STUDY DESIGN AND METHODS Anti-ADAMTS13 antibodies were cloned from four patients with acquired TTP using phage display and characterized with respect to genetic origin, inhibition of ADAMTS13 proteolytic activity, and epitope specificity. Anti-idiotypic antisera raised to a subset of autoantibodies enabled comparison of their relatedness to each other and to polyclonal IgG in patient plasma. RESULTS Fifty-one unique antibodies were isolated comprising epitope specificities resembling the diversity found in circulating patient IgG. Antibodies directed to both the amino terminal domains and those requiring the ADAMTS13 cysteine-rich/spacer region for binding inhibited proteolytic activity, while those solely targeting carboxy-terminal domains were non-inhibitory. Anti-idiotypic antisera raised to a subset of antibody clones crossreacted with and reduced the inhibitory activity of polyclonal IgG from a set of unrelated patients. CONCLUSIONS Anti-ADAMTS13 autoantibodies isolated by repertoire cloning display the diversity of epitope specificities found in patient plasma and provide tools for developing animal models of acquired TTP. Shared idiotypes of inhibitory clones with circulating IgG from multiple patients suggest common features of pathogenic autoantibodies that could be exploited for developing more targeted therapies. PMID:27040144
-
Cloned ferrets produced by somatic cell nuclear transfer
Li, Ziyi; Sun, Xingshen; Chen, Juan; Liu, Xiaoming; Wisely, Samantha M.; Zhou, Qi; Renard, Jean-Paul; Leno, Gregory H.; Engelhardt, John F.
2007-01-01
Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (~3–4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink. PMID:16584722
-
Ju, Huiming; Zhang, Jiaqing; Bai, Lijing; Mu, Yulian; Du, Yutao; Yang, Wenxian; Li, Yong; Sheng, Anzhi; Li, Kui
2015-01-01
Sustained expression of the GH gene has been shown to have detrimental effects on the health of animals. In the current study, transgenic founder pigs, with controllable pig growth hormone (pGH) expression, were cloned via the handmade cloning method (HMC), and pGH expression levels were examined at the cellular and organismal levels. The serum pGH levels in 3 founder male pigs were found to be significantly higher after induction with intramuscular injection of doxycycline (DOX) compared to baseline. A daily dose of DOX was administered via feed to these animals for a period of 65 to 155 days. The growth rate, feed efficiency and pGH serum concentration increased in the DOX-induced transgenic group compared with the other groups. 8 numbers of animals were euthanized and the dressing percentage, loin muscle and lean meat percentage were significantly higher in the DOX-induced F1 transgenic group compared with the other groups. In this study a large population of transgenic pigs, with integrated controllable expression of a transgene, was obtained. The transgenic pigs were healthy and normal in terms of reproductive capability. At the same time, feed efficiency was improved, production processes were accelerated and meat yield was increased. PMID:25959098
-
Ju, Huiming; Zhang, Jiaqing; Bai, Lijing; Mu, Yulian; Du, Yutao; Yang, Wenxian; Li, Yong; Sheng, Anzhi; Li, Kui
2015-05-11
Sustained expression of the GH gene has been shown to have detrimental effects on the health of animals. In the current study, transgenic founder pigs, with controllable pig growth hormone (pGH) expression, were cloned via the handmade cloning method (HMC), and pGH expression levels were examined at the cellular and organismal levels. The serum pGH levels in 3 founder male pigs were found to be significantly higher after induction with intramuscular injection of doxycycline (DOX) compared to baseline. A daily dose of DOX was administered via feed to these animals for a period of 65 to 155 days. The growth rate, feed efficiency and pGH serum concentration increased in the DOX-induced transgenic group compared with the other groups. 8 numbers of animals were euthanized and the dressing percentage, loin muscle and lean meat percentage were significantly higher in the DOX-induced F1 transgenic group compared with the other groups. In this study a large population of transgenic pigs, with integrated controllable expression of a transgene, was obtained. The transgenic pigs were healthy and normal in terms of reproductive capability. At the same time, feed efficiency was improved, production processes were accelerated and meat yield was increased.
-
Genotyping of Global Yersinia Pestis Isolates by Using IS285
2006-11-01
clones and to detect their geographical/ animal origin. 1. INTRODUCTION Yersinia pestis is the causative agent of plague circulating in natural...foci among about 200 species of rodents and lagomorphs. Humans usually become infected from animals via fleabites and display the bubonic form of...philogenetic relationships between Y. pestis strains, their potential geographical and animal origin. 2. GENOTYPING OF Y. PESTIS – CLUSTERING
-
Korzh, Vladimir; Strähle, Uwe
2002-08-01
A hundred years ago, Dr. Marshall A. Barber proposed a new technique - the microinjection technique. He developed this method initially to clone bacteria and to confirm the germ theory of Koch and Pasteur. Later on, he refined his approach and was able to manipulate nuclei in protozoa and to implant bacteria into plant cells. Continuous improvement and adaptation of this method to new applications dramatically changed experimental embryology and cytology and led to the formation of several new scientific disciplines including animal cloning as one of its latest applications. Interestingly, microinjection originated as a method at the crossroad of bacteriology and plant biology, demonstrating once again the unforeseen impact that basic research in an unrelated field can have on the development of entirely different disciplines.
-
Handmade Cloned Transgenic Piglets Expressing the Nematode Fat-1 Gene
Zhang, Peng; Zhang, Yidi; Dou, Hongwei; Yin, Jingdong; Chen, Yu; Pang, Xinzhi; Vajta, Gabor; Bolund, Lars
2012-01-01
Abstract Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been adapted worldwide, but this application is somewhat limited by its relatively low efficiency. In this study, we used handmade cloning (HMC) established previously to produce transgenic pigs that express the functional nematode fat-1 gene. Codon-optimized mfat-1 was inserted into eukaryotic expression vectors, which were transferred into primary swine donor cells. Reverse transcriptase PCR (RT-PCR), gas chromatography, and chromosome analyses were performed to select donor clones capable of converting n-6 into n-3 fatty acids. Blastocysts derived from the clones that lowered the n-6/n-3 ratio to approximately 1:1 were transferred surgically into the uteri of recipients for transgenic piglets. By HMC, 37% (n=558) of reconstructed embryos developed to the blastocyst stage after 7 days of culture in vitro, with an average cell number of 81±36 (n=14). Three recipients became pregnant after 408 day-6 blastocysts were transferred into four naturally cycling females, and a total of 14 live offspring were produced. The nematode mfat-1 effectively lowered the n-6/n-3 ratio in muscle and major organs of the transgenic pig. Our results will help to establish a reliable procedure and an efficient option in the production of transgenic animals. PMID:22686479
-
Costa, Lourena Emanuele; Goulart, Luiz Ricardo; Pereira, Nathália Cristina de Jesus; Lima, Mayara Ingrid Sousa; Duarte, Mariana Costa; Martins, Vivian Tamietti; Lage, Paula Sousa; Menezes-Souza, Daniel; Ribeiro, Tatiana Gomes; Melo, Maria Norma; Fernandes, Ana Paula; Soto, Manuel; Tavares, Carlos Alberto Pereira; Chávez-Fumagalli, Miguel Angel; Coelho, Eduardo Antonio Ferraz
2014-01-01
The development of cost-effective prophylactic strategies to prevent leishmaniasis has become a high-priority. The present study has used the phage display technology to identify new immunogens, which were evaluated as vaccines in the murine model of visceral leishmaniasis (VL). Epitope-based immunogens, represented by phage-fused peptides that mimic Leishmania infantum antigens, were selected according to their affinity to antibodies from asymptomatic and symptomatic VL dogs' sera. Twenty phage clones were selected after three selection cycles, and were evaluated by means of in vitro assays of the immune stimulation of spleen cells derived from naive and chronically infected with L. infantum BALB/c mice. Clones that were able to induce specific Th1 immune response, represented by high levels of IFN-γ and low levels of IL-4 were selected, and based on their selectivity and specificity, two clones, namely B10 and C01, were further employed in the vaccination protocols. BALB/c mice vaccinated with clones plus saponin showed both a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with individual clones or L. infantum extracts. Additionally, these animals, when compared to control groups (saline, saponin, wild-type phage plus saponin, or non-relevant phage clone plus saponin), showed significant reductions in the parasite burden in the liver, spleen, bone marrow, and paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, mainly by CD8+ T cells, against parasite proteins. These animals also presented decreased parasite-mediated IL-4 and IL-10 responses, and increased levels of parasite-specific IgG2a antibodies. This study describes two phage clones that mimic L. infantum antigens, which were directly used as immunogens in vaccines and presented Th1-type immune responses, and that significantly reduced the parasite burden. This is the first study that describes phage-displayed peptides as successful immunogens in vaccine formulations against VL.
-
Post-mortem re-cloning of a transgenic red fluorescent protein dog.
Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo; Lee, Byeong-Chun
2011-12-01
Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.
-
Restoration of Viral Immunity in Immunodeficient Humans by the Adoptive Transfer of T Cell Clones
NASA Astrophysics Data System (ADS)
Riddell, Stanley R.; Watanabe, Kathe S.; Goodrich, James M.; Li, Cheng R.; Agha, Mounzer E.; Greenberg, Philip D.
1992-07-01
The adoptive transfer of antigen-specific T cells to establish immunity is an effective therapy for viral infections and tumors in animal models. The application of this approach to human disease would require the isolation and in vitro expansion of human antigen-specific T cells and evidence that such T cells persist and function in vivo after transfer. Cytomegalovirus-specific CD8^+ cytotoxic T cell (CTL) clones could be isolated from bone marrow donors, propagated in vitro, and adoptively transferred to immunodeficient bone marrow transplant recipients. No toxicity developed and the clones provided persistent reconstitution of CD8^+ cytomegalovirus-specific CTL responses.
-
[Media, cloning, and bioethics].
Costa, S I; Diniz, D
2000-01-01
This article was based on an analysis of three hundred articles from mainstream Brazilian periodicals over a period of eighteen months, beginning with the announcement of the Dolly case in February 1997. There were two main objectives: to outline the moral constants in the press associated with the possibility of cloning human beings and to identify some of the moral assumptions concerning scientific research with non-human animals that were published carelessly by the media. The authors conclude that there was a haphazard spread of fear concerning the cloning of human beings rather than an ethical debate on the issue, and that there is a serious gap between bioethical reflections and the Brazilian media.
-
Fujimura, Tatsuya; Kurome, Mayuko; Murakami, Hiroshi; Takahagi, Yoichi; Matsunami, Katsuyoshi; Shimanuki, Shinichi; Suzuki, Kohei; Miyagawa, Shuji; Shirakura, Ryota; Shigehisa, Tamotsu; Nagashima, Hiroshi
2004-01-01
The present paper describes production of cloned pigs from fibroblast cells of transgenic pigs expressing human decay accelerating factor (DAF, CD55) and N-acetylglucosaminyltransferase III (GnT-III) that remodels sugar-chain biosynthesis. Two nuclear transfer protocols were used: a two-step activation (TA) method and a delayed activation (DA) method. Enucleated in vitro-matured oocytes and donor cells were electrically fused in a calcium-containing medium by TA method or in a calcium-free medium by DA method, followed by electrical activation 1-1.5 h later, respectively. In vitro blastocyst formation rates of nuclear transferred embryos reconstructed by TA and DA method were 8% and 14%, respectively. As a result of embryo transfer of the reconstructed embryos made by each method into recipient pigs, both gave rise to cloned piglets. These cloned pigs expressed transgene as much as their nuclear donor cells. In conclusions, (1) pig cloning can be carried out by TA or DA nuclear transfer methods, (2) expression of transgenes can be maintained to cloned pigs from the nuclear donor cells derived from transgenic animals.
-
Cloning, Characterization, and Expression of Animal Toxin Genes for Vaccine Development
1990-04-27
amino acids have been modified to pyroglutamate . The function of this modification is not known. There are additional venom components that are...been constructed from the messenger ribonucleic acid (mRNA) isolated from venom glands of different poisonous animals such as snakes, scorpions, and...ribonucleic acid (mRNA) isolated from venom glands of different poisonous animals such as snakes, scorpions, and snails. The gene banks thus created con
-
Methicillin-resistant Staphylococcus aureus among animals: current overview.
Aires-de-Sousa, M
2017-06-01
Currently, methicillin-resistant Staphylococcus aureus (MRSA) is a universal threat. After being well established in the healthcare setting, it has emerged in the community among people with no risk factors for MRSA acquisition, therefore imposing a new threat. The subsequent detection of MRSA colonizing or infecting animals as well as in food of animal origin was of major concern, revealing new reservoirs for MRSA. The major MRSA clonal lineages circulating in the different settings, i.e. in hospitals, in the community and among animals, are described here, differentiating between clones colonizing companion and food-chain animals. Particular attention is given to the widely spread livestock-associated MRSA clonal complex (CC) 398, which is mainly associated with professional exposure but may be of high pathogenicity. The recent detection of a mecA homologue, designated mecC, with a wide geographical distribution in Europe, and including a large diversity of hosts (food-chain, companion and wildlife animals and also detected in water samples) adds to the threat. Domestication as well as globalization of the livestock industry have intensified exchanges between human and animal bacteria. We report here several cases of transmission of MRSA between companion or food-chain animals and humans, as well as some MRSA clones of human origin that have adapted to new animal hosts eventually by losing useless virulence factors or acquiring new mobile genetic elements. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
-
Genetically engineered livestock for biomedical models.
Rogers, Christopher S
2016-06-01
To commemorate Transgenic Animal Research Conference X, this review summarizes the recent progress in developing genetically engineered livestock species as biomedical models. The first of these conferences was held in 1997, which turned out to be a watershed year for the field, with two significant events occurring. One was the publication of the first transgenic livestock animal disease model, a pig with retinitis pigmentosa. Before that, the use of livestock species in biomedical research had been limited to wild-type animals or disease models that had been induced or were naturally occurring. The second event was the report of Dolly, a cloned sheep produced by somatic cell nuclear transfer. Cloning subsequently became an essential part of the process for most of the models developed in the last 18 years and is stilled used prominently today. This review is intended to highlight the biomedical modeling achievements that followed those key events, many of which were first reported at one of the previous nine Transgenic Animal Research Conferences. Also discussed are the practical challenges of utilizing livestock disease models now that the technical hurdles of model development have been largely overcome.
-
Dasytricha dominance in Surti buffalo rumen revealed by 18S rRNA sequences and real-time PCR assay.
Singh, K M; Tripathi, A K; Pandya, P R; Rank, D N; Kothari, R K; Joshi, C G
2011-09-01
The genetic diversity of protozoa in Surti buffalo rumen was studied by amplified ribosomal DNA restriction analysis, 18S rDNA sequence homology and phylogenetic and Real-time PCR analysis methods. Three animals were fed diet comprised green fodder Napier bajra 21 (Pennisetum purpureum), mature pasture grass (Dicanthium annulatum) and concentrate mixture (20% crude protein, 65% total digestible nutrients). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360 bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. A total of 91 clones were examined and identified 14 different 18S RNA sequences based on PCR-RFLP pattern. These 14 phylotypes were distributed into four genera-based 18S rDNA database sequences and identified as Dasytricha (57 clones), Isotricha (14 clones), Ostracodinium (11 clones) and Polyplastron (9 clones). Phylogenetic analyses were also used to infer the makeup of protozoa communities in the rumen of Surti buffalo. Out of 14 sequences, 8 sequences (69 clones) clustered with the Dasytricha ruminantium-like clone and 4 sequences (13 clones) were also phylogenetically placed with the Isotricha prostoma-like clone. Moreover, 2 phylotypes (9 clones) were related to Polyplastron multivesiculatum-like clone. In addition, the number of 18S rDNA gene copies of Dasytricha ruminantium (0.05% to ciliate protozoa) was higher than Entodinium sp. (2.0 × 10(5) vs. 1.3 × 10(4)) in per ml ruminal fluid.
-
Maiorka, Paulo Cesar; Favaron, Phelipe Oliveira; Mess, Andrea Maria; dos Santos, Caio Rodrigues; Alberto, Miryan Lanca; Meirelles, Flavio Vieira; Miglino, Maria Angelica
2015-01-01
Although assisted reproductive techniques are commonly applied in humans and animals, they are frequently associated with major developmental deficits and reduced viability. To explore abnormalities associated with cloning or nuclear transfer (NT) as the most invasive of these methods, we used a bovine model to characterize abnormalities. Detailed necropsy examinations were done on 13 calves that died soon after birth; in addition, we included data from embryos and fetuses (produced by NT) that terminated prematurely. Bovine clones that survived until the neonatal period differed quantitatively and qualitatively from in-vivo-derived cattle. Although alterations affected a variety of organs (e.g. heart, lung and liver), there was a clear association with abberant vascular developmental during the early intrauterine phase. Therefore, we concluded that vascular problems were key alterations induced by cloning (presumably via epigenetic modifications).
-
Favaron, Phelipe Oliveira; dos Santos, Caio Rodrigues; Alberto, Miryan Lanca; Meirelles, Flavio Vieira; Miglino, Maria Angelica
2015-01-01
Although assisted reproductive techniques are commonly applied in humans and animals, they are frequently associated with major developmental deficits and reduced viability. To explore abnormalities associated with cloning or nuclear transfer (NT) as the most invasive of these methods, we used a bovine model to characterize abnormalities. Detailed necropsy examinations were done on 13 calves that died soon after birth; in addition, we included data from embryos and fetuses (produced by NT) that terminated prematurely. Bovine clones that survived until the neonatal period differed quantitatively and qualitatively from in-vivo-derived cattle. Although alterations affected a variety of organs (e.g. heart, lung and liver), there was a clear association with abberant vascular developmental during the early intrauterine phase. Therefore, we concluded that vascular problems were key alterations induced by cloning (presumably via epigenetic modifications). PMID:25584533
-
Birth of cloned mice from vaginal smear cells after somatic cell nuclear transfer.
Kuwayama, Hiroki; Tanabe, Yoshiaki; Wakayama, Teruhiko; Kishigami, Satoshi
2017-05-01
Less invasive methods for donor cell collection will facilitate reproduction of wild animals using somatic-cell nuclear transfer. Stages of the estrous cycle in mice have long been studies using somatic cells that can be collected from vaginal walls using cotton tipped swabs in a relatively non-invasive manner. In this study, we examined the feasibility of these cells as sources of nuclei for somatic-cell cloning using nuclear transfer. Estrous cycles generally comprise proestrus, estrus, metestrus, and diestrus stages. In the present experiments, more than 60% of cells were nucleated in vaginal smears from all but the estrus stage. However, after somatic-cell nuclear transfer of cells from proestrus, metestrus, and diestrus stages, 66%, 50%, and 72% of cloned embryos developed to the morula/blastocyst, and cloned female mouse birth rates after embryo transfer were 1.5%, 0.3%, and 1%, respectively. These results show that noninvasively collected vaginal smears contain somatic cells that can be used to clone female mice. Copyright © 2017. Published by Elsevier Inc.
-
Production of the first cloned camel by somatic cell nuclear transfer.
Wani, Nisar A; Wernery, U; Hassan, F A H; Wernery, R; Skidmore, J A
2010-02-01
In this study, we demonstrate the use of somatic cell nuclear transfer to produce the first cloned camelid, a dromedary camel (Camelus dromedarius) belonging to the family Camelidae. Donor karyoplasts were obtained from adult skin fibroblasts, cumulus cells, or fetal fibroblasts, and in vivo-matured oocytes, obtained from preovulatory follicles of superstimulated female camels by transvaginal ultrasound guided ovum pick-up, were used as cytoplasts. Reconstructed embryos were cultured in vitro for 7 days up to the hatching/hatched blastocyst stage before they were transferred to synchronized recipients on Day 6 after ovulation. Pregnancies were achieved from the embryos reconstructed from all cell types, and a healthy calf, named Injaz, was born from the pregnancy by an embryo reconstructed with cumulus cells. Genotype analyses, using 25 dromedary camel microsatellite markers, confirmed that the cloned calf was derived from the donor cell line and the ovarian tissue. In conclusion, the present study reports, for the first time, establishment of pregnancies and birth of the first cloned camelid, a dromedary camel (C. dromedarius), by use of somatic cell nuclear transfer. This has opened doors for the amelioration and preservation of genetically valuable animals like high milk producers, racing champions, and males of high genetic merit in camelids. We also demonstrated, for the first time, that adult and fetal fibroblasts can be cultured, expanded, and frozen without losing their ability to support the development of nuclear transfer embryos, a technology that may potentially be used to modify fibroblast genome by homologous recombination so as to generate genetically altered cloned animals.
-
Watanabe, Shinya
2013-09-01
To increase public understanding on cloned cattle derived from somatic cell nuclear transfer (SCNT), the present review describes the effect of calf death loss on an SCNT cattle herd. The incidence of death loss in SCNT cattle surviving more than 200 days reached the same level as that in conventionally bred cattle. This process could be considered as removal of SCNT cattle with congenital defects caused by calf death loss. As a result of comparative studies of SCNT cattle and conventionally bred cattle, the substantial equivalences in animal health status, milk and meat productive performance have been confirmed. Both sexes of SCNT cattle surviving to adulthood were fertile and their reproductive performance, including efficiency of progeny production, was the same as that in conventionally bred cattle. The presence of substantial equivalence between their progeny and conventionally bred cattle also existed. Despite these scientific findings, the commercial use of food products derived from SCNT cattle and their progeny has not been allowed by governments for reasons including the lack of public acceptance of these products and the low efficiency of animal SCNT. To overcome this situation, communication of the low risk of SCNT technology and research to improve SCNT efficiency are required. © 2013 Japanese Society of Animal Science.
-
Keith's MAGIC: Cloning and the Cell Cycle.
Wells, D N
2013-10-01
Abstract Professor Keith Campbell's critical contribution to the discovery that a somatic cell from an adult animal can be fully reprogrammed by oocyte factors to form a cloned individual following nuclear transfer (NT)(Wilmut et al., 1997 ) overturned a dogma concerning the reversibility of cell fate that many scientists had considered to be biologically impossible. This seminal experiment proved the totipotency of adult somatic nuclei and finally confirmed that adult cells could differentiate without irreversible changes to the genetic material.
-
Pantosti, Annalisa
2012-01-01
Staphylococcus aureus is a typical human pathogen. Some animal S. aureus lineages have derived from human strains following profound genetic adaptation determining a change in host specificity. Due to the close relationship of animals with the environmental microbiome and resistome, animal staphylococcal strains also represent a source of resistance determinants. Methicillin-resistant S. aureus (MRSA) emerged 50 years ago as a nosocomial pathogen but in the last decade it has also become a frequent cause of infections in the community. The recent finding that MRSA frequently colonizes animals, especially livestock, has been a reason for concern, as it has revealed an expanded reservoir of MRSA. While MRSA strains recovered from companion animals are generally similar to human nosocomial MRSA, MRSA strains recovered from food animals appear to be specific animal-adapted clones. Since 2005, MRSA belonging to ST398 was recognized as a colonizer of pigs and human subjects professionally exposed to pig farming. The “pig” MRSA was also found to colonize other species of farmed animals, including horses, cattle, and poultry and was therefore designated livestock-associated (LA)-MRSA. LA-MRSA ST398 can cause infections in humans in contact with animals, and can infect hospitalized people, although at the moment this occurrence is relatively rare. Other animal-adapted MRSA clones have been detected in livestock, such as ST1 and ST9. Recently, ST130 MRSA isolated from bovine mastitis has been found to carry a novel mecA gene that eludes detection by conventional PCR tests. Similar ST130 strains have been isolated from human infections in UK, Denmark, and Germany at low frequency. It is plausible that the increased attention to animal MRSA will reveal other strains with peculiar characteristics that can pose a risk to human health. PMID:22509176
-
Bernstein, K E; Pavirani, A; Alexander, C; Jacobsen, F; Fitzmaurice, L; Mage, R
1983-01-01
Rabbits were infected by Trypanosoma equiperdum and the splenic mRNA was isolated. In vitro translation of this RNA and immunoprecipitation with anti-light chain, anti-heavy chain, anti-mu and anti-VH antibodies demonstrated that T. equiperdum infection elicits large quantities of splenic mRNA encoding mu and kappa chains. The mu and gamma heavy chains and the kappa light chains synthesized in the cell-free translation system were specifically immunoprecipitated by antisera to heavy chain VHa and light chain kappa b allotypes. In vitro labeling of spleen cells from trypanosome-infected animals demonstrated that the biosynthetically labeled IgM has a mu chain of higher molecular weight than the mu chain synthesized by in vitro translation, a difference that is largely abolished when cellular glycosylation is blocked with the antibiotic tunicamycin. Enrichment for heavy chain or light chain mRNA was achieved by fractionating mRNA from trypanosome-infected animals on a sucrose gradient. cDNA clones carrying mu heavy chain sequences were produced using a 'one tube' protocol and identified by cross species hybridization and hybridization selection. Infection of rabbits with T. equiperdum followed by sucrose gradient enrichment of splenic mRNA has provided sufficient quantities of mRNA encoding mu heavy chain suitable for cDNA cloning.
-
Post-mortem re-cloning of a transgenic red fluorescent protein dog
Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo
2011-01-01
Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification. PMID:22122908
-
Using somatic-cell nuclear transfer to study aging.
Kishigami, Satoshi; Lee, Ah Reum; Wakayama, Teruhiko
2013-01-01
In mammals, a diploid genome following fertilization of haploid cells, an egg, and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual's inevitable demise. Since it was first reported in 1997 that Dolly the sheep had been cloned, many mammalian species have been cloned successfully using somatic-cell nuclear transfer (SCNT). The success of SCNT in mammals enables us not only to reproduce offspring without germ cells, that is, to "passage" a unique diploid genome, but also to address valuable biological questions on development, nuclear reprogramming, and epigenetic memory. Successful cloning can also support epigenetic reprogramming where the aging clock is reset or reversed. Recent work using iPS cell technology has explored the practicality and led to the recapitulation of premature aging with iPSCs from progeroid laminopathies. As a result, reprogramming tools are also expected to contribute to studying biological age. However, the efficiency of animal cloning is still low in most cases and the mechanism of reprogramming in cloned embryos is still largely unclear. Here, based on recent advances, we describe an improved, more efficient mouse cloning protocol using histone deacetylase inhibitors (HDACis) and latrunculin A, which increases the success rates of producing cloned mice or establishing ES cells fivefold. This improved method of cloning will provide a strong tool to address many issues including biological aging more easily and with lower cost.
-
Assessment of mitochondrial functions in Daphnia pulex clones using high-resolution respirometry.
Kake-Guena, Sandrine A; Touisse, Kamal; Vergilino, Roland; Dufresne, France; Blier, Pierre U; Lemieux, Hélène
2015-06-01
The objectives of our study were to adapt a method to measure mitochondrial function in intact mitochondria from the small crustacean Daphnia pulex and to validate if this method was sensitive enough to characterize mitochondrial metabolism in clones of the pulex complex differing in ploidy levels, mitochondrial DNA haplotypes, and geographic origins. Daphnia clones belonging to the Daphnia pulex complex represent a powerful model to delineate the link between mitochondrial DNA evolution and mitochondrial phenotypes, as single genotypes with divergent mtDNA can be grown under various experimental conditions. Our study included two diploid clones from temperate environments and two triploid clones from subarctic environments. The whole animal permeabilization and measurement of respiration with high-resolution respirometry enabled the measurement of the functional capacity of specific mitochondrial complexes in four clones. When expressing the activity as ratios, our method detected significant interclonal variations. In the triploid subarctic clone from Kuujjurapik, a higher proportion of the maximal physiological oxidative phosphorylation (OXPHOS) capacity of mitochondria was supported by complex II, and a lower proportion by complex I. The triploid subarctic clone from Churchill (Manitoba) showed the lowest proportion of the maximal OXPHOS supported by complex II. Additional studies are required to determine if these differences in mitochondrial functions are related to differences in mitochondrial haplotypes or ploidy level and if they might be associated with fitness divergences and therefore selective value. © 2015 Wiley Periodicals, Inc.
-
Nikiforuk, Aidan M; Leung, Anders; Cook, Bradley W M; Court, Deborah A; Kobasa, Darwyn; Theriault, Steven S
2016-10-01
Viral Infectious clone systems serve as robust platforms to study viral gene or replicative function by reverse genetics, formulate vaccines and adapt a wild type-virus to an animal host. Since the development of the first viral infectious clone system for the poliovirus, novel strategies of viral genome construction have allowed for the assembly of viral genomes across the identified viral families. However, the molecular profiles of some viruses make their genome more difficult to construct than others. Two factors that affect the difficulty of infectious clone construction are genome length and genome complexity. This work examines the available strategies for overcoming the obstacles of assembling the long and complex RNA genomes of coronaviruses and reports one-step construction of an infectious clone system for the Middle East Respiratory Syndrome coronavirus (MERS-CoV) by homologous recombination in S. cerevisiae. Future use of this methodology will shorten the time between emergence of a novel viral pathogen and construction of an infectious clone system. Completion of a viral infectious clone system facilitates further study of a virus's biology, improvement of diagnostic tests, vaccine production and the screening of antiviral compounds. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.
-
How to improve the success rate of mouse cloning technology.
Thuan, Nguyen Van; Kishigami, Satoshi; Wakayama, Teruhiko
2010-02-01
It has now been 13 years since the first cloned mammal Dolly the sheep was generated from somatic cells using nuclear transfer (SCNT). Since then, this technique has been considered an important tool not only for animal reproduction but also for regenerative medicine. However, the success rate is still very low and the mechanisms involved in genomic reprogramming are not yet clear. Moreover, the NT technique requires donated fresh oocyte, which raises ethical problems for production of human cloned embryo. For this reason, the use of induced pluripotent stem cells for genomic reprogramming and for regenerative medicine is currently a hot topic in this field. However, we believe that the NT approach remains the only valid way for the study of reproduction and basic biology. For example, only the NT approach can reveal dynamic and global modifications in the epigenome without using genetic modification, and it can generate offspring from a single cell or even a frozen dead body. Thanks to much hard work by many groups, cloning success rates are increasing slightly year by year, and NT cloning is now becoming a more applicable method. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.
-
A New, Dynamic Era for Somatic Cell Nuclear Transfer?
Loi, Pasqualino; Iuso, Domenico; Czernik, Marta; Ogura, Atsuo
2016-10-01
Cloning animals by somatic cell nuclear transfer (SCNT) has remained an uncontrollable process for many years. High rates of embryonic losses, stillbirths, and postnatal mortality have been typical outcomes. These developmental problems arise from abnormal genomic reprogramming: the capacity of the oocyte to reset the differentiated memory of a somatic cell. However, effective reprogramming strategies are now available. These target the whole genome or single domains such as the Xist gene, and their effectiveness has been validated with the ability of experimental animals to develop to term. Thus, SCNT has become a controllable process that can be used to 'rescue' endangered species, and for biomedical research such as therapeutic cloning and the isolation of induced pluripotent stem cells (iPSCs). Copyright © 2016 Elsevier Ltd. All rights reserved.
-
2011-01-01
Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool. PMID:21314965
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Chae, Jung-Il; Cho, Young Keun; Cho, Seong-Keun
The potential medical applications of animal cloning include xenotransplantation, but the complex molecular cascades that control porcine organ development are not fully understood. Still, it has become apparent that organs derived from cloned pigs may be suitable for transplantation into humans. In this study, we examined the pancreas of an adult cloned pig developed through somatic cell nuclear transfer (SCNT) using two-dimensional electrophoresis (2-DE) and Western blotting. Proteomic analysis revealed 69 differentially regulated proteins, including such apoptosis-related species as annexins, lamins, and heat shock proteins, which were unanimously upregulated in the SCNT sample. Among the downregulated proteins in SCNT pancreasmore » were peroxiredoxins and catalase. Western blot results indicate that several antioxidant enzymes and the anti-apoptotic protein were downregulated in SCNT pancreas, whereas several caspases were upregulated. Together, these data suggest that the accumulation of reactive oxygen species (ROS) in the pancreas of an adult cloned pig leads to apoptosis.« less
-
Production of cloned calves using roscovitine-treated adult somatic cells as donors.
Miyoshi, Kazuchika; Arat, Sezen; Stice, Steven L
2006-01-01
The stage of the donor cell cycle is a major factor in the success of cloning. Quiescent cells arrested in the G0/G1 phases of the cell cycle by either serum starvation or growth arrest when cultured cells reach confluence have been used as donors to produce cloned animals. Recently, we have developed a novel and effective method using roscovitine to synchronize adult bovine granulosa cells in the G0/G1 cell cycle stage. The resulting fetal and calf survival after transfer of cloned embryos was enhanced in the roscovitine-treated group compared with serum-starved controls. The methods described in this chapter outline (1) the preparation of donor cells, (2) the preparation of recipient oocytes, and (3) the production of cloned embryos. The first section involves methods for the preparation of donor cell stocks from isolated granulosa cells and the roscovitine treatment of the cells before nuclear transfer. The second section explains procedures of in vitro maturation of recipient oocytes. The last section involves methods for the production of cell-oocyte complexes, the fusion of the complexes, and the activation, in vitro culture, and transfer into recipient females of cloned embryos.
-
Salmonella and antimicrobial resistance in an animal-based agriculture river system.
Palhares, Julio Cesar Pascale; Kich, Jalusa D; Bessa, Marjo C; Biesus, Luiza L; Berno, Lais G; Triques, Nelise J
2014-02-15
The aim of this study was to examine the Salmonella serovars and antimicrobial resistance within an animal-based agriculture river system. The study area consisted of a 1,345 ha upper part of Pinhal catchment. A total of 384 samples were collected in four years of monitoring. Salmonella was isolated from 241 samples (62.7%), resulting in 324 isolates. The highest number of Salmonella sp. occurred in samples associated with sites with high stoking density animal unit per hectare. It was possible to demonstrate the variability of serovars in the study area: 30 different serovars were found and at least 11 per monitoring site. Thirty-three potentially related isolates were genotyped by PFGE, one major clone was observed in serovar Typhimurium, which occurred in animal feces (swine and bovine), and different sites and samplings proving the cross-contamination and persistence of this specific clone. Among 180 isolates submitted to an antimicrobial susceptibility test, 50.5% were susceptible to all 21 antimicrobials tested and 54 different profiles were found. In the current study, 49.5% of the tested isolates were resistant to at least one antimicrobial, and multi-resistance occurred in 18% of isolates. Results indicate a close interaction between animal-based agriculture, Salmonella, and antimicrobial resistance. Copyright © 2013 Elsevier B.V. All rights reserved.
-
Costa, Lourena Emanuele; Goulart, Luiz Ricardo; Pereira, Nathália Cristina de Jesus; Lima, Mayara Ingrid Sousa; Duarte, Mariana Costa; Martins, Vivian Tamietti; Lage, Paula Sousa; Menezes-Souza, Daniel; Ribeiro, Tatiana Gomes; Melo, Maria Norma; Fernandes, Ana Paula; Soto, Manuel; Tavares, Carlos Alberto Pereira; Chávez-Fumagalli, Miguel Angel; Coelho, Eduardo Antonio Ferraz
2014-01-01
Background The development of cost-effective prophylactic strategies to prevent leishmaniasis has become a high-priority. The present study has used the phage display technology to identify new immunogens, which were evaluated as vaccines in the murine model of visceral leishmaniasis (VL). Epitope-based immunogens, represented by phage-fused peptides that mimic Leishmania infantum antigens, were selected according to their affinity to antibodies from asymptomatic and symptomatic VL dogs' sera. Methodology/Main Findings Twenty phage clones were selected after three selection cycles, and were evaluated by means of in vitro assays of the immune stimulation of spleen cells derived from naive and chronically infected with L. infantum BALB/c mice. Clones that were able to induce specific Th1 immune response, represented by high levels of IFN-γ and low levels of IL-4 were selected, and based on their selectivity and specificity, two clones, namely B10 and C01, were further employed in the vaccination protocols. BALB/c mice vaccinated with clones plus saponin showed both a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with individual clones or L. infantum extracts. Additionally, these animals, when compared to control groups (saline, saponin, wild-type phage plus saponin, or non-relevant phage clone plus saponin), showed significant reductions in the parasite burden in the liver, spleen, bone marrow, and paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, mainly by CD8+ T cells, against parasite proteins. These animals also presented decreased parasite-mediated IL-4 and IL-10 responses, and increased levels of parasite-specific IgG2a antibodies. Conclusions/Significance This study describes two phage clones that mimic L. infantum antigens, which were directly used as immunogens in vaccines and presented Th1-type immune responses, and that significantly reduced the parasite burden. This is the first study that describes phage-displayed peptides as successful immunogens in vaccine formulations against VL. PMID:25333662
-
Rescue of a Porcine Anellovirus (Torque Teno Sus Virus 2) from Cloned Genomic DNA in Pigs
Huang, Yao-Wei; Patterson, Abby R.; Opriessnig, Tanja; Dryman, Barbara A.; Gallei, Andreas; Harrall, Kylie K.; Vaughn, Eric M.; Roof, Michael B.
2012-01-01
Anelloviruses are a group of single-stranded circular DNA viruses infecting humans and other animal species. Animal models combined with reverse genetic systems of anellovirus have not been developed. We report here the construction and initial characterization of full-length DNA clones of a porcine anellovirus, torque teno sus virus 2 (TTSuV2), in vitro and in vivo. We first demonstrated that five cell lines, including PK-15 cells, are free of TTSuV1 or TTSuV2 contamination, as determined by a real-time PCR and an immunofluorescence assay (IFA) using anti-TTSuV antibodies. Recombinant plasmids harboring monomeric or tandem-dimerized genomic DNA of TTSuV2 from the United States and Germany were constructed. Circular TTSuV2 genomic DNA with or without introduced genetic markers and tandem-dimerized TTSuV2 plasmids were transfected into PK-15 cells, respectively. Splicing of viral mRNAs was identified in transfected cells. Expression of TTSuV2-specific open reading frame 1 (ORF1) in cell nuclei, especially in nucleoli, was detected by IFA. However, evidence of productive TTSuV2 infection was not observed in 12 different cell lines transfected with the TTSuV2 DNA clones. Transfection with circular DNA from a TTSuV2 deletion mutant did not produce ORF1 protein, suggesting that the observed ORF1 expression is driven by TTSuV2 DNA replication in cells. Pigs inoculated with either the tandem-dimerized clones or circular genomic DNA of U.S. TTSuV2 developed viremia, and the introduced genetic markers were retained in viral DNA recovered from the sera of infected pigs. The availability of an infectious DNA clone of TTSuV2 will facilitate future study of porcine anellovirus pathogenesis and biology. PMID:22491450
-
Sahin, Orhan; Terhorst, Samantha A.; Burrough, Eric R.; Shen, Zhangqi; Wu, Zuowei; Dai, Lei; Tang, Yizhi; Plummer, Paul J.; Ji, Ju; Yaeger, Michael J.
2017-01-01
ABSTRACT Campylobacter jejuni is a zoonotic pathogen, and a hypervirulent clone, named clone SA, has recently emerged as the predominant cause of ovine abortion in the United States. To induce abortion, orally ingested Campylobacter must translocate across the intestinal epithelium, spread systemically in the circulation, and reach the fetoplacental tissue. Bacterial factors involved in these steps are not well understood. C. jejuni is known to produce capsular polysaccharide (CPS), but the specific role that CPS plays in systemic infection and particularly abortion in animals remains to be determined. In this study, we evaluated the role of CPS in bacteremia using a mouse model and in abortion using a pregnant guinea pig model following oral challenge. Compared with C. jejuni NCTC 11168 and 81-176, a clone SA isolate (IA3902) resulted in significantly higher bacterial counts and a significantly longer duration of bacteremia in mice. The loss of capsule production via gene-specific mutagenesis in IA3902 led to the complete abolishment of bacteremia in mice and abortion in pregnant guinea pigs, while complementation of capsule expression almost fully restored these phenotypes. The capsule mutant strain was also impaired for survival in guinea pig sera and sheep blood. Sequence-based analyses revealed that clone SA possesses a unique CPS locus with a mosaic structure, which has been stably maintained in all clone SA isolates derived from various hosts and times. These findings establish CPS as a key virulence factor for the induction of systemic infection and abortion in pregnant animals and provide a viable candidate for the development of vaccines against hypervirulent C. jejuni. PMID:28373351
-
Intracerebral infection of Cebus apella with the XJ-Clone 3 strain of Junín virus.
Carballal, G; Oubiña, J R; Molinas, F C; Nagle, C; de la Vega, M T; Videla, C; Elsner, B
1987-03-01
To assess the usefulness of the South American primate Cebus apella as a model for neurovirulence of Junín virus, eight monkeys were inoculated with 10(5) LD50 of the attenuated XJ-Clone 3 Junín virus strain by the intrathalamic route. After the second week, weight loss and polyadenopathies were observed in most animals, one-half of which had a transient leukothrombocytopenia. Moderate clinical central nervous system (CNS) involvement was present in four of eight monkeys, while the rest had only mild neurologic signs. All recovered except one, which developed a deep coma and was killed in a pre-mortem stage at 18 days post-infection (pi). Junín virus was isolated from the throat from five, from the blood from three, and from the brain from two monkeys. In the most severely ill animal, virus titers higher than viremia were detected in both inoculated and contralateral brain hemispheres, as well as in lung, lymph node, and small intestine. Junín antigens and "in vivo" bound immunoglobulins were detected by immunofluorescence (IF) in the brain of four animals at 18, 21, 40, and 155 days pi. Moderate lymphocytic parenchymal and meningeal infiltration were observed in the brain of four animals, and gliosis was also present in the most affected monkey. Although the clinical response to infection was not uniform, all infected monkeys developed high IF antibodies. Cebus apella cannot be used as a highly sensitive model for Argentine hemorrhagic fever (AHF). However, the results obtained show that the XJ-Clone 3 strain can replicate in the primate CNS and to induce lesions and immunoglobulin deposition. In addition, viral persistence is suggested by the late detection of viral antigens in brain at 40 and 155 days pi.
-
Myohara, Maroko; Niva, Cintia Carla; Lee, Jae Min
2006-08-01
To identify genes specifically activated during annelid regeneration, suppression subtractive hybridization was performed with cDNAs from regenerating and intact Enchytraeus japonensis, a terrestrial oligochaete that can regenerate a complete organism from small body fragments within 4-5 days. Filter array screening subsequently revealed that about 38% of the forward-subtracted cDNA clones contained genes that were upregulated during regeneration. Two hundred seventy-nine of these clones were sequenced and found to contain 165 different sequences (79 known and 86 unknown). Nine clones were fully sequenced and four of these sequences were matched to known genes for glutamine synthetase, glucosidase 1, retinal protein 4, and phosphoribosylaminoimidazole carboxylase, respectively. The remaining five clones encoded an unknown open-reading frame. The expression levels of these genes were highest during blastema formation. Our present results, therefore, demonstrate the great potential of annelids as a new experimental subject for the exploration of unknown genes that play critical roles in animal regeneration.
-
Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs
Matsunari, Hitomi; Nagashima, Hiroshi; Watanabe, Masahito; Umeyama, Kazuhiro; Nakano, Kazuaki; Nagaya, Masaki; Kobayashi, Toshihiro; Yamaguchi, Tomoyuki; Sumazaki, Ryo; Herzenberg, Leonard A.; Nakauchi, Hiromitsu
2013-01-01
In the field of regenerative medicine, one of the ultimate goals is to generate functioning organs from pluripotent cells, such as ES cells or induced pluripotent stem cells (PSCs). We have recently generated functional pancreas and kidney from PSCs in pancreatogenesis- or nephrogenesis-disabled mice, providing proof of principle for organogenesis from PSCs in an embryo unable to form a specific organ. Key when applying the principles of in vivo generation to human organs is compensation for an empty developmental niche in large nonrodent mammals. Here, we show that the blastocyst complementation system can be applied in the pig using somatic cell cloning technology. Transgenic approaches permitted generation of porcine somatic cell cloned embryos with an apancreatic phenotype. Complementation of these embryos with allogenic blastomeres then created functioning pancreata in the vacant niches. These results clearly indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize a cloned dysorganogenetic embryo. The feasibility of blastocyst complementation using cloned porcine embryos allows experimentation toward the in vivo generation of functional organs from xenogenic PSCs in large animals. PMID:23431169
-
Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs.
Matsunari, Hitomi; Nagashima, Hiroshi; Watanabe, Masahito; Umeyama, Kazuhiro; Nakano, Kazuaki; Nagaya, Masaki; Kobayashi, Toshihiro; Yamaguchi, Tomoyuki; Sumazaki, Ryo; Herzenberg, Leonard A; Nakauchi, Hiromitsu
2013-03-19
In the field of regenerative medicine, one of the ultimate goals is to generate functioning organs from pluripotent cells, such as ES cells or induced pluripotent stem cells (PSCs). We have recently generated functional pancreas and kidney from PSCs in pancreatogenesis- or nephrogenesis-disabled mice, providing proof of principle for organogenesis from PSCs in an embryo unable to form a specific organ. Key when applying the principles of in vivo generation to human organs is compensation for an empty developmental niche in large nonrodent mammals. Here, we show that the blastocyst complementation system can be applied in the pig using somatic cell cloning technology. Transgenic approaches permitted generation of porcine somatic cell cloned embryos with an apancreatic phenotype. Complementation of these embryos with allogenic blastomeres then created functioning pancreata in the vacant niches. These results clearly indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize a cloned dysorganogenetic embryo. The feasibility of blastocyst complementation using cloned porcine embryos allows experimentation toward the in vivo generation of functional organs from xenogenic PSCs in large animals.
-
Adeno-associated virus–targeted disruption of the CFTR gene in cloned ferrets
Sun, Xingshen; Yan, Ziying; Yi, Yaling; Li, Ziyi; Lei, Diana; Rogers, Christopher S.; Chen, Juan; Zhang, Yulong; Welsh, Michael J.; Leno, Gregory H.; Engelhardt, John F.
2008-01-01
Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene–deficient model in the domestic ferret using recombinant adeno-associated virus–mediated gene targeting in fibroblasts, followed by nuclear transfer cloning. As part of this approach, we developed a somatic cell rejuvenation protocol using serial nuclear transfer to produce live CFTR-deficient clones from senescent gene-targeted fibroblasts. We transferred 472 reconstructed embryos into 11 recipient jills and obtained 8 healthy male ferret clones heterozygous for a disruption in exon 10 of the CFTR gene. To our knowledge, this study represents the first description of genetically engineered ferrets and describes an approach that may be of substantial utility in modeling not only CF, but also other genetic diseases. PMID:18324338
-
Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos
Rodriguez-Osorio, Nelida; Wang, Zhongde; Kasinathan, Poothappillai; Page, Grier P; Robl, James M; Memili, Erdogan
2009-01-01
Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT). Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively) have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF) than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively). However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research. PMID:19393066
-
Somatic cell nuclear transfer in horses.
Galli, Cesare; Lagutina, Irina; Duchi, Roberto; Colleoni, Silvia; Lazzari, Giovanna
2008-07-01
The cloning of equids was achieved in 2003, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay was because of the limited development in the horse of more classical-assisted reproductive techniques required for successful cloning, such as oocyte maturation and in vitro embryo production. When these technologies were developed, the application of cloning also became possible and cloned horse offspring were obtained. This review summarizes the main technical procedures that are required for cloning equids and the present status of this technique. The first step is competent oocyte maturation, this is followed by oocyte enucleation and reconstruction, using either zona-enclosed or zona-free oocytes, by efficient activation to allow high cleavage rates and finally by a suitable in vitro embryo culture technique. Cloning of the first equid, a mule, was achieved using an in vivo-matured oocytes and immediate transfer of the reconstructed embryo, i.e. at the one cell stage, to the recipient oviduct. In contrast, the first horse offspring was obtained using a complete in vitro procedure from oocyte maturation to embryo culture to the blastocyst stage, followed by non-surgical transfer. Later studies on equine cloning report high efficiency relative to that for other species. Cloned equid offspring reported to date appear to be normal and those that have reached puberty have been confirmed to be fertile. In summary, horse cloning is now a reproducible technique that offers the opportunity to preserve valuable genetics and notably to generate copies of castrated champions and therefore, offspring from those champions that would be impossible to obtain otherwise.
-
Human therapeutic cloning (NTSC): applying research from mammalian reproductive cloning.
French, Andrew J; Wood, Samuel H; Trounson, Alan O
2006-01-01
Human therapeutic cloning or nuclear transfer stem cells (NTSC) to produce patient-specific stem cells, holds considerable promise in the field of regenerative medicine. The recent withdrawal of the only scientific publications claiming the successful generation of NTSC lines afford an opportunity to review the available research in mammalian reproductive somatic cell nuclear transfer (SCNT) with the goal of progressing human NTSC. The process of SCNT is prone to epigenetic abnormalities that contribute to very low success rates. Although there are high mortality rates in some species of cloned animals, most surviving clones have been shown to have normal phenotypic and physiological characteristics and to produce healthy offspring. This technology has been applied to an increasing number of mammals for utility in research, agriculture, conservation, and biomedicine. In contrast, attempts at SCNT to produce human embryonic stem cells (hESCs) have been disappointing. Only one group has published reliable evidence of success in deriving a cloned human blastocyst, using an undifferentiated hESC donor cell, and it failed to develop into a hESC line. When optimal conditions are present, it appears that in vitro development of cloned and parthenogenetic embryos, both of which may be utilized to produce hESCs, may be similar to in vitro fertilized embryos. The derivation of ESC lines from cloned embryos is substantially more efficient than the production of viable offspring. This review summarizes developments in mammalian reproductive cloning, cell-to-cell fusion alternatives, and strategies for oocyte procurement that may provide important clues facilitating progress in human therapeutic cloning leading to the successful application of cell-based therapies utilizing autologous hESC lines.
-
Kishigami, Satoshi; Mizutani, Eiji; Ohta, Hiroshi; Hikichi, Takafusa; Thuan, Nguyen Van; Wakayama, Sayaka; Bui, Hong-Thuy; Wakayama, Teruhiko
2006-02-03
The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently, we elucidated by using round spermatids that, after nuclear transfer, treatment of zygotes with trichostatin A (TSA), an inhibitor of histone deacetylase, can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions [S. Kishigami, N. Van Thuan, T. Hikichi, H. Ohta, S. Wakayama. E. Mizutani, T. Wakayama, Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids, Dev. Biol. (2005) in press]. Here, we found that 5-50 nM TSA-treatment for 10 h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells, spleen cells, neural stem cells, and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further, we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus, our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques.
-
Sotomaru, Yusuke; Hirakawa, Reiko; Shimada, Akiko; Shiozawa, Seiji; Sugawara, Ayako; Oiwa, Ryo; Nobukiyo, Asako; Okano, Hideyuki; Tamaoki, Norikazu; Nomura, Tatsuji; Hiyama, Eiso; Sasaki, Erika
2009-12-01
The somatic cell nuclear transfer technique has been applied to various mammals to produce cloned animals; however, a standardized method is not applicable to all species. We aimed here to develop optimum procedures for somatic cell cloning in nonhuman primates, using common marmosets. First, we confirmed that parthenogenetic activation of in vitro matured oocytes was successfully induced by electrical stimulation (three cycles of 150 V/mm, 50 microsec x 2, 20 min intervals), and this condition was applied to the egg activation procedure in the subsequent experiments. Next, nuclear transfer to recipient enucleated oocytes was performed 1 h before, immediately after, or 1 h after egg activation treatment. The highest developmental rate was observed when nuclear transfer was performed 1 h before activation, but none of the cloned embryos developed beyond the eight-cell stage. To investigate the causes of the low developmental potential of cloned embryos, a study was performed to determine whether the presence of metaphase II (MII) chromosome in recipient ooplasm has an effect on developmental potential. As a result, only tetraploid cloned embryos produced by transferring a donor cell into a recipient bearing the MII chromosome developed into blastocysts (66.7%). In contrast, neither parthenogenetic embryos nor cloned embryos (whether diploid or tetraploid) produced using enucleated oocytes developed past the eight-cell stage. These results suggest that MII chromosome, or cytoplasm proximal to the MII chromosome, plays a major role in the development of cloned embryos in common marmosets.
-
Reproduction (II): Human Control of Reproductive Processes
ERIC Educational Resources Information Center
Jost, Alfred
1970-01-01
Describes methods of intervening in reproduction of animals and humans (artificial insemination, contraception, ovular and blastodisc transplants, pre selection of sex, cloning) and discusses the social implications of their use with humans. (AL)
-
Food from cloned animals is part of our brave old world.
Miller, Henry I
2007-05-01
When confronted by pressure from activists and Congress, the US Food and Drug Administration (FDA) has not always adopted policies and made decisions about individual products that accord with the scientific evidence. An example was the unnecessarily and markedly prolonged review of the veterinary drug bovine somatotropin (bST), or bovine growth hormone, during the 1980s. The FDA now faces a similar situation surrounding the question of whether meat and milk from cloned animals and their offspring are safe for human consumption. Having made a preliminary decision in the affirmative - based on an exhaustive analysis of scientific articles, health records, blood samples and studies of the composition of meat and milk - the agency has been beleaguered by criticisms. It remains to be seen whether, ultimately, science will trump anti-technology, anti-consumer activism.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kishigami, Satoshi; Mizutani, Eiji; Ohta, Hiroshi
The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently, we elucidated by using round spermatids that, after nuclear transfer, treatment of zygotes with trichostatin A (TSA), an inhibitor of histone deacetylase, can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions [S. Kishigami, N. Van Thuan, T. Hikichi, H. Ohta, S. Wakayama. E. Mizutani, T. Wakayama, Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids, Dev. Biol. (2005) in press]. Here,more » we found that 5-50 nM TSA-treatment for 10 h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells, spleen cells, neural stem cells, and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further, we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus, our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques.« less
-
Lei, Bei; Cao, Jie; Shen, Jie; Zhao, Lanxiang; Liang, Sheng; Meng, Qinggang; Xie, Wenhui; Yang, Shunfang
2013-08-20
Lung cancer is the leading cause of cancer-related death in men and women. It is also the most common cause of brain metastases. A brain metastasis model is difficult to be established because of the presence of the blood-brain barrier (BBB) and the lack of optimal methods for detecting brain metastasis in nude mice. Thus, the establishment of a Chinese lung adenocarcinoma cell line and its animal model with brain metastasis potency and in vivo research is of great significance. CPA-Yang1 cells were obtained from a patient with human lung adenocarcinoma by lentiviral vector-mediated transfection of green fluorescence protein. Intracardiac inoculation of the cells was performed in nude mice, and brain metastatic lesions were detected using micro ¹⁸F FDG-PET/CT scanners, small animal in vivo imaging system for fluorescence, radionuclide and X ray fused imaging, magnetic resonance imaging (MRI) with sense body detection, and resection. The samples were divided into two parts for cell culture and histological diagnosis. The process was repeated in vivo and in vitro for four cycles to obtain a novel cell clone, CPA-Yang1-BR. A novel cell clone, CPA-Yang1-BR, was obtained with a brain metastatic rate of 50%. The use of MRI for the detection of brain metastases has obvious advantages. An experimental Chinese lung adenocarcinoma cell clone (CPA-Yang1-BR) and its animal model with brain metastasis potency in nude mice were established. MRI with sense body or micro MRI may be used as a sensitive, accurate, and noninvasive method to detect experimental brain metastases in intact live immunodeficient mice. The results of this study may serve as a technical platform for brain metastases from lung adenocarcinoma.
-
[Mystery and problems of cloning].
Nikitin, V A
2010-01-01
The attention of investigators is attracted to the fact that, in spite of great efforts in mammalian cloning, advances that have been made in this area of research are not great, and cloned animals have developmental pathologies often incompatible with life and/or reproduction ability. It is yet not clear what technical or biological factors underlie this, and how they are connected or interact with each other, which is more realistic strategically. There is a great number of articles dealing with the influence of cloning with the nuclear transfer on genetic and epigenetic reprogramming of donor cells. At the same time we can see the practical absence of analytical investigations concerning the technology of cloning as such, its weak points, and possible sources of cellular trauma in the course of microsurgery of nuclear transfer or twinning. This article discusses step by step several nuclear transfer techniques and the methods of dividing early preimplanted embryos for twinning with the aim to reveal possible sources of cell damage during micromanipulation that may have negative influence on the development of cloned organisms. Several new author's technologies based on the study of cell biophysical characteristics are described, which allow one to avoid cellular trauma during manipulation and minimize the possibility of cell damage at any rate.
-
Sheep polyclonal antibody to map Haemonchus contortus mimotopes using phage display library.
Buzatti, Andréia; Fernandez, Arnielis Diaz; Arenal, Amilcar; Pereira, Erlán; Monteiro, Alda Lucia Gomes; Molento, Marcelo Beltrão
2018-05-24
The aim of this study was to evaluate phage display technology for mapping Haemonchus contortus mimotopes. We screened the PhD-7 Phage Display Peptide Library Kit with a sheep polyclonal antibody against H. contortus. After four rounds of selection, 50 phage peptide clones were selected by biopanning and sequenced. Two clones displaying peptide mimotopes of H. contortus proteins were chosen for sheep immunization: clone 6 - mimotope of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and clone 17 - mimotope of a disorganized muscle family member (Dim 1). Twelve sheep were allocated into 3 groups of 4 animals as follow: G1: control group; G2/GAPDH: immunized with clone 6; and G3/Dim1: immunized with clone 17. Four immunizations were performed at intervals of seven days (0, 7, 14, and 21 days). On day 28 post initial vaccination, all groups were orally challenged with 2500 H. contortus infective larvae. The mimotope peptides selected by phage display were recognized by IgG from sheep naturaly infected with H. contortus. The immunization protocol showed an increasein IgG anti-M13 phage titers, but no effect was observed in IgG-specific for the anti-mimotope peptides. This is the first report of successful use of a phage display library for the identification of mimotopes of H. contortus proteins.
-
Reproductive performance and expression of imprinted genes in somatic cell cloned boars.
Kawarasaki, Tatsuo; Enya, Satoko; Otake, Masayoshi; Shibata, Masatoshi; Mikawa, Satoshi
2017-11-01
To assess the performance of boars derived by somatic cell cloning, we analyzed various aspects of their reproductive characteristics and the expression of two imprinted genes. Cloned boars (cloned Duroc × Jinhua) were analyzed for birth weight, growth rate, age at first ejaculation, semen characteristics and fertility, in comparison with naturally bred control boars of the same strain. The expression of imprinted genes was analyzed using the microsatellite marker SWC9 for the paternally expressed gene insulin-like growth factor -2 (IGF2) and with single nucleotide polymorphisms (SNPs) for the gene maternally expressed 3 (MEG3). The cloned boars had high production of semen and were nearly equal in level of fertility to conventional pigs; they showed similar characteristics as naturally bred boars of the same strains. The expression of IGF2 was partially disturbed, but this disturbed expression was not linked to a change in developmental fate or reproductive performance. These results indicate that use of cloned boars could be highly effective for proliferation of pigs with desirable characteristics, preservation of genetic resources and risk reduction against epidemic diseases, such as foot-and-mouth disease, through storage of somatic cells as a precautionary measure for use in regenerating pig populations after a future pandemic. © 2017 Japanese Society of Animal Science.
-
Jia, P; Zhang, C; Huang, X P; Poda, M; Akbas, F; Lemanski, S L; Erginel-Unaltuna, N; Lemanski, L F
2008-11-01
The discovery of the naturally occurring cardiac non-function (c) animal strain in Ambystoma mexicanum (axolotl) provides a valuable animal model to study cardiomyocyte differentiation. In homozygous mutant animals (c/c), rhythmic contractions of the embryonic heart are absent due to a lack of organized myofibrils. We have previously cloned a partial sequence of a peptide cDNA (N1) from an anterior-endoderm-conditioned-medium RNA library that had been shown to be able to rescue the mutant phenotype. In the current studies we have fully cloned the N1 full length cDNA sequence from the library. N1 protein has been detected in both adult heart and skeletal muscle but not in any other adult tissues. GFP-tagged expression of the N1 protein has revealed localization of the N1 protein in the endoplasmic reticulum (ER). Results from in situ hybridization experiments have confirmed the dramatic decrease of expression of N1 mRNA in mutant (c/c) embryos indicating that the N1 gene is involved in heart development.
-
Recent progress in bovine somatic cell nuclear transfer.
Akagi, Satoshi; Geshi, Masaya; Nagai, Takashi
2013-03-01
Bovine somatic cell nuclear transfer (SCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization. However, the full-term developmental rate of SCNT embryos is very low, owing to the high embryonic and fetal losses after embryo transfer. In addition, increased birth weight and postnatal mortality are observed at high rates in cloned calves. The low efficiency of SCNT is probably attributed to incomplete reprogramming of the donor nucleus and most of the developmental problems of clones are thought to be caused by epigenetic defects. Applications of SCNT will depend on improvement in the efficiency of production of healthy cloned calves. In this review, we discuss problems and recent progress in bovine SCNT. © 2013 Japanese Society of Animal Science.
-
Mitochondrial DNA transmission and confounding mitochondrial influences in cloned cattle and pigs.
Takeda, Kumiko
2013-04-01
Although somatic cell nuclear transfer (SCNT) is a powerful tool for production of cloned animals, SCNT embryos generally have low developmental competency and many abnormalities. The interaction between the donor nucleus and the enucleated ooplasm plays an important role in early embryonic development, but the underlying mechanisms that negatively impact developmental competency remain unclear. Mitochondria have a broad range of critical functions in cellular energy supply, cell signaling, and programmed cell death; thus, affect embryonic and fetal development. This review focuses on mitochondrial considerations influencing SCNT techniques in farm animals. Donor somatic cell mitochondrial DNA (mtDNA) can be transmitted through what has been considered a "bottleneck" in mitochondrial genetics via the SCNT maternal lineage. This indicates that donor somatic cell mitochondria have a role in the reconstructed cytoplasm. However, foreign somatic cell mitochondria may affect the early development of SCNT embryos. Nuclear-mitochondrial interactions in interspecies/intergeneric SCNT (iSCNT) result in severe problems. A major biological selective pressure exists against survival of exogenous mtDNA in iSCNT. Yet, mtDNA differences in SCNT animals did not reflect transfer of proteomic components following proteomic analysis. Further study of nuclear-cytoplasmic interactions is needed to illuminate key developmental characteristics of SCNT animals associated with mitochondrial biology.
-
Positional cloning in mice and its use for molecular dissection of inflammatory arthritis.
Abe, Koichiro; Yu, Philipp
2009-02-01
One of the upcoming next quests in the field of genetics might be molecular dissection of the genetic and environmental components of human complex diseases. In humans, however, there are certain experimental limitations for identification of a single component of the complex interactions by genetic analyses. Experimental animals offer simplified models for genetic and environmental interactions in human complex diseases. In particular, mice are the best mammalian models because of a long history and ample experience for genetic analyses. Forward genetics, which includes genetic screen and subsequent positional cloning of the causative genes, is a powerful strategy to dissect a complex phenomenon without preliminarily molecular knowledge of the process. In this review, first, we describe a general scheme of positional cloning in mice. Next, recent accomplishments on the patho-mechanisms of inflammatory arthritis by forward genetics approaches are introduced; Positional cloning effort for skg, Ali5, Ali18, cmo, and lupo mutants are provided as examples for the application to human complex diseases. As seen in the examples, the identification of genetic factors by positional cloning in the mouse have potential in solving molecular complexity of gene-environment interactions in human complex diseases.
-
Reprogramming towards totipotency is greatly facilitated by synergistic effects of small molecules
Tajima, Yosuke; Yoshida, Koki; Oikawa, Mami; Azuma, Rika; Allen, George E.; Tsujikawa, Tomomi; Tsukaguchi, Tomomasa; Bradshaw, Charles R.; Jullien, Jerome; Yamagata, Kazuo; Matsumoto, Kazuya; Anzai, Masayuki; Imai, Hiroshi; Gurdon, John B.; Yamada, Masayasu
2017-01-01
ABSTRACT Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency. PMID:28412714
-
Selander, R K; Beltran, P; Smith, N H; Helmuth, R; Rubin, F A; Kopecko, D J; Ferris, K; Tall, B D; Cravioto, A; Musser, J M
1990-01-01
Multilocus enzyme electrophoresis was employed to measure chromosomal genotypic diversity and evolutionary relationships among 761 isolates of the serovars Salmonella typhi, S. paratyphi A, S. paratyphi B, S. paratyphi C, and S. sendai, which are human-adapted agents of enteric fever, and S. miami and S. java, which are serotypically similar to S. sendai and S. paratyphi B, respectively, but cause gastroenteritis in both humans and animals. To determine the phylogenetic positions of the clones of these forms within the context of the salmonellae of subspecies I, comparative data for 22 other common serovars were utilized. Except for S. paratyphi A and S. sendai, the analysis revealed no close phylogenetic relationships among clones of different human-adapted serovars, which implies convergence in host adaptation and virulence factors. Clones of S. miami are not allied with those of S. sendai or S. paratyphi A, being, instead, closely related to strains of S. panama. Clones of S. paratyphi B and S. java belong to a large phylogenetic complex that includes clones of S. typhimurium, S. heidelberg, S. saintpaul, and S. muenchen. Most strains of S. paratyphi B belong to a globally distributed clone that is highly polymorphic in biotype, bacteriophage type, and several other characters, whereas strains of S. java represent seven diverse lineages. The flagellar monophasic forms of S. java are genotypically more similar to clones of S. typhimurium than to other clones of S. java or S. paratyphi B. Clones of S. paratyphi C are related to those of S. choleraesuis. DNA probing with a segment of the viaB region specific for the Vi capsular antigen genes indicated that the frequent failure of isolates of S. paratyphi C to express Vi antigen is almost entirely attributable to regulatory processes rather than to an absence of the structural determinant genes themselves. Two clones of S. typhisuis are related to those of S. choleraesuis and S. paratyphi C, but a third clone is not. Although the clones of S. decatur and S. choleraesuis are serologically and biochemically similar, they are genotypically very distinct. Two clones of S. typhi were distinguished, one globally distributed and another apparently confined to Africa; both clones are distantly related to those of all other serovars studied. Images PMID:1973153
-
DNA cloning: A personal view after 40 years
Cohen, Stanley N.
2013-01-01
In November 1973, my colleagues A. C. Y. Chang, H. W. Boyer, R. B. Helling, and I reported in PNAS that individual genes can be cloned and isolated by enzymatically cleaving DNA molecules into fragments, linking the fragments to an autonomously replicating plasmid, and introducing the resulting recombinant DNA molecules into bacteria. A few months later, Chang and I reported that genes from unrelated bacterial species can be combined and propagated using the same approach and that interspecies recombinant DNA molecules can produce a biologically functional protein in a foreign host. Soon afterward, Boyer’s laboratory and mine published our collaborative discovery that even genes from animal cells can be cloned in bacteria. These three PNAS papers quickly led to the use of DNA cloning methods in multiple areas of the biological and chemical sciences. They also resulted in a highly public controversy about the potential hazards of laboratory manipulation of genetic material, a decision by Stanford University and the University of California to seek patents on the technology that Boyer and I had invented, and the application of DNA cloning methods for commercial purposes. In the 40 years that have passed since publication of our findings, use of DNA cloning has produced insights about the workings of genes and cells in health and disease and has altered the nature of the biotechnology and biopharmaceutical industries. Here, I provide a personal perspective of the events that led to, and followed, our report of DNA cloning. PMID:24043817
-
Gorskaya, Yu F; Danilova, T A; Mezentseva, M V; Shapoval, I M; Narovlyanskii, A N; Nesterenko, V G
2011-06-01
Injection of S. typhimurium antigens significantly (9-fold) increased cloning efficiency and, hence, the content of stromal precursor cells in the spleen as soon as after 24 h. These parameters returned to normal by days 6-15 after immunization. Cultured splenocytes collected from immune (but not intact) animals expressed the genes of proinflammatory cytokines IL-1β (on days 1, 6, 15) and IL-6 (on days 1 and 6), TNF-α (on days 6 and 15), and of IFN-α and IL-18 (on days 6 and 15). The expression of IL-4 gene was suppressed on day 6 after immunization, of IL-10 gene on days 1 and 6, of IL-6 gene on day 15. Hence, no signs of immune response suppression by stromal cells were found in this system. The spectrum and dynamics of the expression of pro- and anti-inflammatory cytokine genes in stromal cell cultures from the spleen of immunized mice seemed to correspond to those needed for support of the immune response to S. typhimurium antigens, observed in immunized animals. The results indicate possible involvement of stromal cells in the realization of immune response in vivo. The increase of stromal precursor cells cloning efficiency in response to antigen injection could not be reproduced in vitro: the presence of S. typhimurium antigens in primary cultures of intact mouse bone marrow and spleen throughout the entire period of culturing ≈ 20-fold reduced cloning efficiency in cultures.
-
Lopker, Michael J.; Del Prete, Gregory Q.; Estes, Jacob D.; Li, Hui; Reid, Carolyn; Newman, Laura; Lipkey, Leslie; Camus, Celine; Easlick, Juliet L.; Wang, Shuyi; Decker, Julie M.; Bar, Katharine J.; Learn, Gerald; Pal, Ranajit; Weiss, Deborah E.; Hahn, Beatrice H.; Lifson, Jeffrey D.; Shaw, George M.
2016-01-01
ABSTRACT Currently available simian immunodeficiency virus (SIV) infectious molecular clones (IMCs) and isolates used in nonhuman primate (NHP) models of AIDS were originally derived from infected macaques during chronic infection or end stage disease and may not authentically recapitulate features of transmitted/founder (T/F) genomes that are of particular interest in transmission, pathogenesis, prevention, and treatment studies. We therefore generated and characterized T/F IMCs from genetically and biologically heterogeneous challenge stocks of SIVmac251 and SIVsmE660. Single-genome amplification (SGA) was used to identify full-length T/F genomes present in plasma during acute infection resulting from atraumatic rectal inoculation of Indian rhesus macaques with low doses of SIVmac251 or SIVsmE660. All 8 T/F clones yielded viruses that were infectious and replication competent in vitro, with replication kinetics similar to those of the widely used chronic-infection-derived IMCs SIVmac239 and SIVsmE543. Phenotypically, the new T/F virus strains exhibited a range of neutralization sensitivity profiles. Four T/F virus strains were inoculated into rhesus macaques, and each exhibited typical SIV replication kinetics. The SIVsm T/F viruses were sensitive to TRIM5α restriction. All T/F viruses were pathogenic in rhesus macaques, resulting in progressive CD4+ T cell loss in gastrointestinal tissues, peripheral blood, and lymphatic tissues. The animals developed pathological immune activation; lymphoid tissue damage, including fibrosis; and clinically significant immunodeficiency leading to AIDS-defining clinical endpoints. These T/F clones represent a new molecular platform for the analysis of virus transmission and immunopathogenesis and for the generation of novel “bar-coded” challenge viruses and next-generation simian-human immunodeficiency viruses that may advance the HIV/AIDS vaccine agenda. IMPORTANCE Nonhuman primate research has relied on only a few infectious molecular clones for a myriad of diverse research projects, including pathogenesis, preclinical vaccine evaluations, transmission, and host-versus-pathogen interactions. With new data suggesting a selected phenotype of the virus that causes infection (i.e., the transmitted/founder virus), we sought to generate and characterize infectious molecular clones from two widely used simian immunodeficiency virus lineages (SIVmac251 and SIVsmE660). Although the exact requirements necessary to be a T/F virus are not yet fully understood, we generated cloned viruses with all the necessary characteristic of a successful T/F virus. The cloned viruses revealed typical acute and set point viral-load dynamics with pathological immune activation, lymphoid tissue damage progressing to significant immunodeficiency, and AIDS-defining clinical endpoints in some animals. These T/F clones represent a new molecular platform for studies requiring authentic T/F viruses. PMID:27412591
-
Cloning, Codon Optimization, and Expression of Yersinia intermedia Phytase Gene in E. coli.
Mirzaei, Maryam; Saffar, Behnaz; Shareghi, Behzad
2016-06-01
Phytate is an anti-nutritional factor in plants, which catches the most phosphorus contents and some vital minerals. Therefore, Phytase is added mainly as an additive to the monogastric animals' foods to hydrolyze phytate and increase absorption of phosphorus. Y. intermedia phytase is a new phytase with special characteristics such as high specific activity, pH stability, and thermostability. Our aim was to clone, express, and characterizea codon optimized Y. intermedia phytase gene in E. coli . The Y. intermedia phytase gene was optimized according to the codon usage in E. coli . The sequence was synthesized and sub-cloned in pET-22b (+) vector and transformed into E. coli Bl21 (DE3). The protein was expressed in the presence of IPTG at a final concentration of 1 mM at 30°C. The purification of recombinant protein was performed by Ni 2+ affinity chromatography. Phytase activity and stability were determined in various pH and temperatures. The codon optimized Y. intermedia phytase gene was sub-cloned successfully.The expression was confirmed by SDS-PAGE and Western blot analysis. The recombinant enzyme (approximately 45 kDa) was purified. Specific activity of enzyme was 3849 (U.mg -1 ) with optimal pH 5 and optimal temperature of 55°C. Thermostability (80°C for 15 min) and pH stability (3-6) of the enzyme were 56 and more than 80%, respectively. The results of the expression and enzyme characterization revealed that the optimized Y. intermedia phytase gene has a good potential to be produced commercially andto be applied in animals' foodsindustry.
-
Refined human artificial chromosome vectors for gene therapy and animal transgenesis
Kazuki, Y; Hoshiya, H; Takiguchi, M; Abe, S; Iida, Y; Osaki, M; Katoh, M; Hiratsuka, M; Shirayoshi, Y; Hiramatsu, K; Ueno, E; Kajitani, N; Yoshino, T; Kazuki, K; Ishihara, C; Takehara, S; Tsuji, S; Ejima, F; Toyoda, A; Sakaki, Y; Larionov, V; Kouprina, N; Oshimura, M
2011-01-01
Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis. PMID:21085194
-
Asseged, B D; Habtemariam, T; Tameru, B; Nganwa, D
2012-01-15
Deriving horse oocytes in the USA is hampered by the lack of abattoirs processing horse carcasses which could provide abundant quantities of ovaries from slaughtered mares. Therefore, several cloning industries in the USA are attempting to import cloned horse embryos from Canada. Like any agricultural commodity, cloned embryos pose a risk of introduction of exotic animal diseases into the importing country. Under such circumstances, risk assessment could provide an objective, transparent, and internationally accepted means for evaluating the risk. This quantitative risk assessment (QRA) was initiated to determine the risk of introduction of Equine infectious anemia virus (EIAV) into the USA via cloned horse embryos imported from Canada. In assessing the risk, a structured knowledge base regarding cloning in relation to Equine infectious anemia (EIA) was first developed. Based on the knowledge base, a scenario tree was developed to determine conditions (with mathematical probabilities) that could lead to the introduction and maintenance of EIAV along the cloning pathway. Parameters for the occurrence of the event at each node were estimated using published literature. Using @Risk software and setting Monte Carlo simulation at 50,000 iterations, the probability of importing an EIAV-infected cloned horse embryo was 1.8 × 10(-9) (R = 1.5 × 10(-12) to 2.9 × 10(-8)). Taking into account the current protocol for equine cloning and assuming the yield of 5 to 30 clones per year, the possible number of EIAV-infected cloned horse embryos ranged from 2.0 × 10(-10) to 9.1 × 10(-5) (Mean = 1.4×10(-6)) per year. Consequently, it would take up to 1.5 × 10(7) (R = 1.6 × 10(4) to 5.1 × 10(10)) years for EIAV to be introduced into the USA. Based on the knowledge base and our critical pathway analysis, the biological plausibility of introducing EIAV into USA via cloned horse embryos imported from Canada is extremely low. Copyright © 2012 Elsevier Inc. All rights reserved.
-
Driss, Virginie; Leprêtre, Frédéric; Briche, Isabelle; Mopin, Alexia; Villenet, Céline; Figeac, Martin; Quesnel, Bruno; Brinster, Carine
2017-12-01
In acute myeloid leukaemia (AML)-affected patients, the presence of heterogeneous sub-clones at diagnosis has been shown to be responsible for minimal residual disease and relapses. The role played by the immune system in this leukaemic sub-clonal hierarchy and maintenance remains unknown. As leukaemic sub-clone immunogenicity could not be evaluated in human AML xenograft models, we assessed the sub-clonal diversity of the murine C1498 AML cell line and the immunogenicity of its sub-clones in immune-competent syngeneic mice. The murine C1498 cell line was cultured in vitro and sub-clonal cells were generated after limiting dilution. The genomic profiles of 6 different sub-clones were analysed by comparative genomic hybridization arrays (CGH). The sub-clones were then injected into immune-deficient and - competent syngeneic mice. The immunogenicities of the sub-clones was evaluated through 1) assessment of mouse survival, 2) determination of leukaemic cell infiltration into organs by flow cytometry and the expression of a fluorescent reporter gene, 3) assessment of the CTL response ex vivo and 4) detection of residual leukaemic cells in the organs via amplification of the genomic reporter gene by real-time PCR (qPCR). Genomic analyses revealed heterogeneity among the parental cell line and its derived sub-clones. When injected individually into immune-deficient mice, all sub-clones induced cases of AML with different kinetics. However, when administered into immune-competent animals, some sub-clones triggered AML in which no mice survived, whereas others elicited reduced lethality rates. The AML-surviving mice presented efficient anti-leukaemia CTL activity ex vivo and eliminated the leukaemic cells in vivo. We showed that C1498 cell sub-clones presented genomic heterogeneity and differential immunogenicity resulting either in immune escape or elimination. Such findings could have potent implications for new immunotherapeutic strategies in patients with AML. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
-
Phenotypic effects of somatic cell cloning in the mouse.
Ogura, A; Inoue, K; Ogonuki, N; Lee, J; Kohda, T; Ishino, F
2002-01-01
Although a variety of phenotypes and epigenetic alterations have been reported in animals cloned from somatic cells, the exact nature and consequences of cloning remain unclear. We cloned mice using fresh or short-term cultures of donor cells (cumulus cells, immature Sertoli cells, and fetal or adult fibroblast cells) with defined genetic backgrounds, and then compared the phenotypic and epigenetic characteristics of the cloned mice with those of fertilization-derived control mice. Irrespective of the nucleus-donor cell type, about 50% of the reconstructed embryos developed to the morula/blastocyst stage, but about 90% of these clones showed arrested development between days 5 and 8, shortly after implantation. Most of the clones were alive at term, readily recovered respiration, and did not show any malformations or overgrowths. However, their placentas were two- to threefold larger than those of the controls, due to hyperplasia of the basal (or spongiotrophoblast) layer. Although there was significant suppression of a subset of both imprinted and non-imprinted placental genes, fetal gene suppression was minimal. The seven imprinted genes that we examined were all expressed correctly from the parental alleles. These findings were consistent for every cell type from the midgestation through term stages. Therefore, cloning by nuclear transfer does not perturb the parent-specific imprinting memory that is established during gametogenesis, and the phenotypic and epigenetic effects of cloning are restricted to placental development at the midgestation and term stages. Twelve male mice that were born in a normal manner following nuclear transfer with immature Sertoli cells (B6D2F1 genetic background) were subjected to long-term observation. They died earlier than the genotype-matched controls (50% survival point: 550 days vs. 1028 days, respectively), most probably due to severe pneumonia, which indicates that unexpected phenotypes can appear as a result of the long-term effects of somatic cell cloning.
-
Fernandes, C C L; Aguiar, L H; Calderón, C E M; Silva, A M; Alves, J P M; Rossetto, R; Bertolini, L R; Bertolini, M; Rondina, D
2018-01-01
Changes in the nutritional plan have been shown to affect oocyte quality, crucial to oocyte donors animals used in cloning. This study aimed to evaluate the impact of diets with increasing nutritional levels (maintenance diet=M; 1.3M; 1.6M; 1.9M) fed to goats for four weeks on follicular fluid composition, gene expression and oocyte competence used to cloning in goats. Donor females were superovulated for the retrieval of matured oocytes and physical measurements reported. After four weeks, groups receiving diets above maintenance increased thickness of subcutaneous adipose tissue and body weight, with higher values in 1.9M Group (P<0.05). Treatments did not affect follicular density, number of aspirated follicles, retrieved and matured oocytes. Animals from 1.3M group had lower (P<0.05) maturation rate (44.0%) and number of viable oocytes (65.3%) than M (68.8%) and 1.9M (76.0%). Follicular fluid glucose concentrations increased with nutritional levels (P=0.010), with a difference (P<0.05) between groups 1.9M (11.4±2.6mg/dL) and M (2.6±0.5mg/dL). The diet did not affect the expression of GDF9, BMP15, and BAX genes in oocytes, but BCL2 and apoptotic index were significantly higher (P<0.05) in the 1.3M and 1.6M groups than the other groups. Following the transfer of cloned embryos, one fetus was born live of a twin pregnancy in the 1.9M Group. The association between energy intake and oocyte quality suggests better nutritional use by oocytes when the maximum flow was used (1.9M), but the optimal feeding level in cloning still needs refinement. Copyright © 2017 Elsevier B.V. All rights reserved.
-
Piña-Aguilar, Raul E; Lopez-Saucedo, Janet; Sheffield, Richard; Ruiz-Galaz, Lilia I; Barroso-Padilla, Jose de J; Gutiérrez-Gutiérrez, Antonio
2009-09-01
Recent accomplishments in the fields of nuclear transfer and genomics, such as the cloned offspring production from frozen mouse cells, cryopreserved at not too low temperatures without cryoprotectors; or the sequencing of wooly mammoth genome, have opened the opportunity for the revival of extinct species. As expected, they are receiving a lot of publicity in the media and also scientific attention. Furthermore, it was recently published the "revival" of the first extinct subspecie: the Pyrenean ibex (Capra pyrenaica pyrenaica), a wild goat extinct in 2000. This strengthens the field of cloning as it had been tarnished by induced pluripotent stem cells (iPS) and other methods of reprogramming. However, for biological conservation purposes, cloning is not generally accepted as an alternative for animal conservation, and there is an ongoing debate between reproductive scientists and conservation specialists. Although we believe that nuclear transfer technologies have an opportunity in conservation efforts for some species that are on the brink of extinction and that population status, geographical isolation, reproductive characteristics, and human pressure create a situation that is almost unsustainable. In this article we discuss the barriers in cloning mammoths and cloning controversies in conservation from a zoological perspective, citing the species that might benefit from nuclear transfer techniques in the arduous journey so as not to disappear forever from this, our world.
-
Salamone, Daniel; Barañao, Lino; Santos, Claudio; Bussmann, Leonardo; Artuso, Jorge; Werning, Carlos; Prync, Aida; Carbonetto, Cesar; Dabsys, Susana; Munar, Carlos; Salaberry, Roberto; Berra, Guillermo; Berra, Ignacio; Fernández, Nahuel; Papouchado, Mariana; Foti, Marcelo; Judewicz, Norberto; Mujica, Ignacio; Muñoz, Luciana; Alvarez, Silvina Fenández; González, Eliseo; Zimmermann, Juan; Criscuolo, Marcelo; Melo, Carlos
2006-07-13
Transgenic farm animals have been proposed as an alternative to current bioreactors for large scale production of biopharmaceuticals. However, the efficiency of both methods in the production of the same protein has not yet been established. Here we report the production of recombinant human growth hormone (hGH) in the milk of a cloned transgenic cow at levels of up to 5 g l(-1). The hormone is identical to that currently produced by expression in E. coli. In addition, the hematological and somatometric parameters of the cloned transgenic cow are within the normal range for the breed and it is fertile and capable of producing normal offspring. These results demonstrate that transgenic cattle can be used as a cost-effective alternative for the production of this hormone.
-
Proudhon, D; Wei, J; Briat, J; Theil, E C
1996-03-01
Ferritin, a protein widespread in nature, concentrates iron approximately 10(11)-10(12)-fold above the solubility within a spherical shell of 24 subunits; it derives in plants and animals from a common ancestor (based on sequence) but displays a cytoplasmic location in animals compared to the plastid in contemporary plants. Ferritin gene regulation in plants and animals is altered by development, hormones, and excess iron; iron signals target DNA in plants but mRNA in animals. Evolution has thus conserved the two end points of ferritin gene expression, the physiological signals and the protein structure, while allowing some divergence of the genetic mechanisms. Comparison of ferritin gene organization in plants and animals, made possible by the cloning of a dicot (soybean) ferritin gene presented here and the recent cloning of two monocot (maize) ferritin genes, shows evolutionary divergence in ferritin gene organization between plants and animals but conservation among plants or among animals; divergence in the genetic mechanism for iron regulation is reflected by the absence in all three plant genes of the IRE, a highly conserved, noncoding sequence in vertebrate animal ferritin mRNA. In plant ferritin genes, the number of introns (n = 7) is higher than in animals (n = 3). Second, no intron positions are conserved when ferritin genes of plants and animals are compared, although all ferritin gene introns are in the coding region; within kingdoms, the intron positions in ferritin genes are conserved. Finally, secondary protein structure has no apparent relationship to intron/exon boundaries in plant ferritin genes, whereas in animal ferritin genes the correspondence is high. The structural differences in introns/exons among phylogenetically related ferritin coding sequences and the high conservation of the gene structure within plant or animal kingdoms of the gene structure within plant or animal kingdoms suggest that kingdom-specific functional constraints may exist to maintain a particular intron/exon pattern within ferritin genes. In the case of plants, where ferritin gene intron placement is unrelated to triplet codons or protein structure, and where ferritin is targeted to the plastid, the selection pressure on gene organization may relate to RNA function and plastid/nuclear signaling.
-
Enterococcus faecalis urinary-tract infections: Do they have a zoonotic origin?
Abat, Cédric; Huart, Michael; Garcia, Vincent; Dubourg, Grégory; Raoult, Didier
2016-10-01
Major human pathogens are frequently isolated from meat-producing animals, particularly poultry. Among them is Enterococcus faecalis, which is known to be one of the main cause of human urinary-tract infections worldwide. Early in 2015, we detected several, consecutive abnormal increases in the weekly number of human E. faecalis infections in various medical settings in the Provence-Alpes-Côte d'Azur region of France, especially including community-acquired urinary-tract infections. Speculating that this region-wide epidemiological event may have originated from animal-based food, we initiated this work to provide an overview of the epidemiology of E. faecalis, with a particular focus on the possible link between E. faecalis clones isolated from food-producing animals and those responsible for human urinary-tract infections. At that time, only one study had clearly identified strong epidemiological links between E. faecalis clones isolated from food-producing animals and human E. faecalis urinary-tract infections. This observation, coupled with our region-wide epidemiological experience, leads us to strongly believe that E. faecalis is a real zoonotic pathogen with potentially highly significant impact on human health. This is of particular concern because of its ability to acquire antibiotic-resistance genes and to infect animals and humans. Various strategies must be urgently implemented to address this public health threat, in particular through the development and implementation of large integrated automated surveillance systems based on animal and human health data to enable us to detect E. faecalis epidemiological events. Copyright © 2016 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
-
Code of Federal Regulations, 2014 CFR
2014-01-01
... Regulations of the Department of Agriculture (Continued) ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE CONTROL OF ILLEGALLY TAKEN PLANTS § 357.2 Definitions. Artificial selection. The process of selecting plants for particular traits, through such means as breeding, cloning, or genetic...
-
Mesquita, Fernando S; Machado, Sergio A; Drnevich, Jenny; Borowicz, Pawel; Wang, Zhongde; Nowak, Romana A
2013-01-30
Poor success rates in somatic cell cloning are often attributed to abnormal early embryonic development as well as late abnormal fetal growth and placental development. Although promising results have been reported following chromatin transfer (CT), a novel cloning method that includes the remodeling of the donor nuclei in vitro prior to their transfer into enucleated oocytes, animals cloned by CT show placental abnormalities similar to those observed following conventional nuclear transfer. We hypothesized that the placental gene expression pattern from cloned fetuses was ontologically related to the frequently observed placental phenotype. The aim of the present study was to compare global gene expression by microarray analysis of Day 44-47 cattle placentas derived from CT cloned fetuses with those derived from in vitro fertilization (i.e. control), and confirm the altered mRNA and protein expression of selected molecules by qRT-PCR and immunohistochemistry, respectively. The differentially expressed genes identified in the present study are known to be involved in a range of activities associated with cell adhesion, cell cycle control, intracellular transport and proteolysis. Specifically, an imprinted gene, involved with cell proliferation and placentomegaly in humans (CDKN1C) and a peptidase that serves as a marker for non-invasive trophoblast cells in human placentas (DPP4), had mRNA and protein altered in CT placentas. It was concluded that the altered pattern of gene expression observed in CT samples may contribute to the abnormal placental development phenotypes commonly identified in cloned offspring, and that expression of imprinted as well as trophoblast invasiveness-related genes is altered in cattle cloned by CT. Copyright © 2012 Elsevier B.V. All rights reserved.
-
Morganti, Marina; Bolzoni, Luca; Scaltriti, Erika; Casadei, Gabriele; Carra, Elena; Rossi, Laura; Gherardi, Paola; Faccini, Fabio; Arrigoni, Norma; Sacchi, Anna Rita; Delledonne, Marco; Pongolini, Stefano
2018-03-01
Background and aimEpidemiology of human non-typhoid salmonellosis is characterised by recurrent emergence of new clones of the pathogen over time. Some clonal lines of Salmonella have shaped epidemiology of the disease at global level, as happened for serotype Enteritidis or, more recently, for Salmonella 4,[5],12:i:-, a monophasic variant of serotype Typhimurium. The same clonal behaviour is recognisable at sub-serotype level where single outbreaks or more generalised epidemics are attributable to defined clones. The aim of this study was to understand the dynamics of a clone of Salmonella 4,[5],12:i:- over a 3-year period (2012-15) in a province of Northern Italy where the clone caused a large outbreak in 2013. Furthermore, the role of candidate outbreak sources was investigated and the accuracy of multilocus variable-number tandem repeat analysis (MLVA) was evaluated. Methods: we retrospectively investigated the outbreak through whole genome sequencing (WGS) and further monitored the outbreak clone for 2 years after its conclusion. Results: The study showed the transient nature of the clone in the population, possibly as a consequence of its occasional expansion in a food-processing facility. We demonstrated that important weaknesses characterise conventional typing methods applied to clonal pathogens such as Salmonella 4,[5],12:i:-, namely lack of accuracy for MLVA and inadequate resolution power for PFGE to be reliably used for clone tracking. Conclusions : The study provided evidence for the remarkable prevention potential of whole genome sequencing used as a routine tool in systems that integrate human, food and animal surveillance.
-
Characterization of a Highly Pathogenic Molecular Clone of Feline Immunodeficiency Virus Clade C
de Rozières, Sohela; Mathiason, Candace K.; Rolston, Matthew R.; Chatterji, Udayan; Hoover, Edward A.; Elder, John H.
2004-01-01
We have derived and characterized a highly pathogenic molecular isolate of feline immunodeficiency virus subtype C (FIV-C) CABCpady00C. Clone FIV-C36 was obtained by lambda cloning from cats that developed severe immunodeficiency disease when infected with CABCpady00C (Abbotsford, British Columbia, Canada). Clone FIV-C36 Env is 96% identical to the noninfectious FIV-C isolate sequence deposited in GenBank (FIV-Cgb; GenBank accession number AF474246) (A. Harmache et al.) but is much more divergent in Env when compared to the subgroup A clones Petaluma (34TF10) and FIV-PPR (76 and 78% divergence, respectively). Clone FIV-C36 was able to infect freshly isolated feline peripheral blood mononuclear cells and primary T-cell lines but failed to productively infect CrFK cells, as is typical of FIV field isolates. Two-week-old specific-pathogen-free cats infected with FIV-C36 tissue culture supernatant became PCR positive and developed severe acute immunodeficiency disease similar to that caused by the uncloned CABCpady00C parent. At 4 to 5 weeks postinfection (PI), 3 of 4 animals developed CD4+-T-cell depletion, fever, weight loss, diarrhea, and opportunistic infections, including ulcerative stomatitis and tonsillitis associated with abundant bacterial growth, pneumonia, and pyelonephritis, requiring euthanasia. Histopathology confirmed severe thymic and systemic lymphoid depletion. Interestingly, the dam also became infected with a high viral load at 5 weeks PI of the kittens and developed a similar disease syndrome, requiring euthanasia at 11 weeks PI of the kittens. This constitutes the first report of a replication-competent, infectious, and pathogenic molecular clone of FIV-C. Clone FIV-C36 will facilitate dissection of the pathogenic determinants of FIV. PMID:15308694
-
Characterization of a highly pathogenic molecular clone of feline immunodeficiency virus clade C.
de Rozières, Sohela; Mathiason, Candace K; Rolston, Matthew R; Chatterji, Udayan; Hoover, Edward A; Elder, John H
2004-09-01
We have derived and characterized a highly pathogenic molecular isolate of feline immunodeficiency virus subtype C (FIV-C) CABCpady00C. Clone FIV-C36 was obtained by lambda cloning from cats that developed severe immunodeficiency disease when infected with CABCpady00C (Abbotsford, British Columbia, Canada). Clone FIV-C36 Env is 96% identical to the noninfectious FIV-C isolate sequence deposited in GenBank (FIV-Cgb; GenBank accession number AF474246) (A. Harmache et al.) but is much more divergent in Env when compared to the subgroup A clones Petaluma (34TF10) and FIV-PPR (76 and 78% divergence, respectively). Clone FIV-C36 was able to infect freshly isolated feline peripheral blood mononuclear cells and primary T-cell lines but failed to productively infect CrFK cells, as is typical of FIV field isolates. Two-week-old specific-pathogen-free cats infected with FIV-C36 tissue culture supernatant became PCR positive and developed severe acute immunodeficiency disease similar to that caused by the uncloned CABCpady00C parent. At 4 to 5 weeks postinfection (PI), 3 of 4 animals developed CD4(+)-T-cell depletion, fever, weight loss, diarrhea, and opportunistic infections, including ulcerative stomatitis and tonsillitis associated with abundant bacterial growth, pneumonia, and pyelonephritis, requiring euthanasia. Histopathology confirmed severe thymic and systemic lymphoid depletion. Interestingly, the dam also became infected with a high viral load at 5 weeks PI of the kittens and developed a similar disease syndrome, requiring euthanasia at 11 weeks PI of the kittens. This constitutes the first report of a replication-competent, infectious, and pathogenic molecular clone of FIV-C. Clone FIV-C36 will facilitate dissection of the pathogenic determinants of FIV.
-
Proliferative lifespan is conserved after nuclear transfer.
Clark, A John; Ferrier, Patricia; Aslam, Samena; Burl, Sarah; Denning, Chris; Wylie, Diana; Ross, Arlene; de Sousa, Paul; Wilmut, Ian; Cui, Wei
2003-06-01
Cultured primary cells exhibit a finite proliferative lifespan, termed the Hayflick limit. Cloning by nuclear transfer can reverse this cellular ageing process and can be accomplished with cultured cells nearing senescence. Here we describe nuclear transfer experiments in which donor cell lines at different ages and with different proliferative capacities were used to clone foetuses and animals from which new primary cell lines were generated. The rederived lines had the same proliferative capacity and rate of telomere shortening as the donor cell lines, suggesting that these are innate, genetically determined, properties that are conserved by nuclear transfer.
-
Turbulence, cleavage, and the naked embryo: a case for coral clones.
Heyward, A J; Negri, A P
2012-03-02
After mass spawning events, coral embryos, lacking the protective capsule of other metazoans, are directly exposed to the environment at the ocean surface. Here, we present evidence that modest turbulence disrupts the integrity of these embryos, which fragment into totipotent cells that develop into proportionately smaller functional larvae. The level of turbulence required to fragment coral embryos can be generated from small wind-generated waves, which occur frequently during coral spawning on the Great Barrier Reef. The formation of planktonic coral clones, through natural embryo fragmentation of broadcast spawn, is a previously unknown mode of reproduction in the animal kingdom.
-
Singh, Mahipal; Ma, Xiaoling; Sharma, Anil
2012-09-01
Animal cloning using somatic cell nuclear transfer technology has renewed the interest in postmortem tissue storage, since these tissues can be used to reintroduce the lost genes back into the breeding pool in animal agriculture, preserve the genetic diversity, and revive the endangered species. However, for successful cloning of animals, integrity of nuclear DNA is essential. Cell viability and their potential to in vitro culture ensure nuclear integrity. The aim of this study was to determine the limits of postmortem time interval within which live cells can be recovered from goat skin tissues. To test the postmortem tissue storage limits, we cultured 2-3 mm(2) skin pieces (n = 70) from the ears of three breeds of goats (n = 7) after 0, 2, 4, and 6 days of postmortem storage at 24°C. After 10 days of culture, outgrowth of fibroblast-like cells (>50 cells) around the explants was scored. All the explants irrespective of breed displayed outgrowth of cells on the dish containing fresh tissues (i.e., day 0 of storage). However, the number of explants exhibiting outgrowth reduced with increasing time interval. Only 53.85 % explants displayed outgrowth after 2 days of tissue storage. The number of explants displaying outgrowth was much smaller after 4 (16.67 %) and 6 days (13.3 %) of storage. In general, the number of outgrowing cells per explant, on a given day, also decreased with increasing postmortem storage time interval. To test the differences between cell cultures, we established secondary cultures from one of the goats exhibiting outgrowth of cells after 6 days of tissue storage and compared them to similar cells from fresh tissues. Comparison of both the cell lines revealed similar cell morphology and growth curves and had doubling times of 23.04 and 22.56 h, respectively. These results suggest that live cells can be recovered from goat (and perhaps other animal) tissues stored at room temperature even after 6 days of their death with comparable growth profiles and, thus, can be used for tissue banking for preservation of superior genetics, genetic diversity, and cloning of animals.
-
[Therapeutic cloning--future medicine or an ethical dead end?].
Rognum, T O
2001-08-30
Treatment with stem cells has given promising results in animal experiments and may be relevant in a variety of diseases, including heart disease, cancer, diabetes, Parkinson's disease and Alzheimer's disease. However, the use of pluripotent stem cells grown from blastocysts available after in vitro fertilization or from fetuses raises difficult issues in medical ethics. The same goes for therapeutic cloning. Attitudes to this mode of treatment differ from country to country. In Britain, Parliament has accepted therapeutic cloning (February 2001), while the German Bundestag has banned the procedure. The EU Parliament has recommended European countries not to allow therapeutic cloning, and President George W. Bush takes a more critical stand than did the Clinton administration. The debate over embryonal cells and therapeutic cloning involves both scientists and politicians. In Norway, the Biotechnology Advisory Board wants to open up for research on fertilized eggs and for therapeutic cloning. The Storting, Norway's legislature, will have to reach a decision on these issues when the Biotechnology Act come up for revision, probably in 2002. In the debate in Norway, it has been claimed that accepting therapeutic cloning would be a violation of a traditional western norm: Man should always be an end onto himself, never be used as an instrument to other ends. Furthermore, some scientists think that one should use adult stem cells from adults--this also because the procedure seems less risky. Bone marrow transplantation with autologous bone marrow stem cells has been used for 30 years, and recent research has disclosed that such stem cells may be reprogrammed, for instance from bone marrow stem cells to nerve cells.
-
Cloning of nascent monkey DNA synthesized early in the cell cycle.
Kaufmann, G; Zannis-Hadjopoulos, M; Martin, R G
1985-04-01
To study the structure and complexity of animal cell replication origins, we have isolated and cloned nascent DNA from the onset of S phase as follows: African green monkey kidney cells arrested in G1 phase were serum stimulated in the presence of the DNA replication inhibitor aphidicolin. After 18 h, the drug was removed, and DNA synthesis was allowed to proceed in vivo for 1 min. Nuclei were then prepared, and DNA synthesis was briefly continued in the presence of Hg-dCTP. The mercury-labeled nascent DNA was purified in double-stranded form by extrusion (M. Zannis-Hadjopoulos, M. Perisco, and R. G. Martin, Cell 27:155-163, 1981) followed by sulfhydryl-agarose affinity chromatography. Purified nascent DNA (ca. 500 to 2,000 base pairs) was treated with mung bean nuclease to remove single-stranded ends and inserted into the NruI site of plasmid pBR322. The cloned fragments were examined for their time of replication by hybridization to cellular DNA fractions synthesized at various intervals of the S phase. Among five clones examined, four hybridized preferentially with early replicating fractions.
-
Han, Il; Congeevaram, Shankar; Ki, Dong-Won; Oh, Byoung-Taek; Park, Joonhong
2011-02-01
Due to the environmental problems associated with disposal of livestock sludge, many stabilization studies emphasizing on the sludge volume reduction were performed. However, little is known about the microbial risk present in sludge and its stabilized products. This study microbiologically explored the effects of anaerobic lagoon fermentation (ALF) and autothermal thermophilic aerobic digestion (ATAD) on pathogen-related risk of raw swine manure by using culture-independent 16S rDNA cloning and sequencing methods. In raw swine manure, clones closely related to pathogens such as Dialister pneumosintes, Erysipelothrix rhusiopathiae, Succinivibrioan dextrinosolvens, and Schineria sp. were detected. Meanwhile, in the mesophilic ALF-treated swine manure, bacterial community clones closely related to pathogens such as Schineria sp. and Succinivibrio dextrinosolvens were still detected. Interestingly, the ATAD treatment resulted in no detection of clones closely related to pathogens in the stabilized thermophilic bacterial community, with the predominance of novel Clostridia class populations. These findings support the superiority of ATAD in selectively reducing potential human and animal pathogens compared to ALF, which is a typical manure stabilization method used in livestock farms.
-
Large-scale production of functional human lysozyme from marker-free transgenic cloned cows.
Lu, Dan; Liu, Shen; Ding, Fangrong; Wang, Haiping; Li, Jing; Li, Ling; Dai, Yunping; Li, Ning
2016-03-10
Human lysozyme is an important natural non-specific immune protein that is highly expressed in breast milk and participates in the immune response of infants against bacterial and viral infections. Considering the medicinal value and market demand for human lysozyme, an animal model for large-scale production of recombinant human lysozyme (rhLZ) is needed. In this study, we generated transgenic cloned cows with the marker-free vector pBAC-hLF-hLZ, which was shown to efficiently express rhLZ in cow milk. Seven transgenic cloned cows, identified by polymerase chain reaction, Southern blot, and western blot analyses, produced rhLZ in milk at concentrations of up to 3149.19 ± 24.80 mg/L. The purified rhLZ had a similar molecular weight and enzymatic activity as wild-type human lysozyme possessed the same C-terminal and N-terminal amino acid sequences. The preliminary results from the milk yield and milk compositions from a naturally lactating transgenic cloned cow 0906 were also tested. These results provide a solid foundation for the large-scale production of rhLZ in the future.
-
Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki
2007-01-01
Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.
-
Lee, Nam-Jin; Yang, Byoung-Chul; Im, Gi-Sun; Lee, Sung-Soo; Seong, Hwan-Hoo; Park, Jin-Ki; Chang, Won-Kyong; Kang, Jong-Koo; Hwang, Seongsoo
2013-08-01
This study was designed to undertake a risk assessment to identify the health status of rats fed with somatic cell nuclear transfer (SCNT)-cloned Korean native beef cattle (Hanwoo) meat for 26 weeks. The rats were randomly divided into 5 groups, each consisting of 12 male (142.6 ± 5.23 g) and 12 female (113.7 ± 6.31 g) rats each. The animals were fed commercial pellets (control), pellets containing 5% (N-5) and 10% (N-10) of normal cattle meat, and diets containing 5% (C-5) and 10% (C-10) of cloned cattle meat. The mortality; clinical signs; body weight; food consumption; urinary, hematology, blood biochemistry, and histopathological analyses; and absolute and relative organ weights were analyzed and compared. During the 26-week test period, health status-related factors of the rats fed on cloned Hanwoo meat were found to have no test substance-related toxicities. The only difference was the increased uterus weight in female C-10 rats as compared to their counterparts counterparts (p < .05). On the basis of these health status results, it can be postulated that no food consumption risks might arise from the long-term feeding of cloned cattle meat in rats.
-
Rodrigues, Ana Catarina; Belas, Adriana; Marques, Cátia; Cruz, Luís; Gama, Luís T; Pomba, Constança
2018-05-01
Methicillin-resistant staphylococci (MRS), namely Staphylococcus aureus (MRSA) and Staphylococcus pseudintermedius (MRSP), are opportunistic agents of great importance in human and veterinary medicine. The aims of this study were to investigate the frequency, persistence, and risk factors associated with nasal colonization by MRS in people in daily contact with animals in Portugal. Seventy-nine out of 129 (61.2%) participants were found to be colonized by, at least, one methicillin-resistant (MR) staphylococci species (MR Staphylococcus epidermidis [n = 68], MRSA [n = 19], MR Staphylococcus haemolyticus [n = 7], MRSP [n = 2], and other coagulase-negative staphylococci [n = 4]). Three lineages were identified among the MRSA isolates (n = 7): the major human healthcare clone in Portugal (ST22-t032-IV, n = 3), the livestock-associated MRSA (ST398-t108-V, n = 3), and the New York-/Japan-related clone (ST105-t002-II, n = 1). MRSP isolates belonged to the European clone ST71-II-III. We identified two risk factors for nasal colonization by MRS in healthy humans: (i) being a veterinary professional (veterinarian and veterinary nurse) (p < 0.0001, odds ratio [OR] = 6.369, 95% confidence interval [CI, 2.683-15.122]) and (ii) have contacted with one MRSA- or MRSP-positive animal (p = 0.0361, OR = 2.742, 95% CI [1.067-7.045]). The follow-up study revealed that the majority (85%) remain colonized. This study shows that MRS in veterinary clinical practice is a professional hazard and highlights the need to implement preventive measures to minimize spread.
-
Nakajima, T; Walkup, R D; Tochigi, A; Shearer, T R; Azuma, M
2007-11-01
Lacritin is a mitogen of human salivary gland cells as well as a stimulator of human corneal epithelial cells. It is expected to be an important factor in maintaining the surrounding ocular surface. The monkey would be a relevant animal model in which to study the role of lacritin in ophthalmic physiology and pathology. However, to our knowledge, no cDNA cloning or functional analysis of monkey lacritin has been performed. Thus, the purposes of this study were: (1) to clone the monkey ortholog of lacritin; (2) to characterize lacritin in tears from several species; and (3) to determine the tissues where lacritin is produced and secreted. cDNA for lacritin from rhesus macaque contained 547 bp, with 411 bp in an open reading frame (ORF) encoding a protein of 137 amino acids. Monkey lacritin showed 89% amino acid homology with human lacritin; one amino acid was deleted in all three monkey strains. The predicted MW of mature lacritin was 12.2 kDa, and the isoelectric point was 4.99. Lacritin showed anomalous migration at approximately 21.0 kDa on SDS-PAGE, as confirmed by immunoblotting and amino acid sequencing. Similar to native lacritin in monkey tears, a 21 kDa band was also detected in human tears. In contrast, no lacritin was observed at a similar position on SDS-PAGE in rat, rabbit and dog tears. In the monkey, lacritin mRNA was expressed highly in the lacrimal gland, moderately in the conjunctiva and the meibomian gland, and weakly in corneal epithelium. In primates, lacritin was produced in the lacrimal gland and secreted into tear fluid. These results suggest that lacritin might be important for the maintenance of the ocular surface in higher animals, such as monkeys and humans.
-
Aires-de-Sousa, Marta; Parente, Carlos E S R; Vieira-da-Motta, Olney; Bonna, Isabel C F; Silva, Denise A; de Lencastre, Hermínia
2007-06-01
Eighty-four staphylococcal isolates were obtained from milk samples from cows, sheep, goats, and buffalo with subclinical mastitis and from colonization samples from ostriches. The animals were hosted in 18 small dairy herds and an ostrich breeding located in 10 municipalities of the state of Rio de Janeiro, Brazil. Thirty isolates were identified as Staphylococcus aureus by biochemical and molecular techniques and were comparatively characterized by phenotypic and genotypic methods. The molecular characterization by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) revealed five clonal types (PFGE A, spa type t359, sequence type 747 [ST747]; PFGE B, spa type t1180, ST750; PFGE C, spa type t605, ST126; PFGE D, spa type t127, ST751; and PFGE F, spa type t002, ST5). None of the isolates harbored the Panton-Valentine leukocidin or exfoliative toxin D gene. The detection of major clone A (in 63% of the isolates) in different herds, among all animal species studied, and in infection and colonization samples evidenced its geographical spread among Rio de Janeiro State and no host preference among the animal species. Comparison with S. aureus from a human origin suggested that all but one clone found in the present study might be animal specific.
-
Wakayama, Sayaka; Tanabe, Yoshiaki; Nagatomo, Hiroaki; Mizutani, Eiji; Kishigami, Satoshi; Wakayama, Teruhiko
2016-11-01
Although animal cloning is becoming increasingly practicable, cloned embryos possess many abnormalities and so there has been a low success rate for producing live animals. This is most likely due to incomplete reprogramming of somatic cell nuclei before they start to develop as the donor nuclei are usually only exposed to the oocyte cytoplasm for 1-2 hours before reconstructed oocytes are activated to avoid oocyte aging. Therefore, in this study, we attempted to extend the exposure period of somatic cell nuclei to the oocyte cytoplasm to determine whether this could enhance reprogramming of donor nuclei. Donor nuclei were transferred into oocytes, following which pseudo-MII spindles (pMIIs) derived from these were extracted and injected into newly collected enucleated oocytes 24 hours after the first nuclear transfer (NT). These serial NT oocytes were then activated and their developmental potential was examined to full term. There was no obvious difference in the pMIIs of reconstructed oocytes at 6 and 24 hours after donor nucleus injection; however, in both of these, the chromosomes were more widely spread inside the spindle than in fresh intact oocytes. Furthermore, a few chromosomes remained in 25% and 47% of these enucleated oocytes at 6 and 24 hours after donor nucleus injection, respectively. When these pMIIs were injected into fresh enucleated oocytes, the developmental rate to blastocyst was significantly lower, but we could still obtain several healthy cloned offspring. Thus, serial NT at intervals of 24 hours using fresh oocytes is possible, but the success rate could not be improved due to loss of chromosomes during the second NT.
-
Shite, Masato; Yamamura, Yoshimi; Hayashi, Toshimitsu; Kurosaki, Fumiya
2008-11-01
A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein alpha-subunits from various biological sources suggested that, unlike in animal cells, classification of Galpha-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Galpha-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level.
-
Jiang, W; Woitach, J T; Gupta, D; Bhavanandan, V P
1998-10-20
Secreted epithelial mucins are extremely large and heterogeneous glycoproteins. We report the 5 kilobase DNA sequence of a second gene, BSM2, which encodes bovine submaxillary mucin. The determined nucleotide and deduced amino acid sequences of BSM2 are 95.2% and 92. 2% identical, respectively, to those of the previously described BSM1 gene isolated from the same cow. Further, the five predicted protein domains of the two genes are 100%, 94%, 93%, 77%, and 88% identical. Based on the above results, we propose that expression of multiple homologous core proteins from a single animal is a factor in generating diversity of saccharides in mucins and in providing resistance of the molecules to proteolysis. In addition, this work raises several important issues in mucin cloning such as assembling sequences from seemingly overlapping clones and deducing consensus sequences for nearly identical tandem repeats. Copyright 1998 Academic Press.
-
Jain, Sudhir Kumar; Jain, Hemlata; Kumar, Dharmendra; Bedekar, Megha Kadam; Pandey, Akhilesh Kumar; Sarkhel, Bikash Chandra
2015-09-01
Skeletal muscle is the major component of lean tissue that is used for consumption, and myostatin is a negative regulator of skeletal muscle growth. Downregulation of this gene therefore offers a strategy for developing superior animals with enhanced muscle growth. Knockdown of myostatin was achieved by RNA interference technology. The anti-myostatin shRNA were designed and stably transfected in caprine fibroblast cells. The reduced expression of target gene was achieved and measured in clonal fibroblast cells by real-time PCR. Two single-cell clones induced significant decrease of myostatin gene expression by 73.96 and 72.66 %, respectively (P < 0.05). To ensure the appropriate growth of transfected cell, seven media were tested. The best suited media was used for transfected fibroblast cell proliferation. The findings suggest that shRNA provides a novel potential tool for gene knockdown and these stably transfected cells can be used as the donor cells for animal cloning.
-
Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source
NASA Astrophysics Data System (ADS)
Kawarasaki, Tatsuo; Uchiyama, Kazuhiko; Hirao, Atsushi; Azuma, Sadahiro; Otake, Masayoshi; Shibata, Masatoshi; Tsuchiya, Seiko; Enosawa, Shin; Takeuchi, Koichi; Konno, Kenjiro; Hakamata, Yoji; Yoshino, Hiroyuki; Wakai, Takuya; Ookawara, Shigeo; Tanaka, Hozumi; Kobayashi, Eiji; Murakami, Takashi
2009-09-01
Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.
-
Dolferus, R.; Osterman, J. C.; Peacock, W. J.; Dennis, E. S.
1997-01-01
This article reports the cloning of the genes encoding the Arabidopsis and rice class III ADH enzymes, members of the alcohol dehydrogenase or medium chain reductase/dehydrogenase superfamily of proteins with glutathione-dependent formaldehyde dehydrogenase activity (GSH-FDH). Both genes contain eight introns in exactly the same positions, and these positions are conserved in plant ethanol-active Adh genes (class P). These data provide further evidence that plant class P genes have evolved from class III genes by gene duplication and acquisition of new substrate specificities. The position of introns and similarities in the nucleic acid and amino acid sequences of the different classes of ADH enzymes in plants and humans suggest that plant and animal class III enzymes diverged before they duplicated to give rise to plant and animal ethanol-active ADH enzymes. Plant class P ADH enzymes have gained substrate specificities and evolved promoters with different expression properties, in keeping with their metabolic function as part of the alcohol fermentation pathway. PMID:9215914
-
Zhang, Jianqiang; Go, Yun Young; Huang, Chengjin M.; Meade, Barry J.; Lu, Zhengchun; Snijder, Eric J.; Timoney, Peter J.
2012-01-01
A stable full-length cDNA clone of the modified live virus (MLV) vaccine strain of equine arteritis virus (EAV) was developed. RNA transcripts generated from this plasmid (pEAVrMLV) were infectious upon transfection into mammalian cells, and the resultant recombinant virus (rMLV) had 100% nucleotide identity to the parental MLV vaccine strain of EAV. A single silent nucleotide substitution was introduced into the nucleocapsid gene (pEAVrMLVB), enabling the cloned vaccine virus (rMLVB) to be distinguished from parental MLV vaccine as well as other field and laboratory strains of EAV by using an allelic discrimination real-time reverse transcription (RT)-PCR assay. In vitro studies revealed that the cloned vaccine virus rMLVB and the parental MLV vaccine virus had identical growth kinetics and plaque morphologies in equine endothelial cells. In vivo studies confirmed that the cloned vaccine virus was very safe and induced high titers of neutralizing antibodies against EAV in experimentally immunized horses. When challenged with the heterologous EAV KY84 strain, the rMLVB vaccine virus protected immunized horses in regard to reducing the magnitude and duration of viremia and virus shedding but did not suppress the development of signs of EVA, although these were reduced in clinical severity. The vaccine clone pEAVrMLVB could be further manipulated to improve the vaccine efficacy as well as to develop a marker vaccine for serological differentiation of EAV naturally infected from vaccinated animals. PMID:22739697
-
Fire coral clones demonstrate phenotypic plasticity among reef habitats.
Dubé, Caroline E; Boissin, Emilie; Maynard, Jeffrey A; Planes, Serge
2017-08-01
Clonal populations are often characterized by reduced levels of genotypic diversity, which can translate into lower numbers of functional phenotypes, both of which impede adaptation. Study of partially clonal animals enables examination of the environmental settings under which clonal reproduction is favoured. Here, we gathered genotypic and phenotypic information from 3,651 georeferenced colonies of the fire coral Millepora platyphylla in five habitats with different hydrodynamic regimes in Moorea, French Polynesia. In the upper slope where waves break, most colonies grew as vertical sheets ("sheet tree") making them more vulnerable to fragmentation. Nearly all fire corals in the other habitats are encrusting or massive. The M. platyphylla population is highly clonal (80% of the colonies are clones), while characterized by the highest genotype diversity ever documented for terrestrial or marine populations (1,064 genotypes). The proportion of clones varies greatly among habitats (≥58%-97%) and clones (328 clonal lineages) are distributed perpendicularly from the reef crest, perfectly aligned with wave energy. There are six clonal lineages with clones dispersed in at least two adjacent habitats that strongly demonstrate phenotypic plasticity. Eighty per cent of the colonies in these lineages are "sheet tree" on the upper slope, while 80%-100% are encrusting or massive on the mid slope and back reef. This is a unique example of phenotypic plasticity among reef-building coral clones as corals typically have wave-tolerant growth forms in high-energy reef areas. © 2017 John Wiley & Sons Ltd.
-
Samiec, M; Skrzyszowska, M
2011-01-01
Somatic cell cloning technology in mammals promotes the multiplication of productively-valuable genetically engineered individuals, and consequently allows also for standardization of transgenic farm animal-derived products, which, in the context of market requirements, will have growing significance. Gene farming is one of the most promising areas in modern biotechnology. The use of live bioreactors for the expression of human genes in the lactating mammary gland of transgenic animals seems to be the most cost-effective method for the production/processing of valuable recombinant therapeutic proteins. Among the transgenic farm livestock species used so far, cattle, goats, sheep, pigs and rabbits are useful candidates for the expression of tens to hundreds of grams of genetically-engineered proteins or xenogeneic biopreparations in the milk. At the beginning of the new millennium, a revolution in the treatment of disease is taking shape due to the emergence of new therapies based on recombinant human proteins. The ever-growing demand for such pharmaceutical or nutriceutical proteins is an important driving force for the development of safe and large-scale production platforms. The aim of this paper is to present an overall survey of the state of the art in investigations which provide the current knowledge for deciphering the possibilities of practical application of the transgenic mammalian species generated by somatic cell cloning in biomedicine, the biopharmaceutical industry, human nutrition/dietetics and agriculture.
-
Identification of mimotopes of Mycobacterium leprae as potential diagnostic reagents.
Alban, Silvana M; de Moura, Juliana Ferreira; Minozzo, João Carlos; Mira, Marcelo Távora; Soccol, Vanete Thomaz
2013-01-25
An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated. Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents. Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5) and peptide 1B (1/5). In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae. The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.
-
Production of Prnp-/- goats by gene targeting in adult fibroblasts.
Zhu, Caihong; Li, Bei; Yu, Guohua; Chen, Jianquan; Yu, Huiqing; Chen, Juan; Xu, Xujun; Wu, Youbing; Zhang, Aimin; Cheng, Guoxiang
2009-04-01
Homozygous mice devoid of functional Prnp are resistant to scrapie and prion propagation, but heterozygous mice for Prnp disruption still suffer from prion disease and prion deposition. We have previously generated heterozygous cloned goats with one allele of Prnp functional disruption. To obtain goats with both alleles of Prnp be disrupted which would be resistant to scrapie completely, a second-round gene targeting was applied to disrupt the wild type allele of Prnp in the heterozygous goats. By second-round gene targeting, we successfully disrupted the wild type allele of Prnp in primary Prnp (+/-) goat skin fibroblasts and obtained a Prnp (-/-) cell line without Prnp expression. This is the first report on successful targeting modification in primary adult somatic cells of animals. These cells were used as nuclear donors for somatic cell cloning to produce Prnp (-/-) goats. A total of 57 morulae or blastocytes developed from the reconstructed embryos were transferred to 31 recipients, which produced 7 pregnancies at day 35. At 73 days of gestation, we obtained one cloned fetus with Prnp (-/-) genotype. Our research not only indicated that multiple genetic modifications could be accomplished by multi-round gene targeting in primary somatic cells, but also provided strong evidence that gene targeting in adult cells other than fetal cells could be applied to introduce precise genetic modifications in animals without destroying the embryos.
-
Friis, Thor Einar; Stephenson, Sally; Xiao, Yin; Whitehead, Jon
2014-01-01
The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes. PMID:24447069
-
Islam, Md Zohorul; Espinosa-Gongora, Carmen; Damborg, Peter; Sieber, Raphael N.; Munk, Rikke; Husted, Louise; Moodley, Arshnee; Skov, Robert; Larsen, Jesper; Guardabassi, Luca
2017-01-01
Denmark is a country with high prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC) 398 in pigs. Even though pig farming is regarded as the main source of human infection or colonization with MRSA CC398, 10–15% of the human cases appear not to be linked to pigs. Following the recent reports of MRSA CC398 in horses in other European countries and the lack of knowledge on S. aureus carriage in this animal species, we carried out a study to investigate whether horses constitute a reservoir of MRSA CC398 in Denmark, and to gain knowledge on the frequency and genetic diversity of S. aureus in horses, including both methicillin-resistant and -susceptible S. aureus (MSSA). Nasal swabs were collected from 401 horses originating from 74 farms, either at their farms or prior to admission to veterinary clinics. Following culture on selective media, species identification by MALDI-TOF MS and MRSA confirmation by standard PCR-based methods, S. aureus and MRSA were detected in 54 (13%) and 17 (4%) horses originating from 30 (40%) and 7 (9%) farms, respectively. Based on spa typing, MSSA differed genetically from MRSA isolates. The spa type prevalent among MSSA isolates was t127 (CC1), which was detected in 12 horses from 11 farms and represents the most common S. aureus clone isolated from human bacteremia cases in Denmark. Among the 17 MRSA carriers, 10 horses from three farms carried CC398 t011 harboring the immune evasion cluster (IEC), four horses from two farms carried IEC-negative CC398 t034, and three horses from two farms carried a mecC-positive MRSA lineage previously associated with wildlife and domestic ruminants (CC130 t528). Based on whole-genome phylogenetic analysis of the 14 MRSA CC398, t011 isolates belonged to the recently identified horse-adapted clone in Europe and were closely related to human t011 isolates from three Danish equine veterinarians, whereas t034 isolates belonged to pig-adapted clones. Our study confirms that horses carry an equine-specific clone of MRSA CC398 that can be transmitted to veterinary personnel, and reveals that these animals are exposed to MRSA and MSSA clones that are likely to originate from livestock and humans, respectively. PMID:28421046
-
Mourão, Joana; Novais, Carla; Machado, Jorge; Peixe, Luísa; Antunes, Patrícia
2015-06-01
The occurrence of acquired metal tolerance genes in emerging MDR Salmonella enterica serotype 4,[5],12:i:- clones was assessed and their associated platforms and tolerance phenotype were characterised. Salmonella 4,[5],12:i:- from different sources belonging to European, Spanish and Southern European clones were studied. Screening for copper (pcoA-pcoD/tcrB), silver/copper (silA-silE), mercury (merA), arsenic (arsB) and tellurite (terF) tolerance genes was performed by PCR/sequencing. CuSO(4)/AgNO(3) MICs were determined in aerobic/anaerobic atmospheres by agar dilution. Conjugation assays, genomic location and plasmid analysis were performed by standard procedures. Most isolates from European (98%) and Spanish (74%) clones carried silA-silE, contrasting with the Southern European clone (26%). merA/62% (European and Spanish clones) and pcoA-pcoD/50% (European clone) were also detected. merA±pco+sil were chromosomally located in the European clone, whereas in Spanish and Southern European clones sil±merA were within plasmids, both with antibiotic resistance genes. The pcoA-pcoD/silA-silE(+) isolates showed higher MICCuSO(4) in anaerobiosis than those without these genes (MIC(50)=24-28 vs. 2 mM). Different MICAgNO(3) of silA-silE(+) (MIC(50)=0.25 mM) and silA-silE(-)(MIC(50)=0.16 mM) isolates were observed in both atmospheres, with an MIC increment after prior exposure to silver (>3 vs. 0.08-0.125 mM) in aerobiosis. A high frequency of copper and silver tolerance, particularly among the two major Salmonella 4,[5],12:i:- MDR clones (European/Spanish) circulating in Europe and causing human infections, might facilitate adaptation/expansion of these strains in metal-contaminated environments, particularly copper in anaerobiosis. Furthermore, metal toxic concentrations in food-animal environments can contribute to persistence of genetic platforms carrying metal/antibiotic resistance genes in this foodborne zoonotic pathogen. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
-
Cloning, expression, and characterization of Giardia duodenalis, beta-giardin
USDA-ARS?s Scientific Manuscript database
Giardiasis is frequently diagnosed in humans and is highly prevalent in many animal species as well. The cumulative prevalence of infection in dairy cattle frequently reaches 100%. The replicating trophozoite stage colonizes the upper small intestine and attaches to the surface epithelium via a vent...
-
Poirier, John T; Reddy, P Seshidhar; Idamakanti, Neeraja; Li, Shawn S; Stump, Kristine L; Burroughs, Kevin D; Hallenbeck, Paul L; Rudin, Charles M
2012-12-01
Seneca Valley virus (SVV-001) is an oncolytic picornavirus with selective tropism for a subset of human cancers with neuroendocrine differentiation. To characterize further the specificity of SVV-001 and its patterns and kinetics of intratumoral spread, bacterial plasmids encoding a cDNA clone of the full-length wild-type virus and a derivative virus expressing GFP were generated. The full-length cDNA of the SVV-001 RNA genome was cloned into a bacterial plasmid under the control of the T7 core promoter sequence to create an infectious cDNA clone, pNTX-09. A GFP reporter virus cDNA clone, pNTX-11, was then generated by cloning a fusion protein of GFP and the 2A protein from foot-and-mouth disease virus immediately following the native SVV-001 2A sequence. Recombinant GFP-expressing reporter virus, SVV-GFP, was rescued from cells transfected with in vitro RNA transcripts from pNTX-11 and propagated in cell culture. The proliferation kinetics of SVV-001 and SVV-GFP were indistinguishable. The SVV-GFP reporter virus was used to determine that a subpopulation of permissive cells is present in small-cell lung cancer cell lines previously thought to lack permissivity to SVV-001. Finally, it was shown that SVV-GFP administered to tumour-bearing animals homes in to and infects tumours whilst having no detectable tropism for normal mouse tissues at 1×10(11) viral particles kg(-1), a dose equivalent to that administered in ongoing clinical trials. These infectious clones will be of substantial value in further characterizing the biology of this virus and as a backbone for the generation of additional oncolytic derivatives.
-
Wang, Wei-Cheng; Zeng, Zhi-Yong; Tang, De-Yuan; Liang, Hai-Ying; Liu, Zhao; Dai, Zhen-Jiang
2015-08-01
Porcine circovirus-associated disease is a highly contagious disease that has significant economic consequences. The disease is prevalent in many countries and regions. To generate a genetic marker strain of PCV2, a Sal I restriction enzyme site was inserted into the PCV2 clone as a genetic marker by applying iDNA infectious clone technology. The iDNA represents plasmids that encode the full-length DNA genome of PCV2 assembled in a pcDNA3.1-based vectors. The mutant PCV2 was rescued by transfecting an infectious clone into PK-15 cells and was characterised by an immunoperoxidase monolayer assay (IPMA). The viral genome could be differentiated from the wild-type parent by PCR and restriction fragment length polymorphism (PCR-RFLP). Kunming mice were inoculated with the PCV2 infectious clone or rescued virus via intranasal and intraperitoneal routes. Seroconversion to PCV2-specific antibody appeared in the majority of mice from the two inoculated groups at 7 days postinoculation (DPI), and the specific antibody level was steady for at least 42 days. Viraemia, beginning at 7 DPI and lasting 4 weeks, was detected in the majority of the pigs from the two inoculated groups. The animal experiments revealed that the PCV2 infectious clone and rescued virus both could replicate in mice and induce mice to generate anti-PCV2 antibodies. The infectious clones of PCV2 will be useful for further research investigating a potential tractable iDNA vaccine by reverse genetics technology for attenuated virulance. Copyright © 2015 Elsevier B.V. All rights reserved.
-
Differential developmental ability of embryos cloned from tissue-specific stem cells.
Inoue, Kimiko; Noda, Shinichi; Ogonuki, Narumi; Miki, Hiromi; Inoue, Shinichi; Katayama, Kazufumi; Mekada, Kazuyuki; Miyoshi, Hiroyuki; Ogura, Atsuo
2007-05-01
Although cloning animals by somatic cell nuclear transfer is generally inefficient, the use of certain nuclear donor cell types may significantly improve or deteriorate outcomes. We evaluated whether two multipotent stem cell lines produced in vitro--neural stem cells (NSCs) and mesenchymal stem cells (MSCs)--could serve as nuclear donors for nuclear transfer cloning. Most (76%) NSC-derived embryos survived the two-cell-to-four-cell transition, the stage when the major zygotic gene activation occurs. Consistent with this observation, the expression patterns of zygotically active genes were better in NSC-derived embryos than in fibroblast clone embryos, which arrested at the two-cell stage more frequently. Embryo transfer experiments demonstrated that at least some of these NSC embryos had the ability to develop to term fetuses (1.6%, 3/189). In contrast, embryos reconstructed using MSCs showed a low rate of in vitro development and never underwent implantation in vivo. Chromosomal analysis of the donor MSCs revealed very frequent aneuploidy, which probably impaired the potential for development of their derived clones. This is the first demonstration that tissue-specific multipotent stem cells produced in vitro can serve as donors of nuclei for cloning mice; however, these cells may be prone to chromosomal aberrations, leading to high embryonic death rates. We found previously that hematopoietic stem cells (HSCs) are very inefficient donor cells because of their failure to activate the genes essential for embryonic development. Taken together, our data led us to conclude that tissue-specific stem cells in mice, namely NSCs, MSCs, and HSCs, exhibited marked variations in the ability to produce cloned offspring and that this ability varies according to both the epigenetic and genetic status of the original genomes. Disclosure of potential conflicts of interest is found at the end of this article.
-
Sanad, Yasser M; Jung, Kwonil; Kashoma, Isaac; Zhang, Xiaoli; Kassem, Issmat I; Saif, Yehia M; Rajashekara, Gireesh
2014-11-25
Campylobacter jejuni is commonly found in the gastrointestinal tract of many food-animals including sheep without causing visible clinical symptoms of disease. However, C. jejuni has been implicated in ovine abortion cases worldwide. Specifically, in the USA, the C. jejuni sheep abortion (SA) clone has been increasingly associated with sheep abortion. In vivo studies in sheep (the natural host) are needed to better characterize the virulence potential and pathogenesis of this clone. Pregnant ewes intravenously (IV) or orally inoculated with ovine or bovine abortion-associated C. jejuni SA clones exhibited partial or complete uterine prolapse with retained placenta, and abortion or stillbirth, whereas delivery of healthy lambs occurred in pregnant ewes inoculated with C. jejuni 81-176 or in the uninfected group. In sheep inoculated with the SA clone, histopathological lesions including suppurative necrotizing placentitis and/or endometritis coincided with: 1) increased apoptotic death of trophoblasts, 2) increased expression of the host genes (e.g. genes encoding interleukin IL-6 and IL-15) related to cellular necrosis and pro-inflammatory responses in uterus, and 3) decreased expression of the genes encoding GATA binding protein 6, chordin, and insulin-like 3 (INSL3) that account for embryonic development in uterus. Immunohistochemistry revealed localization of bacterial antigens in trophoblasts lining the chorioallantoic membrane of ewes inoculated with the C. jejuni SA clone. The results showed that C. jejuni SA clones are capable of causing abortion or stillbirth in experimentally infected sheep. Furthermore, down- or up-regulation of specific genes in the uterus of infected pregnant ewes might implicate host genes in facilitating the disease progression. Since the C. jejuni SA strains share genotypic similarities with clones that have been isolated from human clinical cases of gastroenteritis, these strains might represent a potential public health risk.
-
Sawada, Koichi; Kokeguchi, Susumu; Hongyo, Hiroshi; Sawada, Satoko; Miyamoto, Manabu; Maeda, Hiroshi; Nishimura, Fusanori; Takashiba, Shogo; Murayama, Yoji
1999-01-01
Subtractive hybridization was employed to isolate specific genes from virulent Porphyromonas gingivalis strains that are possibly related to abscess formation. The genomic DNA from the virulent strain P. gingivalis W83 was subtracted with DNA from the avirulent strain ATCC 33277. Three clones unique to strain W83 were isolated and sequenced. The cloned DNA fragments were 885, 369, and 132 bp and had slight homology with only Bacillus stearothermophilus IS5377, which is a putative transposase. The regions flanking the cloned DNA fragments were isolated and sequenced, and the gene structure around the clones was revealed. These three clones were located side-by-side in a gene reported as an outer membrane protein. The three clones interrupt the open reading frame of the outer membrane protein gene. This inserted DNA, consisting of three isolated clones, was designated IS1598, which was 1,396 bp (i.e., a 1,158-bp open reading frame) in length and was flanked by 16-bp terminal inverted repeats and a 9-bp duplicated target sequence. IS1598 was detected in P. gingivalis W83, W50, and FDC 381 by Southern hybridization. All three P. gingivalis strains have been shown to possess abscess-forming ability in animal models. However, IS1598 was not detected in avirulent strains of P. gingivalis, including ATCC 33277. The IS1598 may interrupt the synthesis of the outer membrane protein, resulting in changes in the structure of the bacterial outer membrane. The IS1598 isolated in this study is a novel insertion element which might be a specific marker for virulent P. gingivalis strains. PMID:10531208
-
USDA-ARS?s Scientific Manuscript database
Background: Rhipicephalus (Boophilus) microplus (Rmi) a major cattle ectoparasite and tick borne disease vector, impacts on animal welfare and industry productivity. In arthropod research there is an absence of a complete Chelicerate genome, which includes ticks, mites, spiders, scorpions and crusta...
-
The oxalic acid biosynthetic activity of Burkholderia mallei is encoded by a single locus
USDA-ARS?s Scientific Manuscript database
Although it is known that oxalic acid provides a selective advantage to the secreting microbe, our understanding of how this acid is biosynthesized remains incomplete. This study reports the identification, cloning, and partial characterization of the oxalic acid biosynthetic enzyme from the animal ...
-
Souza, A M; Reis, J K P; Assis, R A; Horta, C C; Siqueira, F F; Facchin, S; Alvarenga, E R; Castro, C S; Salvarani, F M; Silva, R O S; Pires, P S; Contigli, C; Lobato, F C F; Kalapothakis, E
2010-02-18
Epsilon toxin produced by Clostridium perfringens types B and D causes enterotoxemia in sheep, goats and calves. Enterotoxemia can cause acute or superacute disease, with sudden death of the affected animal. It provokes huge economic losses when large numbers of livestock are affected. Therapeutic intervention is challenging, because the disease progresses very rapidly. However, it can be prevented by immunization with specific immunogenic vaccines. We cloned the etx gene, encoding epsilon toxin, into vector pET-11a; recombinant epsilon toxin (rec-epsilon) was expressed in inclusion bodies and was used for animal immunization. Serum protection was evaluated and cross-serum neutralization tests were used to characterize the recombinant toxin. To analyze the potency of the toxin (as an antigen), rabbits were immunized with 50, 100 or 200 microg recombinant toxin, using aluminum hydroxide gel as an adjuvant. Titers of 10, 30 and 40 IU/mL were obtained, respectively. These titers were higher than the minimum level required by the European Pharmacopoeia (5 IU/mL) and by the USA Code of Federal Regulation (2 IU/mL). This rec-epsilon is a good candidate for vaccine production against enterotoxemia caused by epsilon toxin of C. perfringens type D.
-
Taylor-Robinson, Andrew W; Walton, Simon; Swain, David L; Walsh, Kerry B; Vajta, Gábor
2014-08-01
Recent advances in embryology and related research offer considerable possibilities to accelerate genetic improvement in cattle breeding. Such progress includes optimization and standardization of laboratory embryo production (in vitro fertilization - IVF), introduction of a highly efficient method for cryopreservation (vitrification), and dramatic improvement in the efficiency of somatic cell nuclear transfer (cloning) in terms of required effort, cost, and overall outcome. Handmade cloning (HMC), a simplified version of somatic cell nuclear transfer, offers the potential for relatively easy and low-cost production of clones. A potentially modified method of vitrification used at a centrally located laboratory facility could result in cloned offspring that are economically competitive with elite animals produced by more traditional means. Apart from routine legal and intellectual property issues, the main obstacle that hampers rapid uptake of these technologies by the beef cattle industry is a lack of confidence from scientific and commercial sources. Once stakeholder support is increased, the combined application of these methods makes a rapid advance toward desirable traits (rapid growth, high-quality beef, optimized reproductive performance) a realistic goal. The potential impact of these technologies on genetic advancement in beef cattle herds in which improvement of stock is sought, such as in northern Australia, is hard to overestimate. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.
-
Yamada, Kazunari; Tso, Jonathan L.; Menjivar, Jimmy C.; Tian, Jane Y.; Yong, William H.; Schaue, Dörthe; Mischel, Paul S.; Cloughesy, Timothy F.; Nelson, Stanley F.; Liau, Linda M.; McBride, William; Tso, Cho-Lea
2013-01-01
Glioblastoma stem cells (GSC) are a significant cell model for explaining brain tumor recurrence. However, mechanisms underlying their radiochemoresistance remain obscure. Here we show that most clonogenic cells in GSC cultures are sensitive to radiation treatment (RT) with or without temozolomide (TMZ). Only a few single cells survive treatment and regain their self-repopulating capacity. Cells re-populated from treatment-resistant GSC clones contain more clonogenic cells compared to those grown from treatment-sensitive GSC clones, and repeated treatment cycles rapidly enriched clonogenic survival. When compared to sensitive clones, resistant clones exhibited slower tumor development in animals. Upregulated genes identified in resistant clones via comparative expression microarray analysis characterized cells under metabolic stress, including blocked glucose uptake, impaired insulin/Akt signaling, enhanced lipid catabolism and oxidative stress, and suppressed growth and inflammation. Moreover, many upregulated genes highlighted maintenance and repair activities, including detoxifying lipid peroxidation products, activating lysosomal autophagy/ubiquitin-proteasome pathways, and enhancing telomere maintenance and DNA repair, closely resembling the anti-aging effects of caloric/glucose restriction (CR/GR), a nutritional intervention that is known to increase lifespan and stress resistance in model organisms. Although treatment–introduced genetic mutations were detected in resistant clones, all resistant and sensitive clones were subclassified to either proneural (PN) or mesenchymal (MES) glioblastoma subtype based on their expression profiles. Functional assays demonstrated the association of treatment resistance with energy stress, including reduced glucose uptake, fatty acid oxidation (FAO)-dependent ATP maintenance, elevated reactive oxygen species (ROS) production and autophagic activity, and increased AMPK activity and NAD+ levels accompanied by upregulated mRNA levels of SIRT1/PGC-1α axis and DNA repair genes. These data support the view that treatment resistance may arise from quiescent GSC exhibiting a GR-like phenotype, and suggest that targeting stress response pathways of resistant GSC may provide a novel strategy in combination with standard treatment for glioblastoma. PMID:24260384
-
Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media.
Zhu, Jiang; Wooh, Jong Wei; Hou, Jeff Jia Cheng; Hughes, Benjamin S; Gray, Peter P; Munro, Trent P
2012-01-01
Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum. Copyright © 2012 American Institute of Chemical Engineers (AIChE).
-
Universality of clone dynamics during tissue development
NASA Astrophysics Data System (ADS)
Rulands, Steffen; Lescroart, Fabienne; Chabab, Samira; Hindley, Christopher J.; Prior, Nicole; Sznurkowska, Magdalena K.; Huch, Meritxell; Philpott, Anna; Blanpain, Cedric; Simons, Benjamin D.
2018-05-01
The emergence of complex organs is driven by the coordinated proliferation, migration and differentiation of precursor cells. The fate behaviour of these cells is reflected in the time evolution of their progeny, termed clones, which serve as a key experimental observable. In adult tissues, where cell dynamics is constrained by the condition of homeostasis, clonal tracing studies based on transgenic animal models have advanced our understanding of cell fate behaviour and its dysregulation in disease1,2. But what can be learnt from clonal dynamics in development, where the spatial cohesiveness of clones is impaired by tissue deformations during tissue growth? Drawing on the results of clonal tracing studies, we show that, despite the complexity of organ development, clonal dynamics may converge to a critical state characterized by universal scaling behaviour of clone sizes. By mapping clonal dynamics onto a generalization of the classical theory of aerosols, we elucidate the origin and range of scaling behaviours and show how the identification of universal scaling dependences may allow lineage-specific information to be distilled from experiments. Our study shows the emergence of core concepts of statistical physics in an unexpected context, identifying cellular systems as a laboratory to study non-equilibrium statistical physics.
-
Hsu, Cary; Jones, Stephanie A.; Cohen, Cyrille J.; Zheng, Zhili; Kerstann, Keith; Zhou, Juhua; Robbins, Paul F.; Peng, Peter D.; Shen, Xinglei; Gomes, Theotonius J.; Dunbar, Cynthia E.; Munroe, David J.; Stewart, Claudia; Cornetta, Kenneth; Wangsa, Danny; Ried, Thomas; Rosenberg, Steven A.
2007-01-01
Malignancies arising from retrovirally transduced hematopoietic stem cells have been reported in animal models and human gene therapy trials. Whether mature lymphocytes are susceptible to insertional mutagenesis is unknown. We have characterized a primary human CD8+ T-cell clone, which exhibited logarithmic ex vivo growth in the absence of exogenous cytokine support for more than 1 year after transduction with a murine leukemia virus–based vector encoding the T-cell growth factor IL-15. Phenotypically, the clone was CD28−, CD45RA−, CD45RO+, and CD62L−, a profile consistent with effector memory T lymphocytes. After gene transfer with tumor-antigen–specific T-cell receptors, the clone secreted IFN-γ upon encountering tumor targets, providing further evidence that they derived from mature lymphocytes. Gene-expression analyses revealed no evidence of insertional activation of genes flanking the retroviral insertion sites. The clone exhibited constitutive telomerase activity, and the presence of autocrine loop was suggested by impaired cell proliferation following knockdown of IL-15Rα expression. The generation of this cell line suggests that nonphysiologic expression of IL-15 can result in the long-term in vitro growth of mature human T lymphocytes. The cytokine-independent growth of this line was a rare event that has not been observed in other IL-15 vector transduction experiments or with any other integrating vector system. It does not appear that the retroviral vector integration sites played a role in the continuous growth of this cell clone, but this remains under investigation. PMID:17353346
-
Ortegon, H; Betts, D H; Lin, L; Coppola, G; Perrault, S D; Blondin, P; King, W A
2007-01-01
It appears that overt phenotypic abnormalities observed in some domestic animal clones are not transmitted to their progeny. The current study monitored Holstein heifers sired by a bull clone, Starbuck II, from weaning to puberty. Genomic stability was assessed by telomere length status and chromosomal analysis. Growth parameters, blood profiles, physical exams and reproductive parameters were assessed for 12 months (and compared to age-matched control heifers). Progeny sired by the clone bull did not differ (P>0.05) in weight, length and height compared to controls. However, progeny had lower heart rates (HR) (P=0.009), respiratory rates (RR) (P=0.007) and body temperature (P=0.03). Hematological profiles were within normal ranges and did not differ (P>0.05) between both groups. External and internal genitalia were normal and both groups reached puberty at expected ages. Progeny had two or three ovarian follicular waves per estrous cycle and serum progesterone concentrations were similar (P=0.99) to controls. Telomere lengths of sperm and blood cells from Starbuck II were not different (P>0.05) than those of non-cloned cattle; telomere lengths of progeny were not different (P>0.05) from age-matched controls. In addition, progeny had normal karyotypes in peripheral blood leukocytes compared to controls (89.1% versus 86.3% diploid, respectively). In summary, heifers sired by a bull clone had normal chromosomal stability, growth, physical, hematological and reproductive parameters, compared to normal heifers. Furthermore, they had moderate stress responses to routine handling and restraint.
-
Lee, Sangmi; Chen, Yi; Gorski, Lisa; Ward, Todd J; Osborne, Jason; Kathariou, Sophia
2018-04-17
Biodiversity analysis of the foodborne pathogen Listeria monocytogenes recently revealed four serotype 4b major hypervirulent clonal complexes (CCs), i.e., CC1, CC2, CC4, and CC6. Hypervirulence was indicated by overrepresentation of these clones, and serotype 4b as a whole, among human clinical isolates in comparison to food. However, data on potential source-dependent partitioning among serotype 4b clones in diverse regions are sparse. We analyzed a panel of 347 serotype 4b isolates, primarily from North America, to determine the distribution of clones in humans, other animals, food, and water. CC1, CC2, CC4, and CC6 predominated, but surprisingly, only three clones, i.e., CC2 and the singleton sequence types (STs) ST382 and ST639, exhibited significant source-dependent associations, with higher propensity for food (CC2) or water (ST382 and ST639) than other sources. Pairwise comparisons between human and food isolates identified CC4 as the only serotype 4b clone significantly overrepresented among human isolates. Our analysis also revealed several serotype 4b clones emerging in North America. Two such emerging clones, ST382 (implicated in several outbreaks since 2014) and ST639, were primarily encountered among human and water isolates. Findings suggest that in spite of the ubiquity of CC1, CC2, CC4, and CC6, regional heterogeneity in serotype 4b is substantially larger than previously surmised. Analysis of even large strain panels from one region may not adequately predict clones unique to, and emerging in, other areas. Serotype 4b clonal complexes may differ in ecological niche preference, suggesting the need to further elucidate reservoirs and vehicles, especially for emerging clones. IMPORTANCE In Listeria monocytogenes , serotype 4b strains are leading contributors to human disease, but intraserotype distributions among different sources and regions remain poorly elucidated. Analysis of 347 serotype 4b isolates from four different sources, mostly from North America, confirmed the overall predominance of the major clones CC1, CC2, CC4, and CC6 but found that only CC4 was significantly associated with human disease, while CC2 was significantly associated with food. Remarkably, several emerging clones were identified among human isolates from North America, with some of these also exhibiting a propensity for surface water. The latter included the singleton clones ST382, implicated in several outbreaks in the United States since 2014, and ST639. These clones were noticeably underrepresented among much larger panels from other regions. Though associated with North America for the time being, they may eventually become globally disseminated through the food trade or other venues. Copyright © 2018 Lee et al.
-
Vincke, Cécile; Gutiérrez, Carlos; Wernery, Ulrich; Devoogdt, Nick; Hassanzadeh-Ghassabeh, Gholamreza; Muyldermans, Serge
2012-01-01
Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.
-
Musette, P; Galelli, A; Chabre, H; Callard, P; Peumans, W; Truffa-Bachi, P; Kourilsky, P; Gachelin, G
1996-08-01
The V beta 8.3-specific superantigenic lectin Urtica dioica agglutinin (UDA) was used to delete the V beta 8.3+ T cells in MRL lpr/lpr mice. In contrast to the systemic lupus erythematosus-like pathology which progresses with age in the phosphate-buffered saline-injected MRL lpr/lpr controls, UDA-treated animals did not develop overt clinical signs of lupus and nephritis. The pathogenic T cell clones thus reside within the V beta 8.3+ T cell population, which includes an expanded T cell clone described previously. Finally, UDA alters the production of autoantibodies in a sex-dependent manner.
-
Detection and identification of Theileria infection in sika deer ( Cervus nippon ) in China.
He, Lan; Khan, Muhanmad Kasib; Zhang, Wen-Jie; Zhang, Qing-Li; Zhou, Yan-Qin; Hu, Min; Zhao, Junlong
2012-06-01
The sika deer ( Cervus nippon ) is a first-grade state-protected animal in China and designated a threatened species by the World Conservation Union. To detect hemoparasite infection of sika deer, blood samples were collected from 24 animals in the Hubei Province Deer Center. Genomic DNA was extracted, and the V4 hypervariable region encoding 18S rRNA was analyzed by reverse line blot hybridization assay. PCR products hybridized with Babesia / Theileria genus-specific probes but failed to hybridize with any of the Babesia or Theileria species-specific probes, suggesting the presence of a novel, or variant, species. Here 18S rRNA and internal transcribed spacer (ITS) genes were amplified, cloned, and sequenced from 7 isolates. Alignment and BlastN of the cloned sequences revealed high similarities to the homologous 18S rRNA genes and ITS genes of Theileria cervi (AY735122), Theileria sp. CNY1A (AB012194), and Theileria sp. ex Yamaguchi (AF529272). Phylogenetic analysis based on the 18S rRNA gene and ITS sequences showed that all cloned sequences were grouped within the Theileria clade. Phylogeny based on the 18S rRNA gene divided the organisms into 2 groups. Group 1 was closest to Theileria sp. ex Yamaguchi (AF529272), and group 2 was distinct from all other identified Theileria and Babesia species. These results suggest the existence of Theileria sp. infection in sika deer in China. To our knowledge, this is the first report of cervine Theileria sp. in China.
-
Monath, Thomas P; Caldwell, Joseph R; Mundt, Wolfgang; Fusco, Joan; Johnson, Casey S; Buller, Mark; Liu, Jian; Gardner, Bridget; Downing, Greg; Blum, Paul S; Kemp, Tracy; Nichols, Richard; Weltzin, Richard
2004-10-01
The threat of smallpox as a biological weapon has spurred efforts to create stockpiles of vaccine for emergency preparedness. In lieu of preparing vaccine in animal skin (the original method), we cloned vaccinia virus (New York City Board of Health strain, Dryvax by plaque purification and amplified the clone in cell culture. The overarching goal was to produce a modern vaccine that was equivalent to the currently licensed Dryvax in its preclinical and clinical properties, and could thus reliably protect humans against smallpox. A variety of clones were evaluated, and many were unacceptably virulent in animal models. One clonal virus (ACAM1000) was selected and produced at clinical grade in MRC-5 human diploid cells. ACAM1000 was comparable to Dryvax in immunogenicity and protective activity but was less neurovirulent for mice and nonhuman primates. To meet requirements for large quantities of vaccine after the events of September 11th 2001, the ACAM1000 master virus seed was used to prepare vaccine (designated ACAM2000) at large scale in Vero cells under serum-free conditions. The genomes of ACAM1000 and ACAM2000 had identical nucleotide sequences, and the vaccines had comparable biological phenotypes. ACAM1000 and ACAM2000 were evaluated in three Phase 1 clinical trials. The vaccines produced major cutaneous reactions and evoked neutralizing antibody and cell-mediated immune responses in the vast majority of subjects and had a reactogenicity profile similar to that of Dryvax.
-
Xia, Yun; Massé, Daniel I; McAllister, Tim A; Kong, Yunhong; Seviour, Robert; Beaulieu, Carole
2012-04-01
Digestion of raw feathers in anaerobic digesters inoculated with adapted swine manure, slaughterhouse sludge or dairy manure was investigated using twelve 42-L anaerobic digesters at 25°C. After 120days 74%, 49% and 40% added feathers were converted to methane in swine manure, dairy manure and slaughterhouse sludge anaerobic digesters respectively. 16S rRNA gene clone library analyses identified twenty-one operational taxonomic units containing clone sequences from 5 genera, 5 families and 2 phyla of members of the Archaea from 158 sequenced clones. Fluorescence insitu hybridization revealed that methanogens from the Methanomicrobiales, Methanosarcinales and Methanobacteriales constituted a major fraction (>78%) of these Archaea. A high correlation was seen between the distribution of functional archaeal groups and the NH(3)-N levels of digester mixed liquors. The compositions of archaeal communities fed different substrates were statistically significantly different (P<0.05). Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.
-
Cloning of Macaque Monkeys by Somatic Cell Nuclear Transfer.
Liu, Zhen; Cai, Yijun; Wang, Yan; Nie, Yanhong; Zhang, Chenchen; Xu, Yuting; Zhang, Xiaotong; Lu, Yong; Wang, Zhanyang; Poo, Muming; Sun, Qiang
2018-02-08
Generation of genetically uniform non-human primates may help to establish animal models for primate biology and biomedical research. In this study, we have successfully cloned cynomolgus monkeys (Macaca fascicularis) by somatic cell nuclear transfer (SCNT). We found that injection of H3K9me3 demethylase Kdm4d mRNA and treatment with histone deacetylase inhibitor trichostatin A at one-cell stage following SCNT greatly improved blastocyst development and pregnancy rate of transplanted SCNT embryos in surrogate monkeys. For SCNT using fetal monkey fibroblasts, 6 pregnancies were confirmed in 21 surrogates and yielded 2 healthy babies. For SCNT using adult monkey cumulus cells, 22 pregnancies were confirmed in 42 surrogates and yielded 2 babies that were short-lived. In both cases, genetic analyses confirmed that the nuclear DNA and mitochondria DNA of the monkey offspring originated from the nucleus donor cell and the oocyte donor monkey, respectively. Thus, cloning macaque monkeys by SCNT is feasible using fetal fibroblasts. Copyright © 2018 Elsevier Inc. All rights reserved.
-
TsDAF-21/Hsp90 is expressed in all examined stages of Trichinella spiralis
USDA-ARS?s Scientific Manuscript database
Trichinella is an important parasitic nematode of animals worldwide. Heat shock proteins are ubiquitous in nature and allow organisms to quickly respond to environmental stress. A portion of the Tsdaf-21 gene, a Caenorhabditis elegans daf-21 homologue encoding heat-shock protein 90 (Hsp90) was clone...
-
Wilkie, Michael Patrick
2002-08-01
In fresh water fishes, ammonia is excreted across the branchial epithelium via passive NH(3) diffusion. This NH(3) is subsequently trapped as NH(4)(+) in an acidic unstirred boundary layer lying next to the gill, which maintains the blood-to-gill water NH(3) partial pressure gradient. Whole animal, in situ, ultrastructural and molecular approaches suggest that boundary layer acidification results from the hydration of CO(2) in the expired gill water, and to a lesser extent H(+) excretion mediated by apical H(+)-ATPases. Boundary layer acidification is insignificant in highly buffered sea water, where ammonia excretion proceeds via NH(3) diffusion, as well as passive NH(4)(+) diffusion due to the greater ionic permeability of marine fish gills. Although Na(+)/H(+) exchangers (NHE) have been isolated in marine fish gills, possible Na(+)/NH(4)(+) exchange via these proteins awaits evaluation using modern electrophysiological and molecular techniques. Although urea excretion (J(Urea)) was thought to be via passive diffusion, it is now clear that branchial urea handling requires specialized urea transporters. Four urea transporters have been cloned in fishes, including the shark kidney urea transporter (shUT), which is a facilitated urea transporter similar to the mammalian renal UT-A2 transporter. Another urea transporter, characterized but not yet cloned, is the basolateral, Na(+) dependent urea antiporter of the dogfish gill, which is essential for urea retention in ureosmotic elasmobranchs. In ureotelic teleosts such as the Lake Magadi tilapia and the gulf toadfish, the cloned mtUT and tUT are facilitated urea transporters involved in J(Urea). A basolateral urea transporter recently cloned from the gill of the Japanese eel (eUT) may actually be important for urea retention during salt water acclimation. A multi-faceted approach, incorporating whole animal, histological, biochemical, pharmacological, and molecular techniques is required to learn more about the location, mechanism of action, and functional significance of urea transporters in fishes. Copyright 2002 Wiley-Liss, Inc.
-
Oswald, E.; Schmidt, H.; Morabito, S.; Karch, H.; Marchès, O.; Caprioli, A.
2000-01-01
Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) produce the characteristic “attaching and effacing” (A/E) lesion of the brush border. Intimin, an outer membrane protein encoded by eae, is responsible for the tight association of both pathogens with the host cell. Several eae have been cloned from different EPEC and EHEC strains isolated from humans and animals. These sequences are conserved in the N-terminal region but highly variable in the last C-terminal 280 amino acids (aa), where the cell binding activity is localized. Based on these considerations, we developed a panel of specific primers to investigate the eae heterogeneity of the variable 3′ region by using PCR amplification. We then investigated the distribution of the known intimin types in a large collection of EPEC and EHEC strains isolated from humans and different animal species. The existence of a yet-unknown family of intimin was suspected because several EHEC strains, isolated from human and cattle, did not react with any of the specific primer pairs, although these strains were eae positive when primers amplifying the conserved 5′ end were used. We then cloned and sequenced the eae present in one of these strains (EHEC of serotype O103:H2) and subsequently designed a PCR primer that recognizes in a specific manner the variable 3′ region of this new intimin type. This intimin, referred to as “ɛ,” was present in human and bovine EHEC strains of serogroups O8, O11, O45, O103, O121, and O165. Intimin ɛ is the largest intimin cloned to date (948 aa) and shares the greatest overall sequence identity with intimin β, although analysis of the last C-terminal 280 aa suggests a greater similarity with intimins α and γ. PMID:10603369
-
Cavaco, L M; Hasman, H; Stegger, M; Andersen, P S; Skov, R; Fluit, A C; Ito, T; Aarestrup, F M
2010-09-01
We recently reported a phenotypic association between reduced susceptibility to zinc and methicillin resistance in Staphylococcus aureus CC398 isolates from Danish swine (F. M. Aarestrup, L. M. Cavaco, and H. Hasman, Vet. Microbiol. 142:455-457, 2009). The aim of this study was to identify the genetic determinant causing zinc resistance in CC398 and examine its prevalence in isolates of animal and human origin. Based on the sequence of the staphylococcal cassette chromosome mec (SCCmec) element from methicillin-resistant S. aureus (MRSA) CC398 strain SO385, a putative metal resistance gene was identified in strain 171 and cloned in S. aureus RN4220. Furthermore, 81 MRSA and 48 methicillin-susceptible S. aureus (MSSA) strains, isolated from pigs (31 and 28) and from humans (50 and 20) in Denmark, were tested for susceptibility to zinc chloride and for the presence of a putative resistance determinant, czrC, by PCR. The cloning of czrC confirmed that the zinc chloride and cadmium acetate MICs for isogenic constructs carrying this gene were increased compared to those for S. aureus RN4220. No difference in susceptibility to sodium arsenate, copper sulfate, or silver nitrate was observed. Seventy-four percent (n = 23) of the animal isolates and 48% (n = 24) of the human MRSA isolates of CC398 were resistant to zinc chloride and positive for czrC. All 48 MSSA strains from both human and pig origins were found to be susceptible to zinc chloride and negative for czrC. Our findings showed that czrC is encoding zinc and cadmium resistance in CC398 MRSA isolates, and that it is widespread both in humans and animals. Thus, resistance to heavy metals such as zinc and cadmium may play a role in the coselection of methicillin resistance in S. aureus.
-
NASA Astrophysics Data System (ADS)
Li, Chen; Niu, Cuijuan
2015-03-01
Sexual reproduction adversely affects the population growth of cyclic parthenogenetic animals. The density-dependent sexual reproduction of a superior competitor could mediate the coexistence. However, the cost of sex may make the inferior competitor more vulnerable. To investigate the effect of sexual reproduction on the inferior competitor, we experimentally paired the competition of one Brachionus angularis clone against three Brachionus calyciflorus clones. One of the B. calyciflorus clones showed a low propensity for sexual reproduction, while the other two showed high propensities. The results show that all B. calyciflorus clones were excluded in the competition for resources at low food level. The increased food level promoted the competition persistence, but the clones did not show a clear pattern. Both the cumulative population density and resting egg production increased with the food level. The cumulative population density decreased with the mixis investment, while the resting egg production increased with the mixis investment. A trade-off between the population growth and sexual reproduction was observed in this research. The results indicate that although higher mixis investment resulted in a lower population density, it would not determinately accelerate the exclusion process of the inferior competitor. On the contrary, higher mixis investment promoted resting egg production before being excluded and thus promised a long-term benefit. In conclusion, our results suggest that mixis investment, to some extent, favored the excluded inferior competitor under fierce competition or some other adverse conditions.
-
Chattaway, Marie A.; Aboderin, Aaron O.; Fashae, Kayode; Okoro, Chinyere K.; Opintan, Japheth A.; Okeke, Iruka N.
2016-01-01
Fluoroquinolones came into widespread use in African countries in the early 2000s, after patents for the first generation of these drugs expired. By that time, quinolone antibacterial agents had been used intensively worldwide and resistant lineages of many bacterial species had evolved. We sought to understand which Gram negative enteric pandemic lineages have been reported from Africa, as well as the nature and transmission of any indigenous resistant clones. A systematic review of articles indexed in the Medline and AJOL literature databases was conducted. We report on the findings of 43 eligible studies documenting local or pandemic fluoroquinolone-resistant enteric clones in sub-Sahara African countries. Most reports are of invasive non-typhoidal Salmonella and Escherichia coli lineages and there have been three reports of cholera outbreaks caused by fluoroquinolone-resistant Vibrio cholerae O1. Fluoroquinolone-resistant clones have also been reported from commensals and animal isolates but there are few data for non-Enterobacteriaceae and almost none for difficult-to-culture Campylobacter spp. Fluoroquinolone-resistant lineages identified in African countries were universally resistant to multiple other classes of antibacterial agents. Although as many as 972 non-duplicate articles refer to fluoroquinolone resistance in enteric bacteria from Africa, most do not report on subtypes and therefore information on the epidemiology of fluoroquinolone-resistant clones is available from only a handful of countries in the subcontinent. When resistance is reported, resistance mechanisms and lineage information is rarely investigated. Insufficient attention has been given to molecular and sequence-based methods necessary for identifying and tracking resistant clones in Africa and more research is needed in this area. PMID:27148238
-
2012-01-01
Background Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147, basigin) is an inducer of the expression of several matrix metalloproteinases (MMPs). We reported previously that blocking EMMPRIN activity reduced neuroinflammation and severity of disease in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). Methods To improve upon EMMPRIN blockade, and to help unravel the biological functions of EMMPRIN in inflammatory disorders, we have developed several anti-EMMPRIN monoclonal antibodies. Results Of these monoclonal antibodies, a particular one, clone 10, was efficient in binding mouse and human cells using several methods of detection. The specificity of clone 10 was demonstrated by its lack of staining of EMMPRIN-null embryos compared to heterozygous and wild-type mouse samples. Functionally, human T cells activated with anti-CD3 and anti-CD28 elevated their expression of EMMPRIN and the treatment of these T cells with clone 10 resulted in decreased proliferation and matrix metalloproteinase- 9 (MMP-9) production. Activated human T cells were toxic to human neurons in culture and clone 10 pretreatment reduced T cell cytotoxicity correspondent with decrease of granzyme B levels within T cells. In vivo, EAE mice treated with clone 10 had a markedly reduced disease score compared to mice treated with IgM isotype control. Conclusions We have produced a novel anti-EMMPRIN monoclonal antibody that blocks several aspects of T cell activity, thus highlighting the multiple roles of EMMPRIN in T cell biology. Moreover, clone 10 reduces EAE scores in mice compared to controls, and has activity on human cells, potentially allowing for the testing of anti-EMMPRIN treatment not only in EAE, but conceivably also in MS. PMID:22480370
-
Agrawal, Smriti M; Silva, Claudia; Wang, Janet; Tong, Jade Pui-Wai; Yong, V Wee
2012-04-05
Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147, basigin) is an inducer of the expression of several matrix metalloproteinases (MMPs). We reported previously that blocking EMMPRIN activity reduced neuroinflammation and severity of disease in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). To improve upon EMMPRIN blockade, and to help unravel the biological functions of EMMPRIN in inflammatory disorders, we have developed several anti-EMMPRIN monoclonal antibodies. Of these monoclonal antibodies, a particular one, clone 10, was efficient in binding mouse and human cells using several methods of detection. The specificity of clone 10 was demonstrated by its lack of staining of EMMPRIN-null embryos compared to heterozygous and wild-type mouse samples. Functionally, human T cells activated with anti-CD3 and anti-CD28 elevated their expression of EMMPRIN and the treatment of these T cells with clone 10 resulted in decreased proliferation and matrix metalloproteinase- 9 (MMP-9) production. Activated human T cells were toxic to human neurons in culture and clone 10 pretreatment reduced T cell cytotoxicity correspondent with decrease of granzyme B levels within T cells. In vivo, EAE mice treated with clone 10 had a markedly reduced disease score compared to mice treated with IgM isotype control. We have produced a novel anti-EMMPRIN monoclonal antibody that blocks several aspects of T cell activity, thus highlighting the multiple roles of EMMPRIN in T cell biology. Moreover, clone 10 reduces EAE scores in mice compared to controls, and has activity on human cells, potentially allowing for the testing of anti-EMMPRIN treatment not only in EAE, but conceivably also in MS.
-
Clonal success of piliated penicillin nonsusceptible pneumococci
Sjöström, K.; Blomberg, C.; Fernebro, J.; Dagerhamn, J.; Morfeldt, E.; Barocchi, M. A.; Browall, S.; Moschioni, M.; Andersson, M.; Henriques, F.; Albiger, B.; Rappuoli, Rino; Normark, S.; Henriques-Normark, B.
2007-01-01
Antibiotic resistance in pneumococci is due to the spread of strains belonging to a limited number of clones. The Spain9V-3 clone of sequence type (ST)156 is one of the most successful clones with reduced susceptibility to penicillin [pneumococci nonsusceptible to penicillin (PNSP)]. In Sweden during 2000–2003, a dramatic increase in the number of PNSP isolates was observed. Molecular characterization of these isolates showed that a single clone of sequence type ST156 increased from 40% to 80% of all serotype 14, thus causing the serotype expansion. Additionally, during the same time period, we examined the clonal composition of two serotypes 9V and 19F: all 9V and 20% of 19F isolates belonged to the clonal cluster of ST156, and overall ≈50% of all PNSP belonged to the ST156 clonal cluster. Moreover, microarray and PCR analysis showed that all ST156 isolates, irrespective of capsular type, carried the rlrA pilus islet. This islet was also found to be present in the penicillin-sensitive ST162 clone, which is believed to be the drug-susceptible ancestor of ST156. Competitive experiments between related ST156 serotype 19F strains confirmed that those containing the rlrA pilus islet were more successful in an animal model of carriage. We conclude that the pilus island is an important biological factor common to ST156 isolates and other successful PNSP clones. In Sweden, a country where the low antibiotic usage does not explain the spread of resistant strains, at least 70% of all PNSP isolates collected during year 2003 carried the pilus islet. PMID:17644611
-
Livestock-associated Staphylococcus aureus on Polish pig farms
Mroczkowska, Aneta; Żmudzki, Jacek; Marszałek, Natalia; Orczykowska-Kotyna, Monika; Komorowska, Iga; Nowak, Agnieszka; Grzesiak, Anna; Czyżewska-Dors, Ewelina; Dors, Arkadiusz; Pejsak, Zygmunt; Hryniewicz, Waleria; Wyszomirski, Tomasz; Empel, Joanna
2017-01-01
Background Livestock-associated Staphylococcus aureus (LA-SA) draws increasing attention due to its particular ability to colonize farm animals and be transmitted to people, which in turn leads to its spread in the environment. The aim of the study was to determine the dissemination of LA-SA on pig farms selected throughout Poland, characterize the population structure of identified S. aureus, and assess the prevalence of LA-SA carriage amongst farmers and veterinarians being in contact with pigs. Methods and findings The study was conducted on 123 pig farms (89 farrow-to-finish and 34 nucleus herds), located in 15 out of 16 provinces of Poland. Human and pig nasal swabs, as well as dust samples were analyzed. S. aureus was detected on 79 (64.2%) farms from 14 provinces. Amongst these farms LA-SA-positive farms dominated (71/79, 89.9%, 95% CI [81.0%, 95.5%]). The prevalence of LA-MRSA-positive farms was lower than LA-MSSA-positive (36.6% of LA-SA-positive farms, 95% CI [25.5%, 48.9%] vs. 74.6%, 95% CI [62.9%, 84.2%]). In total, 190 S. aureus isolates were identified: 72 (38%) MRSA and 118 (62%) methicillin-susceptible S. aureus (MSSA), of which 174 (92%) isolates were classified to three livestock-associated lineages: CC398 (73%), CC9 (13%), and CC30/ST433 (6%). All CC398 isolates belonged to the animal clade. Four LA-MRSA clones were detected: ST433-IVa(2B) clone (n = 8, 11%), described to the best of our knowledge for the first time, and three ST398 clones (n = 64, 89%) with the most prevalent being ST398-V(5C2&5)c, followed by ST398-V(5C2), and ST398-IVa(2B). Nasal carriage of LA-SA by pig farmers was estimated at 13.2% (38/283), CC398 carriage at 12.7% (36/283) and ST398-MRSA carriage at 3.2% (9/283), whereas by veterinarians at 21.1% (8/38), 18.4% (7/38) and 10.5% (4/38), respectively. Conclusions The prevalence of LA-MRSA-positive pig farms in Poland has increased considerably since 2008, when the first MRSA EU baseline survey was conducted in Europe. On Polish pig farms CC398 of the animal clade predominates, this being also reflected in the prevalence of CC398 nasal carriage in farmers and veterinarians. However, finding a new ST433-IVa(2B) clone provides evidence for the continuing evolution of LA-MRSA and argues for further monitoring of S. aureus in farm animals. PMID:28151984
-
Sequeira, Ana Filipa; Brás, Joana L A; Guerreiro, Catarina I P D; Vincentelli, Renaud; Fontes, Carlos M G A
2016-12-01
Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. In addition, when coupled with efficient gene design algorithms that optimize codon usage, it leads to high levels of recombinant protein expression. Here, we describe the development of an optimized gene synthesis platform that was applied to the large scale production of small genes encoding venom peptides. This improved gene synthesis method uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides and was developed to synthesise multiples genes simultaneously. This technology incorporates an accurate, automated and cost effective ligation independent cloning step to directly integrate the synthetic genes into an effective Escherichia coli expression vector. The robustness of this technology to generate large libraries of dozens to thousands of synthetic nucleic acids was demonstrated through the parallel and simultaneous synthesis of 96 genes encoding animal toxins. An automated platform was developed for the large-scale synthesis of small genes encoding eukaryotic toxins. Large scale recombinant expression of synthetic genes encoding eukaryotic toxins will allow exploring the extraordinary potency and pharmacological diversity of animal venoms, an increasingly valuable but unexplored source of lead molecules for drug discovery.
-
Agabou, Amir; Ouchenane, Zouleikha; Ngba Essebe, Christelle; Khemissi, Salim; Chehboub, Mohamed Tedj Eddine; Chehboub, Ilyes Bey; Dunyach-Remy, Catherine
2017-01-01
The spread of toxinogenic Staphylococcus aureus is a public health problem in Africa. The objectives of the study were to investigate the rate of S. aureus nasal carriage and molecular characteristics of these strains in livestock and humans in three Algerian provinces. Nasal samples were collected from camels, horses, cattle, sheep and monkeys, as well as humans in contact with them. S. aureus isolates were genotyped using DNA microarray. The rate of S. aureus nasal carriage varied between species: camels (53%), humans and monkeys (50%), sheep (44.2%), horses (15.2%) and cattle (15%). Nine methicillin-resistant S. aureus (MRSA) isolates (7.6%) were identified, isolated from camels and sheep. The S. aureus isolates belonged to 15 different clonal complexes. Among them, PVL+ (Panton–Valentine Leukocidin) isolates belonging to ST80-MRSA-IV and ST152-MSSA were identified in camels (n = 3, 13%) and sheep (n = 4, 21.1%). A high prevalence of toxinogenic animal strains was noted containing TSST-1- (22.2%), EDINB- (29.6%) and EtD- (11.1%) encoding genes. This study showed the dispersal of the highly human pathogenic clones ST152-MSSA and ST-80-MRSA in animals. It suggests the ability of some clones to cross the species barrier and jump between humans and several animal species. PMID:28946704
-
Salmonellosis: the role of poultry meat.
Antunes, P; Mourão, J; Campos, J; Peixe, L
2016-02-01
Salmonellosis remains one of the most frequent food-borne zoonoses, constituting a worldwide major public health concern. Currently, at a global level, the main sources of infection for humans include meat products, including the consumption of contaminated poultry meat, in spite of the success of Salmonella control measures implemented in food-animal production of industrialized countries. In recent years, a shift in Salmonella serotypes related to poultry and poultry production has been reported in diverse geographical regions, being particularly associated with the spread of certain well-adapted clones. Moreover, antimicrobial resistance in non-typhoidal Salmonella is considered one of the major public health threats related with food-animal production, including the poultry production chain and poultry meat, which is an additional concern in the management of salmonellosis. The circulation of the same multidrug-resistant Salmonella clones and/or identical mobile genetic elements encoding antibiotic resistance genes from poultry to humans highlights this scenario. The purpose of this review was to provide an overview of the role of poultry meat on salmonellosis at a global scale and the main problems that could hinder the success of Salmonella control measures at animal production level. With the increasing globalization of foodstuffs like poultry meat, new problems and challenges might arise regarding salmonellosis control, making new integrated intervention strategies necessary along the food chain. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
-
Enhance beef cattle improvement by embryo biotechnologies.
Wu, B; Zan, L
2012-10-01
Embryo biotechnology has become one of the prominent high businesses worldwide. This technology has evolved through three major changes, that is, traditional embryo transfer (in vivo embryo production by donor superovulation), in vitro embryo production by ovum pick up with in vitro fertilization and notably current cloning technique by somatic cell nuclear transfer and transgenic animal production. Embryo biotechnology has widely been used in dairy and beef cattle industry and commercial bovine embryo transfer has become a large international business. Currently, many developed biotechnologies during the period from early oocyte stage to pre-implantation embryos can be used to create new animal breeds and accelerate genetic progression. Based on recent advances in embryo biotechnologies and authors current studies, this review will focus on a description of the application of this technology to beef cattle improvement and discuss how to use this technology to accelerate beef cattle breeding and production. The main topics of this presentation include the following: (i) how to increase calf production numbers from gametes including sperm and oocyte; (ii) multiple ovulation and embryo transfer breeding schemes; (iii) in vitro fertilization and intracytoplasm sperm injection in bovine; (iv) pronuclear development and transgenic animals; (v) sex selection from sperm and embryos; (vi) cloning and androgenesis; (vii) blastocyst development and embryonic stem cells; (viii) preservation of beef cattle genetic resources; and (ix) conclusions. © 2011 Blackwell Verlag GmbH.
-
Björnsdóttir, Sigríður; Harris, Simon R.; Svansson, Vilhjálmur; Gunnarsson, Eggert; Sigurðardóttir, Ólöf G.; Gammeljord, Kristina; Steward, Karen F.; Newton, J. Richard; Robinson, Carl; Charbonneau, Amelia R. L.
2017-01-01
ABSTRACT Iceland is free of the major infectious diseases of horses. However, in 2010 an epidemic of respiratory disease of unknown cause spread through the country’s native horse population of 77,000. Microbiological investigations ruled out known viral agents but identified the opportunistic pathogen Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) in diseased animals. We sequenced the genomes of 257 isolates of S. zooepidemicus to differentiate epidemic from endemic strains. We found that although multiple endemic clones of S. zooepidemicus were present, one particular clone, sequence type 209 (ST209), was likely to have been responsible for the epidemic. Concurrent with the epidemic, ST209 was also recovered from a human case of septicemia, highlighting the pathogenic potential of this strain. Epidemiological investigation revealed that the incursion of this strain into one training yard during February 2010 provided a nidus for the infection of multiple horses that then transmitted the strain to farms throughout Iceland. This study represents the first time that whole-genome sequencing has been used to investigate an epidemic on a national scale to identify the likely causative agent and the link to an associated zoonotic infection. Our data highlight the importance of national biosecurity to protect vulnerable populations of animals and also demonstrate the potential impact of S. zooepidemicus transmission to other animals, including humans. PMID:28765219
-
Weiss, Sonja; Kadlec, Kristina; Fessler, Andrea T; Schwarz, Stefan
2013-12-27
The aim of this study was to isolate and characterize methicillin-resistant staphylococci (MRS) in a small animal clinic and to investigate their distribution and possible transmission. Swabs (n=72) were taken from hospitalized pets, the environment and employees of a small animal clinic and screened for the presence of MRS. The staphylococcal species was confirmed biochemically or by 16S rDNA sequencing. Susceptibility to antimicrobial agents was tested by broth dilution. The presence of mecA and other resistance genes was confirmed by PCR. Molecular typing of the isolates followed standard procedures. In total, 34 MRS belonging to the four species Staphylococcus aureus (n=5), Staphylococcus epidermidis (n=21), Staphylococcus haemolyticus (n=6) or Staphylococcus pettenkoferi (n=2) were isolated. All isolates were multidrug-resistant with resistance to at least three classes of antimicrobial agents. Among the five methicillin-resistant S. aureus (MRSA) isolates, four belonged to the clonal complex CC398; two of them were isolated from cats, the remaining two from pet cages. Overall, the MRS isolates differed in their characteristics, except for one S. epidermidis clone (n=9) isolated from hospitalized cats without clinical staphylococcal infections, pet cages, the clinic environment as well as from a healthy employee. This MRSE clone was resistant to 10 classes of antimicrobial agents, including aminocyclitols, β-lactams, fluoroquinolones, lincosamides, macrolides, phenicols, pleuromutilins, sulfonamides, tetracyclines and trimethoprim. These findings suggest a possible transmission of specific MRS isolates between animal patients, employees and the clinic environment. Copyright © 2013 Elsevier B.V. All rights reserved.
-
Bai, Xuelian; Lee, Ji-Yun; Kim, Tae Im; Dai, Fuhong; Lee, Tae-Jin; Hong, Sung-Jong
2014-01-01
Background Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2) is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2) from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. Methodology/Principal Findings A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. Conclusion Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths. PMID:24454892
-
Ovine induced pluripotent stem cells are resistant to reprogramming after nuclear transfer.
German, Sergio D; Campbell, Keith H S; Thornton, Elisabeth; McLachlan, Gerry; Sweetman, Dylan; Alberio, Ramiro
2015-02-01
Induced pluripotent stem cells (iPSCs) share similar characteristics of indefinite in vitro growth with embryonic stem cells (ESCs) and may therefore serve as a useful tool for the targeted genetic modification of farm animals via nuclear transfer (NT). Derivation of stable ESC lines from farm animals has not been possible, therefore, it is important to determine whether iPSCs can be used as substitutes for ESCs in generating genetically modified cloned farm animals. We generated ovine iPSCs by conventional retroviral transduction using the four Yamanaka factors. These cells were basic fibroblast growth factor (bFGF)- and activin A-dependent, showed persistent expression of the transgenes, acquired chromosomal abnormalities, and failed to activate endogenous NANOG. Nonetheless, iPSCs could differentiate into the three somatic germ layers in vitro. Because cloning of farm animals is best achieved with diploid cells (G1/G0), we synchronized the iPSCs in G1 prior to NT. Despite the cell cycle synchronization, preimplantation development of iPSC-NT embryos was lower than with somatic cells (2% vs. 10% blastocysts, p<0.01). Furthermore, analysis of the blastocysts produced demonstrated persistent expression of the transgenes, aberrant expression of endogenous SOX2, and a failure to activate NANOG consistently. In contrast, gene expression in blastocysts produced with the parental fetal fibroblasts was similar to those generated by in vitro fertilization. Taken together, our data suggest that the persistent expression of the exogenous factors and the acquisition of chromosomal abnormalities are incompatible with normal development of NT embryos produced with iPSCs.
-
Cynomolgus Macaque as an Animal Model for Severe Acute Respiratory Syndrome
Lawler, James V; Endy, Timothy P; Hensley, Lisa E; Garrison, Aura; Fritz, Elizabeth A; Lesar, May; Baric, Ralph S; Kulesh, David A; Norwood, David A; Wasieloski, Leonard P; Ulrich, Melanie P; Slezak, Tom R; Vitalis, Elizabeth; Huggins, John W; Jahrling, Peter B; Paragas, Jason
2006-01-01
Background The emergence of severe acute respiratory syndrome (SARS) in 2002 and 2003 affected global health and caused major economic disruption. Adequate animal models are required to study the underlying pathogenesis of SARS-associated coronavirus (SARS-CoV) infection and to develop effective vaccines and therapeutics. We report the first findings of measurable clinical disease in nonhuman primates (NHPs) infected with SARS-CoV. Methods and Findings In order to characterize clinically relevant parameters of SARS-CoV infection in NHPs, we infected cynomolgus macaques with SARS-CoV in three groups: Group I was infected in the nares and bronchus, group II in the nares and conjunctiva, and group III intravenously. Nonhuman primates in groups I and II developed mild to moderate symptomatic illness. All NHPs demonstrated evidence of viral replication and developed neutralizing antibodies. Chest radiographs from several animals in groups I and II revealed unifocal or multifocal pneumonia that peaked between days 8 and 10 postinfection. Clinical laboratory tests were not significantly changed. Overall, inoculation by a mucosal route produced more prominent disease than did intravenous inoculation. Half of the group I animals were infected with a recombinant infectious clone SARS-CoV derived from the SARS-CoV Urbani strain. This infectious clone produced disease indistinguishable from wild-type Urbani strain. Conclusions SARS-CoV infection of cynomolgus macaques did not reproduce the severe illness seen in the majority of adult human cases of SARS; however, our results suggest similarities to the milder syndrome of SARS-CoV infection characteristically seen in young children. PMID:16605302
-
Codner, Gemma F; Lindner, Loic; Caulder, Adam; Wattenhofer-Donzé, Marie; Radage, Adam; Mertz, Annelyse; Eisenmann, Benjamin; Mianné, Joffrey; Evans, Edward P; Beechey, Colin V; Fray, Martin D; Birling, Marie-Christine; Hérault, Yann; Pavlovic, Guillaume; Teboul, Lydia
2016-08-05
Karyotypic integrity is essential for the successful germline transmission of alleles mutated in embryonic stem (ES) cells. Classical methods for the identification of aneuploidy involve cytological analyses that are both time consuming and require rare expertise to identify mouse chromosomes. As part of the International Mouse Phenotyping Consortium, we gathered data from over 1,500 ES cell clones and found that the germline transmission (GLT) efficiency of clones is compromised when over 50 % of cells harbour chromosome number abnormalities. In JM8 cells, chromosomes 1, 8, 11 or Y displayed copy number variation most frequently, whilst the remainder generally remain unchanged. We developed protocols employing droplet digital polymerase chain reaction (ddPCR) to accurately quantify the copy number of these four chromosomes, allowing efficient triage of ES clones prior to microinjection. We verified that assessments of aneuploidy, and thus decisions regarding the suitability of clones for microinjection, were concordant between classical cytological and ddPCR-based methods. Finally, we improved the method to include assay multiplexing so that two unstable chromosomes are counted simultaneously (and independently) in one reaction, to enhance throughput and further reduce the cost. We validated a PCR-based method as an alternative to classical karyotype analysis. This technique enables laboratories that are non-specialist, or work with large numbers of clones, to precisely screen ES cells for the most common aneuploidies prior to microinjection to ensure the highest level of germline transmission potential. The application of this method allows early exclusion of aneuploid ES cell clones in the ES cell to mouse conversion process, thus improving the chances of obtaining germline transmission and reducing the number of animals used in failed microinjection attempts. This method can be applied to any other experiments that require accurate analysis of the genome for copy number variation (CNV).
-
Miguel, Célia; Simões, Marta; Oliveira, Maria Margarida; Rocheta, Margarida
2008-11-01
Retroviruses differ from retrotransposons due to their infective capacity, which depends critically on the encoded envelope. Some plant retroelements contain domains reminiscent of the env of animal retroviruses but the number of such elements described to date is restricted to angiosperms. We show here the first evidence of the presence of putative env-like gene sequences in a gymnosperm species, Pinus pinaster (maritime pine). Using a degenerate primer approach for conserved domains of RNaseH gene, three clones from putative envelope-like retrotransposons (PpRT2, PpRT3, and PpRT4) were identified. The env-like sequences of P. pinaster clones are predicted to encode proteins with transmembrane domains. These sequences showed identity scores of up to 30% with env-like sequences belonging to different organisms. A phylogenetic analysis based on protein alignment of deduced aminoacid sequences revealed that these clones clustered with env-containing plant retrotransposons, as well as with retrotransposons from invertebrate organisms. The differences found among the sequences of maritime pine clones isolated here suggest the existence of different putative classes of env-like retroelements. The identification for the first time of env-like genes in a gymnosperm species may support the ancestrality of retroviruses among plants shedding light on their role in plant evolution.
-
MIYAGAWA, Shuji; MATSUNARI, Hitomi; WATANABE, Masahito; NAKANO, Kazuaki; UMEYAMA, Kazuhiro; SAKAI, Rieko; TAKAYANAGI, Shuko; TAKEISHI, Toki; FUKUDA, Tooru; YASHIMA, Sayaka; MAEDA, Akira; EGUCHI, Hiroshi; OKUYAMA, Hiroomi; NAGAYA, Masaki; NAGASHIMA, Hiroshi
2015-01-01
Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs. PMID:26227017
-
Miyagawa, Shuji; Matsunari, Hitomi; Watanabe, Masahito; Nakano, Kazuaki; Umeyama, Kazuhiro; Sakai, Rieko; Takayanagi, Shuko; Takeishi, Toki; Fukuda, Tooru; Yashima, Sayaka; Maeda, Akira; Eguchi, Hiroshi; Okuyama, Hiroomi; Nagaya, Masaki; Nagashima, Hiroshi
2015-01-01
Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.
-
Yan, Hao; Yan, Zhonghai; Ma, Qingwen; Jiao, Fei; Huang, Shuzhen; Zeng, Fanyi; Zeng, Yitao
2011-01-01
Reconstructed embryos derived from intersubspecies somatic cell nuclear transfer (SCNT) have poorer developmental potential than those from intrasubspecies SCNT. Based on our previous study that Holstein dairy bovine (HD) mitochondrial DNA (mtDNA) haplotype compatibility between donor karyoplast and recipient cytoplast is crucial for SCNT embryo development, we performed intersubspecies SCNT using HD as donor karyoplast and Luxi yellow heifer (LY) as recipient cytoplast according to mtDNA haplotypes determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The results demonstrated that intersubspecies mtDNA homotype SCNT embryos had higher pre- and post-implantation developmental competence than intrasubspecies mtDNA heterotype embryos as well as improved blastocyst reprogramming status, including normal H3K9 dimethylation pattern and promoter hypomethylation of pluripotent genes such as Oct4 and Sox2, suggesting that intersubspecies SCNT using LY oocytes maintains HD cloning efficiency and may reprogram HD nuclei to develop into a normal cloned animal ultimately. Our results indicated that karyoplast-cytoplast interactions and mtDNA haplotype compatibility may affect bovine intersubspecies SCNT efficiency. This study on bovine intersubspecies SCNT is valuable for understanding the mechanisms of mtDNA haplotype compatibility between karyoplast and cytoplast impacting the bovine SCNT efficiency, and provides an alternative and economic resource for HD cloning. Copyright © 2011. Published by Elsevier Ltd.
-
Drougka, Eleanna; Foka, Antigoni; Koutinas, Christos K; Jelastopulu, Eleni; Giormezis, Nikolaos; Farmaki, Ourania; Sarrou, Styliani; Anastassiou, Evangelos D; Petinaki, Efthimia; Spiliopoulou, Iris
2016-04-01
Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) prevalence among companion animals and veterinary personnel (VP) was investigated. Strains' molecular characteristics were evaluated in order to assess S. aureus transmission. Specimens (224) from colonized and infected sites of 102 animals (92 dogs, 10 cats) and 18 VP were collected during 2012 and 2013. Antibiotic susceptibility was performed by the disk diffusion method and Etest. mecA, mecC, tst (toxic shock syndrome toxin) and lukF/lukS-PV (Panton-Valentine leukocidin, PVL) genes were investigated by PCR. Genotypes were identified by Multi Locus Sequence Typing (MLST), Staphylococcal Cassette Chromosome mec (SCCmec), accessory gene regulator group (agr), spa and Pulsed Field Gel Electrophoresis (PFGE). S. aureus prevalence among pets and VP was 36.3% (37/102) and 38.9% (7/18), respectively. Younger companion animals, those living in rural areas, having a disease upon admission or Coagulase-negative staphylococci co-carriage showed significantly higher prevalence of S. aureus isolation (p<0.05). Twenty-six pets and five VP carried PVL-positive S. aureus. In total, 60 S. aureus strains were recovered (53 from pets, seven from VP) of which 16 were MRSA (26.7%), 12 mecA- and four mecC-positive. MRSA showed higher resistance rates against other antimicrobials as compared to methicillin-susceptible ones. Strains were classified by MLST in 13 STs, with the predominance of ST80 and ST15. In MRSA, SCCmec types II, IV and XI were identified. The most frequent spa types were t5559 and t7558. Fifty-six strains were classified into 15 PFGE types. Comparison of genetic markers shows that identical or very similar strains disseminate among animals and VP. Companion animals harbor PVL-positive clones constituting a possible source for transmission to humans. Copyright © 2016 Elsevier B.V. All rights reserved.
-
USDA-ARS?s Scientific Manuscript database
The domestic pig is an ideal “dual purpose” animal model for agricultural and biomedical research. With the availability of genome editing tools [e.g. clustered regularly interspersed short palindromic repeat (CRISPR) and associated nuclease Cas9 (CRISPR/Cas9)] it is now possible to perform site-sp...
-
Two Asian highly pathogenic strains of Type 2 PRRSV in United States swine
USDA-ARS?s Scientific Manuscript database
Highly pathogenic PRRSV (HP-PRRSV) has been circulating in Asia for 7 years. rJXwn06 and rSRV07 were rescued from infectious clones of two HP-PRRSV for investigation at the National Animal Disease Center. The clinical disease and viral replication kinetics of HP-PRRSV were compared to prototype stra...
-
Clonal profile, virulence and resistance of Staphylococcus aureus isolated from sheep milk.
Martins, Katheryne Benini; Faccioli-Martins, Patricia Yoshida; Riboli, Danilo Flávio Moraes; Pereira, Valéria Cataneli; Fernandes, Simone; Oliveira, Aline A; Dantas, Ariane; Zafalon, Luiz Francisco; da Cunha, Maria de Lourdes Ribeiro de Souza
2015-06-01
The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT), somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst), biofilm (icaA, icaC, icaD, bap), leukocidin (luk-PV) oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics). Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene.
-
Zhao, Xueyan; Yang, Qiang; Zhao, Kewei; Jiang, Chao; Ren, Dongren; Xu, Pan; He, Xiaofang; Liao, Rongrong; Jiang, Kai; Ma, Junwu; Xiao, Shijun; Ren, Jun; Xing, Yuyun
2016-07-01
In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive F1 piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive F1 boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive F1 sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. cDNA sequencing and western blot indicated that the exogenous BMPR1B CDS was successfully expressed in host pigs. The transgenic pigs showed normal litter size performance. However, no significant differences in litter size were found between transgene-positive and negative sows. Our study provides new insight into producing cloned transgenic livestock related to reproductive traits.
-
Do, Ltk; Wittayarat, M; Terazono, T; Sato, Y; Taniguchi, M; Tanihara, F; Takemoto, T; Kazuki, Y; Kazuki, K; Oshimura, M; Otoi, T
2016-12-01
The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 μs. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC-transchromosomic embryos, when the duration of fusion was prolonged to 60 μs. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells. © 2016 Blackwell Verlag GmbH.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Chang, Yu-Tzu; Shu, Chung-Li; Lai, Jing-Yang
Mouse embryo fibroblasts (MEFs) grow slowly after cultivation from animals, however, after an extended period of cultivation, their growth accelerates. We found that SWAP-70 deficient MEFs failed to increase growth rates. They maintain normal growth rates and proliferation cycles for at least 5 years. Complementing SWAP-70 deficiency in one of these MEF clones, MEF1F2, by expressing human SWAP-70 resulted in fast growth of the cells after further cultivation for a long period. The resulting cells show a transformation phenotype, since they grow on top of each other and do not show contact inhibition. This phenotype was reverted when sanguinarine, amore » putative SWAP-70 inhibitor, was added. Two SWAP-70 expressing clones were examined in detail. Even after cell density became very high their cdc2 and NFκB were still activated suggesting that they do not stop growing. One of the clones formed colonies in soft agar and formed tumors in nude mice. Lately, one more clone became transformed being able to make colonies in soft agar. We maintain 4 human SWAP-70 expressing MEF1F2 cell lines. Three out of 4 clones exhibited transforming phenotypes. The mouse SWAP-70 gene also promoted transformation of MEFs. Taken together our data suggest that SWAP-70 is not a typical oncogene, but is required for spontaneous transformation of MEFs. - Highlights: • Mouse embryo fibroblasts (MEFs) lacking SWAP-70 do not cause spontaneous transform. • Adding back of SWAP-70 to SWAP-70-deficient MEFs induces spontaneous transformation. • SWAP-70 is required for spontaneous transformation of MEFs.« less
-
Moraxella Species as Potential Sources of MCR-Like Polymyxin Resistance Determinants
Kieffer, Nicolas; Nordmann, Patrice
2017-01-01
ABSTRACT Plasmid-mediated resistance to polymyxins mediated by the MCR-1/2 determinants has been reported in Enterobacteriaceae worldwide. Using PCR-based and cloning strategies, a series of Moraxella spp. were screened for mcr-like genes. Moraxella spp. that are mainly animal pathogens but may also be human pathogens were identified as potential reservoirs of mcr-like genes. PMID:28320720
-
ERIC Educational Resources Information Center
Chattopadhyay, Ansuman
2005-01-01
Since the work of Watson and Crick in the mid-1950s, the science of genetics has become increasingly molecular. The development of recombinant DNA technologies by the agricultural and pharmaceutical industries led to the introduction of genetically modified organisms (GMOs). By the end of the twentieth century, reports of animal cloning and recent…
-
Shen, Zhicheng; Denton, Michael; Mutti, Navdeep; Pappan, Kirk; Kanost, Michael R.; Reese, John C.; Reeck, Gerald R.
2003-01-01
Endo-polygalacturonase, one of the group of enzymes known collectively as pectinases, is widely distributed in bacteria, plants and fungi. The enzyme has also been found in several weevil species and a few other insects, such as aphids, but not in Drosophila melanogaster, Anopheles gambiae, or Caenorhabditis elegans or, as far as is known, in any more primitive animal species. What, then, is the genetic origin of the polygalacturonases in weevils? Since some weevil species harbor symbiotic microorganisms, it has been suggested, reasonably, that the symbionts' genomes of both aphids and weevils, rather than the insects' genomes, could encode polygalacturonase. We report here the cloning of a cDNA that encodes endo-polygalacturonase in the rice weevil, Sitophilus oryzae (L.), and investigations based on the cloned cDNA. Our results, which include analysis of genes in antibiotic-treated rice weevils, indicate that the enzyme is, in fact, encoded by the insect genome. Given the apparent absence of the gene in much of the rest of the animal kingdom, it is therefore likely that the rice weevil polygalacturonase gene was incorporated into the weevil's genome by horizontal transfer, possibly from a fungus. PMID:15841240
-
Shen, Zhicheng; Denton, Michael; Mutti, Navdeep; Pappan, Kirk; Kanost, Michael R; Reese, John C; Reeck, Gerald R
2003-01-01
Endo-polygalacturonase, one of the group of enzymes known collectively as pectinases, is widely distributed in bacteria, plants and fungi. The enzyme has also been found in several weevil species and a few other insects, such as aphids, but not in Drosophila melanogaster, Anopheles gambiae, or Caenorhabditis elegans or, as far as is known, in any more primitive animal species. What, then, is the genetic origin of the polygalacturonases in weevils? Since some weevil species harbor symbiotic microorganisms, it has been suggested, reasonably, that the symbionts' genomes of both aphids and weevils, rather than the insects' genomes, could encode polygalacturonase. We report here the cloning of a cDNA that encodes endo-polygalacturonase in the rice weevil, Sitophilus oryzae (L.), and investigations based on the cloned cDNA. Our results, which include analysis of genes in antibiotic-treated rice weevils, indicate that the enzyme is, in fact, encoded by the insect genome. Given the apparent absence of the gene in much of the rest of the animal kingdom, it is therefore likely that the rice weevil polygalacturonase gene was incorporated into the weevil's genome by horizontal transfer, possibly from a fungus.
-
Chandramore, Kalpana; Ito, Yuzuro; Takahashi, Shuji; Asashima, Makoto; Ghaskadbi, Surendra
2010-01-01
Hydra, a member of phylum Cnidaria that arose early in evolution, is endowed with a defined axis, organized nervous system, and active behavior. It is a powerful model system for the elucidation of evolution of developmental mechanisms in animals. Here, we describe the identification and cloning of noggin-like gene from hydra. Noggin is a secreted protein involved at multiple stages of vertebrate embryonic development including neural induction and is known to exert its effects by inhibiting the bone morphogenetic protein (BMP)-signaling pathway. Sequence analysis revealed that hydra Noggin shows considerable similarity with its orthologs at the amino acid level. When microinjected in the early Xenopus embryos, hydra noggin mRNA induced a secondary axis in 100% of the injected embryos, demonstrating functional conservation of hydra noggin in vertebrates. This was further confirmed by the partial rescue of Xenopus embryos by hydra noggin mRNA from UV-induced ventralization. By using animal cap assay in Xenopus embryos, we demonstrate that these effects of hydra noggin in Xenopus embryos are because of inhibition of BMP signaling by Noggin. Our data indicate that BMP/Noggin antagonism predates the bilaterian divergence and is conserved during the evolution.
-
Nephronophthisis: Disease Mechanisms of a Ciliopathy
Hildebrandt, Friedhelm; Attanasio, Massimo; Otto, Edgar
2009-01-01
Nephronophthisis (NPHP), a recessive cystic kidney disease, is the most frequent genetic cause of end-stage kidney disease in children and young adults. Positional cloning of nine genes (NPHP1-9) and functional characterization of their encoded proteins (nephrocystins) has contributed to a unifying theory that defines cystic kidney diseases as “ciliopathies”. The theory is based on the finding that all proteins mutated in cystic kidney diseases of humans or animal models are expressed in primary cilia or centrosomes of renal epithelial cells. Primary cilia are sensory organelles that connect mechanosensory, visual, and other stimuli to mechanisms of epithelial cell polarity and cell cycle control. Mutations in NPHP genes cause defects in signaling mechanisms that involve the non-canonical Wnt signaling pathway and the sonic hedgehog signaling pathway, resulting in defects of planar cell polarity and tissue maintenance. The ciliary theory explains the multiple organ involvement in NPHP, which includes retinal degeneration, cerebellar hypoplasia, liver fibrosis, situs inversus, and mental retardation. Positional cloning of dozens of unknown genes that cause NPHP will elucidate further signaling mechanisms involved. Nephrocystins are highly conserved in evolution, thus allowing the use of animal models to develop future therapeutic approaches. PMID:19118152
-
Nilsson, Oskar; Börjesson, Stefan; Landén, Annica; Bengtsson, Björn
2014-06-01
In Sweden the prevalence of Enterobacteriaceae with transferable resistance to extended-spectrum cephalosporins (ESCs) is low. However, in broilers ESC-resistant Escherichia coli is common, with a clear dominance of blaCMY-2. Antimicrobials are rarely used in broiler production in Sweden and cephalosporins are never used. Introduction through imported breeding stock and subsequent vertical transmission of the bacteria through the production pyramid could be one explanation for this high prevalence. To test this hypothesis, paper linings from imported flocks of grandparent animals were screened for the presence of ESC-resistant E. coli and a positive flock, together with its progeny, was followed longitudinally through the production pyramid using boot swabs. The relationship of isolated ESC-resistant E. coli was investigated using multiple-locus variable number tandem repeat analysis (MLVA). ESC-resistant E. coli carrying blaCMY-2 was isolated from six out of eight imported flocks of grandparent animals. One clone of E. coli carrying blaCMY-2 occurred in all levels of the production pyramid and in flocks of imported grandparent animals. E. coli carrying blaCMY-2 is frequently present among grandparent animals imported to Sweden for breeding purposes. The occurrence of one clone in all levels of the production pyramid indicates that its introduction through imported breeding stock and vertical transmission through the production pyramid could be one explanation for the high proportion of Swedish broilers colonized with ESC-resistant E. coli. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
-
kgp, rgpA, and rgpB DNA vaccines induce antibody responses in experimental peri-implantitis.
Guo, Meihua; Wang, Zhifeng; Fan, Xin; Bian, Yuanyuan; Wang, Tiantian; Zhu, Lina; Lan, Jing
2014-11-01
Peri-implantitis is the key factor for implant failure. This study aims to evaluate kgp, rgpA, and rgpB DNA vaccines to induce an immune response and prevent peri-implantitis. The kgp, rgpA, and rgpB genes were amplified by polymerase chain reaction (PCR) from Porphyromonas gingivalis (Pg) ATCC 33277 and cloned into the pVAX1 vector. Titanium implants were placed into the mandibular bone of dogs. Three months later, the animals were divided into four groups, immunized with pVAX1-kgp, pVAX1-rgpA, pVAX1-rgpB, or pVAX1. Cotton ligatures infiltrated with Pg were tied around the neck of the implants. Immunoglobulin (Ig)G and IgA antibodies were detected by enzyme-linked immunosorbent assay before and after immunization. The kgp, rgpA, and rgpB genes were successfully cloned into the pVAX1 plasmid. Animals immunized with pVAX1-kgp and pVAX1-rgpA showed higher titers of IgG and IgA antibodies compared to those before immunization (P <0.05) and compared to those that were immunized with pVAX1 and pVAX1-rgpB, whereas there were no significant differences in the animals treated with pVAX1 and pVAX1-rgpB. Furthermore, among these, the kgp DNA vaccine was more effective. The bone losses of the groups with pVAX1-kgp and pVAX1-rgpA were significantly attenuated. pVAX1-kgp and pVAX1-rgpA DNA vaccines enhanced immunity responses and significantly retarded bone loss in experimental peri-implantitis animal models, whereas pVAX1-rgpB was ineffective.
-
Cloning of the short-tailed Gyeongju Donggyeong dog via SCNT: conserving phenotypic inheritance.
Choi, Yoo Bin; Kim, Geon A; Oh, Hyun Ju; Kim, Min Jung; Jo, Young Kwang; Setyawan, Erif Maha Nugraha; Lee, Seok Hee; Lee, Byeong Chun
2016-02-01
Somatic cell nuclear transfer is a useful tool to maintain genetic information of animals. The Gyeongju Donggyeong dog is a breed registered as natural monument in Korea. The unique feature of the Donggyeong dog is its tail, as the Donggyeong dog can be classified as either short tailed or tailless. The aim of this study was to preserve the Donggyeong dog's unique feature by cloning. Fibroblasts were obtained from a short-tailed Donggyeong dog. In vivo matured oocytes were enucleated, microinjected with a donor cell and fused electrically. Reconstructed embryos were transferred to six recipient dogs. One surrogate became pregnant, and one short-tailed Donggyeong dog was delivered. This study demonstrated that the phenotype of the Donggyeong dog could be conserved by somatic cell nuclear transfer.
-
Automated Cell-Cutting for Cell Cloning
NASA Astrophysics Data System (ADS)
Ichikawa, Akihiko; Tanikawa, Tamio; Matsukawa, Kazutsugu; Takahashi, Seiya; Ohba, Kohtaro
We develop an automated cell-cutting technique for cell cloning. Animal cells softened by the cytochalasin treatment are injected into a microfluidic chip. The microfluidic chip contains two orthogonal channels: one microchannel is wide, used to transport cells, and generates the cutting flow; the other is thin and used for aspiration, fixing, and stretching of the cell. The injected cell is aspirated and stretched in the thin microchannel. Simultaneously, the volumes of the cell before and after aspiration are calculated; the volumes are used to calculate the fluid flow required to aspirate half the volume of the cell into the thin microchannel. Finally, we apply a high-speed flow in the orthogonal microchannel to bisect the cell. This paper reports the cutting process, the cutting system, and the results of the experiment.
-
The Arabidopsis thaliana ortholog of a purported maize cholinesterase gene encodes a GDSL-lipase
Muralidharan, Mrinalini; Buss, Kristina; Larrimore, Katherine E.; Segerson, Nicholas A.; Kannan, Latha
2013-01-01
Acetylcholinesterase is an enzyme that is intimately associated with regulation of synaptic transmission in the cholinergic nervous system and in neuromuscular junctions of animals. However the presence of cholinesterase activity has been described also in non-metazoan organisms such as slime molds, fungi and plants. More recently, a gene purportedly encoding for acetylcholinesterase was cloned from maize. We have cloned the Arabidopsis thaliana homolog of the Zea mays gene, At3g26430, and studied its biochemical properties. Our results indicate that the protein encoded by the gene exhibited lipase activity with preference to long chain substrates but did not hydrolyze choline esters. The At3g26430 protein belongs to the SGNH clan of serine hydrolases, and more specifically to the GDS(L) lipase family. PMID:23430565
-
Del Prete, Sonia; De Luca, Viviana; Vullo, Daniela; Osman, Sameh M; AlOthman, Zeid; Carginale, Vincenzo; Supuran, Claudiu T; Capasso, Clemente
2016-12-01
Malassezia yeasts are almost exclusively the single eukaryotic members of the fungal flora of the skin. Malassezia globosa and Malassezia restricta are found on the skin of practically all humans. Malassezia globosa is highly implicated in the pathogenesis of dandruff and its genome encodes for only one carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the β-class (MgCA). It has been indeed demonstrated that in many pathogenic microorganisms, CAs are essential for their life cycle and their inhibition can lead to growth impairment and defects. In the previous work, the recombinant MgCA was investigated for its inhibition profile with sulfonamides, which in models of dandruff infection were able to protect animals from the fungal infection, allowing us to propose this enzyme as a new antidandruff target. MgCA was cloned as GST-fusion protein, but the yield was rather low and the protein was often found in inclusion bodies. Here, we propose an alternative procedure consisting in cloning the recombinant MgCA as His-Tag fusion protein. This procedure resulted in a good method to express and purify the active recombinant MgCA, and the protein recovery was better with respect to that used for preparing MG-CA (β-CA cloned as GST-fusion protein).
-
A Unique Report: Development of Super Anti-Human IgG Monoclone with Optical Density Over Than 3
Aghebati Maleki, Leili; Baradaran, Behzad; Abdolalizadeh, Jalal; Ezzatifar, Fatemeh; Majidi, Jafar
2013-01-01
Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity with IgM & IgA by ELISA and confirmed by immunoblotting. The subclasses of the selected mAbs were determined. The best clone was injected intraperitoneally to some pristane-injected mice. Anti-IgG mAb was purified from the animals' ascitic fluid by Ion exchange chromatography and then, mAb was conjugated with HRP. Results: In the present study, over than 50 clones were obtained that 1 clone had optical density over than 3. We named this clone as supermonoclone which was selected for limiting dilution. The result of the immunoblotting, showed sharp band in IgG position and did not show any band in IgM&IgA position. Conclusion: Based on the findings of this study, the conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases. PMID:24312857
-
Geng-Sheng, Cao; Yu, Gao; Kun, Wang; Fang-Rong, Ding; Ning, Li
2009-08-01
X inactivation is the process of a chromosome-wide silencing of the majority of genes on the X chromosome during early mammalian development. This process may be aberrant in cloned animals. Here we show that repressive modifications, such as methylation of DNA, and the presence of methylated histones, H3K9me2 and H3K27me3, exhibit distinct aberrance on the inactive X chromosome in live clones. In contrast, H3K4me3, an active gene marker, is obviously missing from the inactive X chromosome in all cattle studied. This suggests that the disappearance of active histone modifications (H3K4me3) seems to be more important for X inactivation than deposition of marks associated with heterochromatin (DNA methylation, H3K27me3 and H3K9me2). It also implies that even apparently normal clones may have subtle abnormalities in repressive, but not activating epigenetic modifications on the inactive X when they survive to term. We also found that the histone H3 methylations were enriched and co-localized at q21-31 of the active X chromosome, which may be associated with an abundance of LINE1 repeat elements. © 2009 The Authors. Journal compilation © 2009 Japanese Society of Developmental Biologists.
-
Zhou, Cui; Wang, Jian Wu; Huang, Kun Lun; He, XiaoYun; Chen, Xiu Ping; Sun, Hong; Yu, Tian; Che, Hui Lian
2011-10-01
Transgenic cloned animals expressing beneficial human nutritional traits offer a new strategy for large-scale production of some kinds of functional substances. In some cases, the required safety testing for genetically modified (GM) foods do not seem appropriate for human food safety, though regulations do not seem to provide alternatives. A 90-day rat feeding study is the core study for the safety assessment of GM foods. The test material in this 90-day study was prepared nonfat milk powder containing recombinant human lactoferrin (rhLF), which was expressed in transgenic cloned cattle. Groups of 10 male and female Sprague-Dawley rats were given a nutritionally balanced purified diet containing 7.5, 15, or 30% transgenic or conventional milk powder for 90 days. A commercial AIN93G diet was used as an additional control group. Clinical, biological, and pathological parameters were compared between groups. The only significant effect of treatment was higher mean ferritin and Fe(+) concentrations for both male and female rats fed the transgenic milk powder diets, as compared to rats fed nontransgenic milk diets or the commercial diet. The results of the present study are consistent with previous research, which indicates that milk powder containing rhLF derived from healthy transgenic cloned cattle is as safe as conventional milk powder.
-
The Influence of Polyploidy on the Evolution of Yeast Grown in a Sub-Optimal Carbon Source
Scott, Amber L.; Richmond, Phillip A.; Dowell, Robin D.; Selmecki, Anna M.
2017-01-01
Abstract Polyploidization events have occurred during the evolution of many fungi, plant, and animal species and are thought to contribute to speciation and tumorigenesis, however little is known about how ploidy level contributes to adaptation at the molecular level. Here we integrate whole genome sequencing, RNA expression analysis, and relative fitness of ∼100 evolved clones at three ploidy levels. Independent haploid, diploid, and tetraploid populations were grown in a low carbon environment for 250 generations. We demonstrate that the key adaptive mutation in the evolved clones is predicted by a gene expression signature of just five genes. All of the adaptive mutations identified encompass a narrow set of genes, however the tetraploid clones gain a broader spectrum of adaptive mutations than haploid or diploid clones. While many of the adaptive mutations occur in genes that encode proteins with known roles in glucose sensing and transport, we discover mutations in genes with no canonical role in carbon utilization (IPT1 and MOT3), as well as identify novel dominant mutations in glucose signal transducers thought to only accumulate recessive mutations in carbon limited environments (MTH1 and RGT1). We conclude that polyploid cells explore more genotypic and phenotypic space than lower ploidy cells. Our study provides strong evidence for the beneficial role of polyploidization events that occur during the evolution of many species and during tumorigenesis. PMID:28957510
-
Bovine somatic cell nuclear transfer.
Ross, Pablo J; Cibelli, Jose B
2010-01-01
Somatic cell nuclear transfer (SCNT) is a technique by which the nucleus of a differentiated cell is introduced into an oocyte from which its genetic material has been removed by a process called enucleation. In mammals, the reconstructed embryo is artificially induced to initiate embryonic development (activation). The oocyte turns the somatic cell nucleus into an embryonic nucleus. This process is called nuclear reprogramming and involves an important change of cell fate, by which the somatic cell nucleus becomes capable of generating all the cell types required for the formation of a new individual, including extraembryonic tissues. Therefore, after transfer of a cloned embryo to a surrogate mother, an offspring genetically identical to the animal from which the somatic cells where isolated, is born. Cloning by nuclear transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. Cattle were the second mammalian species to be cloned after Dolly the sheep, and it is probably the most widely used species for SCNT experiments. This is, in part due to the high availability of bovine oocytes and the relatively higher efficiency levels usually obtained in cattle. Given the wide utilization of this species for cloning, several alternatives to this basic protocol can be found in the literature. Here we describe a basic protocol for bovine SCNT currently being used in our laboratory, which is amenable for the use of the nuclear transplantation technique for research or commercial purposes.
-
Germline modification of domestic animals
Tang, L.; González, R.; Dobrinski, I.
2016-01-01
Genetically-modified domestic animal models are of increasing significance in biomedical research and agriculture. As authentic ES cells derived from domestic animals are not yet available, the prevailing approaches for engineering genetic modifications in those animals are pronuclear microinjection and somatic cell nuclear transfer (SCNT, also known as cloning). Both pronuclear microinjection and SCNT are inefficient, costly, and time-consuming. In animals produced by pronuclear microinjection, the exogenous transgene is usually inserted randomly into the genome, which results in highly variable expression patterns and levels in different founders. Therefore, significant efforts are required to generate and screen multiple founders to obtain animals with optimal transgene expression. For SCNT, specific genetic modifications (both gain-of-function and loss-of-function) can be engineered and carefully selected in the somatic cell nucleus before nuclear transfer. SCNT has been used to generate a variety of genetically modified animals such as goats, pigs, sheep and cattle; however, animals resulting from SCNT frequently suffer from developmental abnormalities associated with incomplete nuclear reprogramming. Other strategies to generate genetically-modified animals rely on the use of the spermatozoon as a natural vector to introduce genetic material into the female gamete. This sperm mediated DNA transfer (SMGT) combined with intracytoplasmatic sperm injection (ICSI) has relatively high efficiency and allows the insertion of large DNA fragments, which, in turn, enhance proper gene expression. An approach currently being developed to complement SCNT for producing genetically modified animals is germ cell transplantation using genetically modified male germline stem cells (GSCs). This approach relies on the ability of GSCs that are genetically modified in vitro to colonize the recipient testis and produce donor derived sperm upon transplantation. As the genetic change is introduced into the male germ line just before the onset of spermatogenesis, the time required for the production of genetically modified sperm is significantly shorter using germ cell transplantation compared to cloning or embryonic stem (ES) cell based technology. Moreover, the GSC-mediated germline modification circumvents problems associated with embryo manipulation and nuclear reprogramming. Currently, engineering targeted mutations in domestic animals using GSCs remains a challenge as GSCs from those animals are difficult to maintain in vitro for an extended period of time. Recent advances in genome editing techniques such as Zinc-Finger Nucleases (ZFNs), Transcription Activator-like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) greatly enhance the efficiency of engineering targeted genetic change in domestic animals as demonstrated by the generation of several gene knock-out pig and cattle models using those techniques. The potential of GSC-mediated germline modification in making targeted genetic modifications in domestic animal models will be maximized if those genome editing techniques can be applied in GSCs. PMID:27390591
-
Jeevan, Amminikutty; Yoshimura, Teizo; Ly, Lan H; Dirisala, Vijaya R; McMurray, David N
2011-01-01
Interleukin-4 (IL-4), a pleiotropic cytokine produced by T-helper type 2 (Th2) cells, is involved in promoting humoral immune responses, allergic reactions and asthma. Previous studies suggested an important role for IL-4 in susceptibility to pulmonary tuberculosis; however, the role of IL-4 has not been studied in the guinea pig, a highly relevant model for this disease. In the present study, we cloned a cDNA for guinea pig IL-4 and examined, for the first time, mRNA expression by real-time RT-PCR in cultured guinea pig cells. High levels of IL-4 mRNA expression were detected in spleen T cells of naïve animals after in vitro stimulation with PMA plus ionomycin for 4-24 h. The expression of IL-4 mRNA was low in spleen and lymph node cells immunized with ovalbumin (OVA) plus Complete Freund's Adjuvant (CFA) in response to OVA (Th1), but significantly higher in the guinea pigs immunized with OVA plus alum (Th2). BCG vaccination reduced the expression of IL-4 mRNA in both spleen and lung digest cells compared to naïve guinea pigs, while levels of IFN-γ were similar in both groups. Furthermore, lung cells from Mycobacterium tuberculosis-infected guinea pigs stimulated in vitro with PPD or MPT64 showed low levels of IL-4 mRNA expression. Thus, BCG vaccination or M. tuberculosis infection modulates IL-4 mRNA expression in the guinea pig. Cloning of guinea pig IL-4 will allow us to address the role of IL-4 in vaccine-induced resistance to pulmonary TB in a highly relevant animal model. Copyright © 2010 Elsevier Ltd. All rights reserved.
-
Pietsch, Michael; Eller, Christoph; Wendt, Constanze; Holfelder, Martin; Falgenhauer, Linda; Fruth, Angelika; Grössl, Tobias; Leistner, Rasmus; Valenza, Giuseppe; Werner, Guido; Pfeifer, Yvonne
2017-02-01
The increase of Escherichia coli producing extended-spectrum β-lactamases (ESBL) in hospitals and their emergence as intestinal colonisers of healthy humans is of concern. Transmission ways and the extent of spread of distinct E. coli clones or ESBL genes among humans and animals via the food chain or the environment is a matter of debate. In this study we determined ESBL genotypes in E. coli isolates (n=233) resistant to 3rd generation cephalosporins from hospitals and medical practices using PCR and sequencing. Bacterial strain typing was performed by PCR-based phylogrouping, multilocus sequence typing (MLST) and a ST131-specific PCR. Results showed that CTX-M-15 (50.4%), CTX-M-1 (28.4%) and CTX-M-14 (5.6%) were the most common ESBL types. Especially, CTX-M-15 was associated with E. coli ST131 of phylogenetic group B2, which was the dominant sequence type among our isolates (35.8%). MLST typing revealed 40 different sequence types (STs), with ST131, ST410, ST10 and ST38 as the most prevalent ones. Our findings give an overview of the current distribution of ESBL-producing E. coli isolates from humans in Germany. E. coli O25b:H4-ST131 was confirmed to be the most common clone, which is known for its successful dissemination worldwide. Although heterogeneity among the isolates was found, several successful clones previously described in animals (ST410, ST10) also occurred in our isolate collection. Further detailed investigations of ESBL-producing isolates from different habitats are needed to evaluate possible transfer ways. Copyright © 2015 Elsevier B.V. All rights reserved.
-
Genome engineering in cattle: recent technological advancements.
Wang, Zhongde
2015-02-01
Great strides in technological advancements have been made in the past decade in cattle genome engineering. First, the success of cloning cattle by somatic cell nuclear transfer (SCNT) or chromatin transfer (CT) is a significant advancement that has made obsolete the need for using embryonic stem (ES) cells to conduct cell-mediated genome engineering, whereby site-specific genetic modifications can be conducted in bovine somatic cells via DNA homologous recombination (HR) and whereby genetically engineered cattle can subsequently be produced by animal cloning from the genetically modified cells. With this approach, a chosen bovine genomic locus can be precisely modified in somatic cells, such as to knock out (KO) or knock in (KI) a gene via HR, a gene-targeting strategy that had almost exclusively been used in mouse ES cells. Furthermore, by the creative application of embryonic cloning to rejuvenate somatic cells, cattle genome can be sequentially modified in the same line of somatic cells and complex genetic modifications have been achieved in cattle. Very recently, the development of designer nucleases-such as zinc finger nucleases (ZFNs) and transcription activator-like effector nuclease (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-has enabled highly efficient and more facile genome engineering in cattle. Most notably, by employing such designer nucleases, genomes can be engineered at single-nucleotide precision; this process is now often referred to as genome or gene editing. The above achievements are a drastic departure from the traditional methods of creating genetically modified cattle, where foreign DNAs are randomly integrated into the animal genome, most often along with the integrations of bacterial or viral DNAs. Here, I review the most recent technological developments in cattle genome engineering by highlighting some of the major achievements in creating genetically engineered cattle for agricultural and biomedical applications.
-
Kwon, Hyuk Moo; LeRoith, Tanya; Pudupakam, R S; Pierson, F William; Huang, Yao-Wei; Dryman, Barbara A; Meng, Xiang-Jin
2011-01-27
A genetically distinct strain of avian hepatitis E virus (avian HEV-VA strain) was isolated from a healthy chicken in Virginia, and thus it is important to characterize and compare its pathogenicity with the prototype strain (avian HEV-prototype) isolated from a diseased chicken. Here we first constructed an infectious clone of the avian HEV-VA strain. Capped RNA transcripts from the avian HEV-VA clone were replication-competent after transfection of LMH chicken liver cells. Chickens inoculated intrahepatically with RNA transcripts of avian HEV-VA clone developed active infection as evidenced by fecal virus shedding, viremia, and seroconversion. To characterize the pathogenicity, RNA transcripts of both avian HEV-VA and avian HEV-prototype clones were intrahepatically inoculated into the livers of chickens. Avian HEV RNA was detected in feces, serum and bile samples from 10/10 avian HEV-VA-inoculated and 9/9 avian HEV-prototype-inoculated chickens although seroconversion occurred only in some chickens during the experimental period. The histopathological lesion scores were lower for avian HEV-VA group than avian HEV-prototype group in the liver at 3 and 5 weeks post-inoculation (wpi) and in the spleen at 3 wpi, although the differences were not statistically significant. The liver/body weight ratio, indicative of liver enlargement, of both avian HEV-VA and avian HEV-prototype groups were significantly higher than that of the control group at 5 wpi. Overall, the avian HEV-VA strain still induces histological liver lesions even though it was isolated from a healthy chicken. The results also showed that intrahepatic inoculation of chickens with RNA transcripts of avian HEV infectious clone may serve as an alternative for live virus in animal pathogenicity studies. Copyright © 2010 Elsevier B.V. All rights reserved.
-
[Therapeutic cloning: far from application at this stage].
De Both, N J
2001-11-03
Therapeutic cloning has become possible since the discovery that nuclei from somatic cells of adult animal tissue can successfully be used for cloning and the fact that human embryonic stem cell lines have been established from preimplantation embryos. When nuclei from healthy tissue of a patient are transplanted into enucleated oocytes, these oocytes can be artificially activated so that embryos develop from which embryonic stem cells of the donor can be derived. These embryonic stem cells can be cultured as permanent lines in unlimited numbers and remain pluripotent, i.e. they can be induced to differentiate into the required cell type by adding one or more specific factors. These cells can then be transplanted back into the patient suffering from either a lack or dysfunction of these cells. This approach prevents the rejection of the transplanted cells by the patient's immunological system. As this type of cloning has a very low efficiency, a large number of unfertilized donor oocytes is required. It is questionable whether enough donors are or will be available for this purpose. The cultured cells must satisfy certain conditions before they can be used for transplantation. They must be checked for chromosomal abnormalities, and a complete differentiation of the embryonic stem cells into the cells types needed by the patient is necessary as after the transplantation, undifferentiated stem cells will form teratomas. Furthermore, it is difficult to ensure that the cells end up in the right place and to ensure that they fully integrate into the existing tissue to form functional connections. Due to this array of technical problems the question remains as to whether therapeutic cloning will become feasible in the near future.
-
Akshey, Yogesh S; Malakar, Dhruba; De, Arun K; Jena, Manoj K; Garg, Shweta; Dutta, Rahul; Pawar, Sachin Kumar; Mukesh, Manisha
2010-10-01
Nuclear transfer is a very effective method for propagation of valuable, extinct, and endangered animals. Hand-made cloning (HMC) is an efficient alternative to the conventional micromanipulator-based technique in some domestic species. The present study was carried out for the selection of suitable somatic cells as a nuclear donor and development of an optimum culture system for in vitro culture of zona-free goat cloned embryos. Cleavage and blastocyst rates were observed 72.06 ± 2.94% and 0% for fresh cumulus cells, 81.95 ± 3.40% and 12.74 ± 2.12% for cultured cumulus cells, and 92.94 ± 0.91% and 23.78 ± 3.33% for fetal fibroblast cells, respectively. There was a significant (p < 0.05) increase in blastocyst production in goats when cultured on a flat surface (FS) (23.78 ± 3.33 %) than well of wells (WOW) (15.84 ± 2.12 %) and microdrops (MD) (0.7 ± 0.7%). Furthermore, cleavage and blastocyst production rates were significantly (p < 0.05) more in the WOW (15.84 ± 2.12%) than the MD (0.7 ± 0.7%) system. The quality of HMC blastocysts was studied by differential staining. Genetic similarity was confirmed by polymerase chain reaction (PCR)-based amplification of the second exon of the MHC class II DRB gene, which gave similar bands in electrophoresis (286 bp) both in cloned embryos and donor cells. In conclusion, the present study describes that the fetal fibroblast cell is a suitable candidate as nuclear donor, and the flat surface culture system is suitable for zona-free blastocyst development by the hand-made cloning technique in the goat.
-
Washington, O R; Deslauriers, M; Stevens, D P; Lyford, L K; Haque, S; Yan, Y; Flood, P M
1993-01-01
Fimbrillin is the major subunit protein of fimbriae from the human periodontal pathogen Porphyromonas (Bacteroides) gingivalis. We describe here the generation and initial characterization of recombinant fimbrillin (r-fimbrillin) isolated from P. gingivalis 381. A fragment of DNA encoding the gene for fimbrillin was generated by polymerase chain reaction and cloned into the expression vector pET11b. Plasmids containing the recombinant gene were transfected into Escherichia coli. Clones were selected on plates for ampicillin resistance and individually screened by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein production after activation with IPTG (isopropyl-beta-D- thiogalactopyranoside). One clone, OW0.2, produced significant amounts of a 42-kDa protein after induction with IPTG. This clone contained the pET11b plasmid with a 1-kb insert that had sequence homology to the gene encoding fimbrillin. The majority of recombinant protein from clone OW0.2 was found in the cytoplasm within inclusion bodies. Protein aggregates were solubilized in 8 M urea, and SDS-PAGE analysis showed two major protein bands, one at 42 kDa and the other at 17 kDa. These two proteins coeluted from a DEAE-Sepharose column at 0.15 M NaCl and were reactive to rabbit antiserum to fimbrillin in a Western blot (immunoblot). A preparation giving a single protein band at 42 kDa in SDS-PAGE was obtained by size fractionation by using continuous-elution electrophoresis. Lymph node cells from animals immunized with either fimbrillin from P. gingivalis or r-fimbrillin showed antigen-specific proliferation to both P. gingivalis fimbrillin and r-fimbrillin in an in vitro recall assay. Therefore, it appears that r-fimbrillin is chemically, antigenically, and serologically identical to fimbrillin isolated from P. gingivalis 381. Images PMID:8094377
-
Future and applications of cloning.
Trounson, Alan O
2006-01-01
The birth of viable offspring from somatic cell nuclear transfer (SCNT) in mammals caused a major re-examination of the understanding of the commitment of cells to specific tissue lineages during differentiation. The questions of whether cells undergo dedifferentiation or transdifferentiation during the development of offspring and how these changes are controlled is a source of ongoing debate that is yet to be resolved. Irrespective of the outcome of this debate, it is clear that cloning using SCNT has a place and purpose in the future of research and animal breeding. The future uses of SCNT could include the production of transgenic mice, the production of transgenic livestock and assisting with the re-establishment of endangered species. Human medicine also would benefit from future use of SCNT because it would allow the production of patient-specific embryonic stem cells.
-
Gene cloning and prokaryotic expression of recombinant flagellin A from Vibrio parahaemolyticus
NASA Astrophysics Data System (ADS)
Yuan, Ye; Wang, Xiuli; Guo, Sheping; Liu, Yang; Ge, Hui; Qiu, Xuemei
2010-11-01
The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene ( flaA) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can be used for further functional and structural studies.
-
NASA Astrophysics Data System (ADS)
Yuan, Ye; Wang, Xiuli; Guo, Sheping; Qiu, Xuemei
2011-06-01
Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V. parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.
-
NASA Astrophysics Data System (ADS)
Sun, S. M.; Slightom, J. L.; Hall, T. C.
1981-01-01
A plant gene coding for the major storage protein (phaseolin, G1-globulin) of the French bean was isolated from a genomic library constructed in the phage vector Charon 24A. Comparison of the nucleotide sequence of part of the gene with that of the cloned messenger RNA (cDNA) revealed the presence of three intervening sequences, all beginning with GTand ending with AG. The 5' and 3' boundaries of intervening sequences TVS-A (88 base pairs) and IVS-B (124 base pairs) are similar to those described for animal and viral genes, but the 3' boundary of IVS-C (129 base pairs) shows some differences. A sequence of 185 amino acids deduced from the cloned DMAs represents about 40% of a phaseolin polypeptide.
-
Modification of the Genome of Domestic Animals.
Lotti, Samantha N; Polkoff, Kathryn M; Rubessa, Marcello; Wheeler, Matthew B
2017-07-03
In the past few years, new technologies have arisen that enable higher efficiency of gene editing. With the increase ease of using gene editing technologies, it is important to consider the best method for transferring new genetic material to livestock animals. Microinjection is a technique that has proven to be effective in mice but is less efficient in large livestock animals. Over the years, a variety of methods have been used for cloning as well as gene transfer including; nuclear transfer, sperm mediated gene transfer (SMGT), and liposome-mediated DNA transfer. This review looks at the different success rate of these methods and how they have evolved to become more efficient. As well as gene editing technologies, including Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the most recent clustered regulatory interspaced short palindromic repeats (CRISPRs). Through the advancements in gene-editing technologies, generating transgenic animals is now more accessible and affordable. The goals of producing transgenic animals are to 1) increase our understanding of biology and biomedical science; 2) increase our ability to produce more efficient animals; and 3) produce disease resistant animals. ZFNs, TALENs, and CRISPRs combined with gene transfer methods increase the possibility of achieving these goals.
-
Dooley, Helen; Stanfield, Robyn L; Brady, Rebecca A; Flajnik, Martin F
2006-02-07
The cartilaginous fish are the oldest phylogenetic group in which Igs have been found. Sharks produce a unique Ig isotype, IgNAR, a heavy-chain homodimer that does not associate with light chains. Instead, the variable (V) regions of IgNAR bind antigen as soluble single domains. Our group has shown that IgNAR plays an integral part in the humoral response of nurse sharks (Ginglymostoma cirratum) upon antigen challenge. Here, we generated phage-displayed libraries of IgNAR V regions from an immunized animal and found a family of clones derived from the same rearrangement event but differentially mutated during expansion. Because of the cluster organization of shark Ig genes and the paucicopy nature of IgNAR, we were able to construct the putative ancestor of this family. By studying mutations in the context of clone affinities, we found evidence that affinity maturation occurs for this isotype. Subsequently, we were able to identify mutations important in the affinity improvement of this family. Because the family clones were all obtained after immunization, they provide insight into the in vivo maturation mechanisms, in general, and for single-domain antibody fragments.
-
Interspecies Somatic Cell Nuclear Transfer: Advancements and Problems
Lagutina, Irina; Fulka, Helena; Lazzari, Giovanna
2013-01-01
Abstract Embryologists working with livestock species were the pioneers in the field of reprogramming by somatic cell nuclear transfer (SCNT). Without the “Dolly experiment,” the field of cellular reprogramming would have been slow and induced plutipotent cells (iPSCs) would not have been conceived. The major drive of the work in mammalian cloning was the interest of the breeding industry to propagate superior genotypes. Soon it was realized that the properties of oocytes could be used also to clone endangered mammalian species or to reprogram the genomes of unrelated species through what is known as interspecies (i) SCNT, using easily available oocytes of livestock species. iSCNT for cloning animals works only for species that can interbreed, and experiments with taxonomically distant species have not been successful in obtaining live births or deriving embryonic stem cell (ESC) lines to be used for regenerative medicine. There are controversial reports in the literature, but in most cases these experiments have underlined some of the cellular and molecular mechanisms that are incomplete during cell nucleus reprogramming, including the failure to organize nucleoli, silence somatic cell genes, activate the embryonic genome, and resume mitochondrial replication and function, thus indicating nucleus–cytoplasmic incompatibility. PMID:24033141
-
The origin of mesoderm in phoronids
NASA Technical Reports Server (NTRS)
Freeman, Gary; Martindale, Mark Q.
2002-01-01
Descriptive studies of phoronid development have concluded that the mesoderm of these animals originates from the endoderm during gastrulation. This interpretation has been tested by labeling one blastomere of 4- through 16-cell embryos and examining the position and germ layers occupied by the labeled clones of cells in the larva. No 2 injections gave rise to identical clones of cells, suggesting that the cleavage program does not generate cells of unique identity and that cell fates are established at later developmental time points. In many cases, a relatively large sector composed of ectodermal cells was labeled. When these labeled cells were adjacent to the mouth or anus of the larva, muscle and mesenchyme cells originated from the labeled clones. Under these circumstances, nerve cells also originated from these labeled sectors. These labeling studies also showed that endodermal cells can give rise to mesodermal and neural cells. These results suggest that nerve and muscle cells are induced to form at ectodermal-endodermal boundaries from both germ layers. These marking experiments also confirmed the observation that nerve cells originate both from the apical organ and the trunk region and show for the first time that the intestine originates by ingression of posterior ectoderm.
-
Detection of Different DNA Animal Species in Commercial Candy Products.
Muñoz-Colmenero, Marta; Martínez, Jose Luis; Roca, Agustín; Garcia-Vazquez, Eva
2016-03-01
Candy products are consumed all across the world, but there is not much information about their composition. In this study we have used a DNA-based approach for determining the animal species occurring in 40 commercial candies of different types. We extracted DNA and performed PCR amplification, cloning and sequencing for obtaining species-informative DNA sequences. Eight species were identified including fish (hake and anchovy) in 22% of the products analyzed. Bovine and porcine were the most abundant appearing in 27 samples each one. Most products contained a mixture of species. Marshmallows (7), jelly-types, and gummies (20) contained a significantly higher number of species than hard candies (9). We demonstrated the presence of DNA animal species in candy product which allow consumers to make choices and prevent allergic reaction. © 2016 Institute of Food Technologists®
-
Contribution of radiation hybrids to genome mapping in domestic animals.
Faraut, T; de Givry, S; Hitte, C; Lahbib-Mansais, Y; Morisson, M; Milan, D; Schiex, T; Servin, B; Vignal, A; Galibert, F; Yerle, M
2009-01-01
Radiation hybrid mapping has emerged in the end of the 1990 s as a successful and complementary approach to map genomes, essentially because of its ability to bridge the gaps between genetic and clone-based physical maps, but also using comparative mapping approaches, between 'gene-rich' and 'gene-poor' maps. Since its early development in human, radiation hybrid mapping played a pivotal role in the process of mapping animal genomes, especially mammalian ones. We review here all the different steps involved in radiation hybrid mapping from the constitution of panels to the construction of maps. A description of its contribution to whole genome maps with a special emphasis on domestic animals will also be presented. Finally, current applications of radiation hybrid mapping in the context of whole genome assemblies will be described. (c) 2009 S. Karger AG, Basel.
-
Tashakkori, Maryam Mohammadi; Tebianian, Majid; Tabatabaei, Mohammad; Mosavari, Nader
2016-12-01
Tuberculosis (TB) is a zoonotic infectious disease common to humans and animals that is caused by the rod-shaped acid-fast bacterium Mycobacterium bovis. Rapid and sensitive detection of TB is promoted by specific antigens. Virulent strains of the TB complex from M. bovis contain 16 regions of difference (RD) in their genome that encode important proteins, including major protein of Mycobacterium Tuberculosis 64 (MBT-64, which is a primary immune-stimulating antigen encoded by RD-2. In this study, we cloned, expressed, and purified MPT-64 as a potent M. bovis antigen in a prokaryotic system for use in future diagnostic studies. The antigenic region of the Mpt64 gene was investigated by bioinformatics methods, cloned into the PQE-30 plasmid, and expressed in Escherichia coli M15 cells, followed by isopropyl β-d-1-thiogalactopyranoside induction. The expressed protein was analyzed sodium dodecyl sulfate polyacrylamide gel electrophoresis and purified using a nickel-affinity column. Biological activity was confirmed by western blot using specific antibodies. Our data verified the successful cloning of the Mpt64 gene (687-bp segment) via the expression vector and purification of recombinant MPT-64 as a 24-kDa protein. These results indicated successful expression and purification of recombinant MPT-64 protein in a prokaryotic system. This protein can be used for serological diagnosis, improved detection of pathogenicity and non-pathogenicity between infected cattle, and for verification of suspected cases of bovine TB. Copyright © 2016.
-
Predominance of Gram-positive bacteria in house dust in the low-allergy risk Russian Karelia.
Pakarinen, Jaakko; Hyvärinen, Anne; Salkinoja-Salonen, Mirja; Laitinen, Sirpa; Nevalainen, Aino; Mäkelä, Mika J; Haahtela, Tari; von Hertzen, Leena
2008-12-01
Simple living conditions and farming environment have been associated with reduced risk for allergic diseases such as atopy and asthma but the factors responsible for this effect remain unresolved. We examined the bacterial composition of house dusts obtained from Finnish and Russian Karelia, two adjacent areas with high and low occurrence of atopic diseases respectively. Two dust mixes, both composed of 10 randomly selected dust samples from 349 Finnish and 417 Russian Karelian households were studied for bacterial biomarkers (DNA, Limulus-active endotoxin, 3-OH fatty acids, muramic acid) and for 16S rRNA gene sequences. Overall, the DNA cloning revealed more taxons (94 different genera) of dustborne bacteria than seen in any previous study on residential environments. Majority (67%) of the bacterial DNA clones in house dust from the low-allergy Russian Kareliarepresented Gram-positive bacteria (Firmicutes and Actinobacteria), predominantly Staphylococcaceae and Corynebacteriaceae. Russian Karelian dust showed up to 20-fold higher contents of muramic acid (marker of Gram-positive bacteria) and a sevenfold higher number of clones of animal-associated species, whereas in Finnish Karelian dust Gram-negatives (mainly Proteobacteria) predominated. Clones of plant-associated bacterial species and of chloroplast, indicating plant biomass, were more numerous in Finnish than in Russian Karelian dust. In conclusion, this study revealed major disparities between Finnish and Russian house dusts. The higher bacterial content and the predominance of Gram-positive bacteria in Russian dust may have implications for occurrence of atopy.
-
Liu, Changqing; Bai, Chunyu; Guo, Yu; Liu, Dan; Lu, Taofeng; Li, Xiangchen; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun
2014-01-01
Bacterial artificial chromosome (BAC) libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12), consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger. PMID:24608928
-
Viable calves produced by somatic cell nuclear transfer using meiotic-blocked oocytes.
De Bem, Tiago H C; Chiaratti, Marcos R; Rochetti, Raquel; Bressan, Fabiana F; Sangalli, Juliano R; Miranda, Moysés S; Pires, Pedro R L; Schwartz, Kátia R L; Sampaio, Rafael V; Fantinato-Neto, Paulo; Pimentel, José R V; Perecin, Felipe; Smith, Lawrence C; Meirelles, Flávio V; Adona, Paulo R; Leal, Cláudia L V
2011-10-01
Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585±34,775 vs. 595,579±31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179±45,617 vs. 498,771±33,231) and blastocysts (816,627±40,235 vs. 765,332±51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.
-
Insect Optic Glomeruli-Exploration of a Universal Circuit for Sensorimotor Processing
2010-02-03
laboratory of the small palisade output neurons from the lobula of Drosophila melanogaster, using in vivo targeting of green fluorescent protein...animals that reveal cohorts of lobula outputs to identified optic glomeruli. Using infrared illumination and optics, the cell bodies of such clones...surface of one of these neurons. Once contiguity between the neuron and electrolyte of the electrode has been established, the cell is recorded
-
Somatic cell nuclear transfer: pros and cons.
Sumer, Huseyin; Liu, Jun; Tat, Pollyanna; Heffernan, Corey; Jones, Karen L; Verma, Paul J
2009-01-01
Even though the technique of mammalian SCNT is just over a decade old it has already resulted in numerous significant advances. Despite the recent advances in the reprogramming field, SCNT remains the bench-mark for the generation of both genetically unmodified autologous pluripotent stem cells for transplantation and for the production of cloned animals. In this review we will discuss the pros and cons of SCNT, drawing comparisons with other reprogramming methods.
-
Construction of Rabbit Immune Antibody Libraries.
Nguyen, Thi Thu Ha; Lee, Jong Seo; Shim, Hyunbo
2018-01-01
Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice, and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage-display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from nonmurine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.
-
Baldassarre, H; Wang, B; Pierson, J; Neveu, N; Sneek, L; Lapointe, J; Cote, F; Kafidi, N; Keefer, C L; Lazaris, A; Karatzas, C N
2004-01-01
The use of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production was evaluated in the early propagation of cloned goats. Ten kinder goats produced by somatic cell nuclear transfer technology were used as oocyte donors. Half of the donor animals were subjected to LOPU at 2-3 months of age (prior to induction of lactation), whereas the other five goats were subjected to LOPU at 6-7 months of age (following induction to lactation). They were stimulated with 80 mg NIH-FSH-P1 (Folltropin, Vetrepharm, Canada) together with 300 IU eCG (Novormon, Vetrepharm, Canada) administered intramuscularly 36 h prior to LOPU. The number of follicles aspirated and oocytes recovered was higher in the younger group of donors (57 +/- 7 and 41 +/- 4 vs. 28 +/- 2 and 25.8 +/- 2, p < 0.05), however, oocytes from animals in the late prepubertal age showed higher developmental capacity resulting in higher transferable embryo yield (81.4% vs. 67.8%, p < 0.01), pregnancy rate (80% vs. 40%, p < 0.05) and total kids born (27 vs. 15, p < 0.01). In conclusion, LOPU in combination with in vitro embryo production techniques is an efficient method for the early propagation of valuable goats produced by somatic cell nuclear transfer.
-
Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis
Sun, Xingshen; Sui, Hongshu; Fisher, John T.; Yan, Ziying; Liu, Xiaoming; Cho, Hyung-Ju; Joo, Nam Soo; Zhang, Yulong; Zhou, Weihong; Yi, Yaling; Kinyon, Joann M.; Lei-Butters, Diana C.; Griffin, Michelle A.; Naumann, Paul; Luo, Meihui; Ascher, Jill; Wang, Kai; Frana, Timothy; Wine, Jeffrey J.; Meyerholz, David K.; Engelhardt, John F.
2010-01-01
Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments. PMID:20739752
-
Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis.
Sun, Xingshen; Sui, Hongshu; Fisher, John T; Yan, Ziying; Liu, Xiaoming; Cho, Hyung-Ju; Joo, Nam Soo; Zhang, Yulong; Zhou, Weihong; Yi, Yaling; Kinyon, Joann M; Lei-Butters, Diana C; Griffin, Michelle A; Naumann, Paul; Luo, Meihui; Ascher, Jill; Wang, Kai; Frana, Timothy; Wine, Jeffrey J; Meyerholz, David K; Engelhardt, John F
2010-09-01
Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments.
-
Lebedinskaia, O V; Gorskaia, Iu F; Shuklina, E Iu; Latsinik, N V; Nesterenko, V G
2005-01-01
The objective of this study was to analyze the species differences in the numbers of stromal precursor cell (CFU-f), their cloning efficiency (CFE-0 and their dynamics in different organs during aging, using the mathematical gradient decrease method. Age changes of CFU-f numbers and of their CFE-f were studied in the thymus and the spleen of mice and guinea pigs. The study was performed using CFU-f cloning in monolayer cultures. CFU-f numbers and CFE-f were found to decrease with aging both in the thymus and the spleen of mice and guinea pigs. However these changes were different in each species and were variable in different organs of the animals of the same species, which, probably was associated with the physiological characteristics and aging peculiarities of the animals of different species and with the functional role of organs studied. The process of reduction was more significant in the thymus of guinea pigs and mice - the numbers of CFU-f were decreased 75- and 12-fold, respectively. Since it is known that the population of CFU-f in the thymus and the spleen includes inducible osteogenic precursor cells, the data obtained indicate the possibility of a reduction in numbers of this category ofstromal precursors, that could be one of the reasons of osteoporosis of aging. The application of a mathematical analysis using the gradient decrease method allows to predict the time-course of age changes and to evaluate the dynamics of CFU-f numbers and of CFE-f in association with organism aging.
-
Evolutionary perspectives on clonal reproduction in vertebrate animals
Avise, John C.
2015-01-01
A synopsis is provided of different expressions of whole-animal vertebrate clonality (asexual organismal-level reproduction), both in the laboratory and in nature. For vertebrate taxa, such clonal phenomena include the following: human-mediated cloning via artificial nuclear transfer; intergenerational clonality in nature via parthenogenesis and gynogenesis; intergenerational hemiclonality via hybridogenesis and kleptogenesis; intragenerational clonality via polyembryony; and what in effect qualifies as clonal replication via self-fertilization and intense inbreeding by simultaneous hermaphrodites. Each of these clonal or quasi-clonal mechanisms is described, and its evolutionary genetic ramifications are addressed. By affording an atypical vantage on standard vertebrate reproduction, clonality offers fresh perspectives on the evolutionary and ecological significance of recombination-derived genetic variety. PMID:26195735
-
Tollrian, Ralph
1995-02-01
Juvenile Daphnia pulex form neckteeth in reponse to chemicals released by predatory Chaoborus crystallinus larvae. Formation of neckteeth is strongest in the second instar followed by the third instar, whereas only small neckteeth are found in the first and fourth instar of experimental clones. Predation experiments showed that body-size-dependent vulnerability of animals without neckteeth to fourth instar C. crystallinus larvae matched the pattern of neckteeth formation over the four juvenile instars. Predation experiments on D. pulex of the same clone with neckteeth showed that vulnerability to C. crystallinus predation is reduced, and that the induced protection is correlated with the degree of neckteeth formation. The pattern of neckteeth formation in successive instars is probably adaptive, and it can be concluded that neckteeth are formed to different degrees in successive instars as an evolutionary compromise to balance prediation risk and protective costs.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Woodhead, A.D.; Setlow, R.B.; Hart, R.W.
1977-01-01
It is suggested that the viviparous teleost, Poecilia formosa, (Amazon molly) may have wide potential use for aquatic radiation studies. The Amazon molly is a naturally occurring gynogenetic species, in which the eggs are activated after mating with the males of closely related species, without the subsequent genetic contribution from the male. The offspring of a single original female constitute a clone, having identical genotypes. Clones of the genetically homogeneous Amazon molly may prove to be equally as valuable to aquatic radiobiologists as the inbred rodent lines have been to mammalian studies. In many other respects the Amazon molly ismore » a satisfactory laboratory animal. It is robust, easy to rear, and has large broods of young when fully grown. Maintenance costs are low. Details are given of the conditions under which colonies reproduce.« less
-
An efficient transgenic system by TA cloning vectors and RNAi for C. elegans
DOE Office of Scientific and Technical Information (OSTI.GOV)
Gengyo-Ando, Keiko; CREST, JST, 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012; Yoshina, Sawako
2006-11-03
In the nematode, transgenic analyses have been performed by microinjection of DNA from various sources into the syncytium gonad. To expedite these transgenic analyses, we solved two potential problems in this work. First, we constructed an efficient TA-cloning vector system which is useful for any promoter. By amplifying the genomic DNA fragments which contain regulatory sequences with or without the coding region, we could easily construct plasmids expressing fluorescent protein fusion without considering restriction sites. We could dissect motor neurons with three colors in a single animal. Second, we used feeding RNAi to isolate transgenic strains which express lag-2::venus fusionmore » gene. We found that the fusion protein is toxic when ectopically expressed in embryos but is functional to rescue a loss of function mutant in the lag-2 gene. Thus, the transgenic system described here should be useful to examine the protein function in the nematode.« less
-
ERIC Educational Resources Information Center
Kano, Kei; Yahata, Saiko; Muroi, Kaori; Kawakami, Masahiro; Tomoda, Mari; Miyaki, Koichi; Nakayama, Takeo; Kosugi, Shinji; Kato, Kazuto
2008-01-01
Genome science, including topics such as gene recombination, cloning, genetic tests, and gene therapy, is now an established part of our daily lives; thus we need to learn genome science to better equip ourselves for the present day. Learning from topics directly related to the human has been suggested to be more effective than learning from…
-
Tumor immunology viewed from alternative animal models—the Xenopus story
Banach, Maureen; Robert, Jacques
2017-01-01
a) Purpose of review Nonmammalian comparative animal models are important not only to gain fundamental evolutionary understanding of the complex interactions of tumors with the immune system, but also to better predict the applicability of novel immunotherapeutic approaches to humans. After reviewing recent advances in developing alternative models, we focus on the amphibian Xenopus laevis and its usefulness in deciphering the perplexing roles of MHC class I-like molecules and innate (i)T cells in tumor immunity. b) Recent findings Experiments using MHC-defined inbred and cloned animals, tumor cell lines, effective reagents, sequenced genomes, and adapted gene editing techniques in Xenopus, have revealed that the critical involvement of class I-like molecules and iT cells in tumor immunity has been conserved during evolution. c) Summary Comparative studies with the X. laevis tumor immunity model can contribute to the development of better and more efficient cancer immunotherapies. PMID:28944105
-
The Clock gene clone and its circadian rhythms in Pelteobagrus vachelli
NASA Astrophysics Data System (ADS)
Qin, Chuanjie; Shao, Ting
2015-05-01
The Clock gene, a key molecule in circadian systems, is widely distributed in the animal kingdom. We isolated a 936-bp partial cDNA sequence of the Clock gene ( Pva-clock) from the darkbarbel catfish Pelteobagrus vachelli that exhibited high identity with Clock genes of other species of fish and animals (65%-88%). The putative domains included a basic helix-loop-helix (bHLH) domain and two period-ARNT-single-minded (PAS) domains, which were also similar to those in other species of fish and animals. Pva-Clock was primarily expressed in the brain, and was detected in all of the peripheral tissues sampled. Additionally, the pattern of Pva-Clock expression over a 24-h period exhibited a circadian rhythm in the brain, liver and intestine, with the acrophase at zeitgeber time 21:35, 23:00, and 23:23, respectively. Our results provide insight into the function of the molecular Clock of P. vachelli.
-
2012-01-01
Background Anatomic and physiological similarities to the human make swine an excellent large animal model for human health and disease. Methods Cloning from a modified somatic cell, which can be determined in cells prior to making the animal, is the only method available for the production of targeted modifications in swine. Results Since some strains of swine are similar in size to humans, technologies that have been developed for swine can be readily adapted to humans and vice versa. Here the importance of swine as a biomedical model, current technologies to produce genetically enhanced swine, current biomedical models, and how the completion of the swine genome will promote swine as a biomedical model are discussed. Conclusions The completion of the swine genome will enhance the continued use and development of swine as models of human health, syndromes and conditions. PMID:23151353
-
Subtyping Listeria monocytogenes isolates genetically related to the Swiss epidemic clone.
Boerlin, P; Bannerman, E; Jemmi, T; Bille, J
1996-01-01
Macrorestriction analysis by pulsed-field gel electrophoresis was used to assess the diversity of strains within the epidemic-associated electrophoretic type 1 (ET1) clone of Listeria monocytogenes. For this purpose, a total of 144 isolates from Switzerland shown by multilocus enzyme electrophoresis to belong to the ET1 were examined. These isolates were subtyped by macrorestriction analysis using the enzymes ApaI and SmaI and field inversion gel electrophoresis. Among these 144 isolates, 45 were isolated in human listeriosis cases of the postepidemic period of 1988 to 1993 and 44 were isolated in animal listeriosis cases of the same period. Forty-seven isolates were from the epidemic period of 1983 to 1987, and eight additional isolates were from cattle from two different farms. Twenty-nine different subtypes could be identified among the 144 isolates tested. Five major subtypes were found more frequently than the others during the postepidemic period, both in humans and in animals. Two of these subtypes had been previously implicated in outbreaks of listeriosis, thus suggesting that particular pulsed-field gel electrophoresis subtypes may be frequently associated with disease in humans and animals. Two of these frequent subtypes were also suspected to be related to small clusters of listeriosis cases during the postepidemic period. The results obtained by typing epidemiologically related isolates from different animals within the same farms and from different body sites of a given patient confirmed the potential of macrorestriction analysis for epidemiological studies restricted to short periods of time and to small number of isolates. The analysis of 47 isolates related to the Swiss listeriosis epidemic period of 1983 to 1987 and the use of Southern blotting and hybridization experiments show that the interpretation of relatedness between isolates presenting slightly different macrorestriction patterns may be more complex than commonly accepted. In such cases, careful interpretation of the potential molecular mechanisms leading to the differences observed between patterns is necessary. PMID:8862575
-
Pseudomonas aeruginosa Population Structure Revisited
Pirnay, Jean-Paul; Bilocq, Florence; Pot, Bruno; Cornelis, Pierre; Zizi, Martin; Van Eldere, Johan; Deschaght, Pieter; Vaneechoutte, Mario; Jennes, Serge; Pitt, Tyrone; De Vos, Daniel
2009-01-01
At present there are strong indications that Pseudomonas aeruginosa exhibits an epidemic population structure; clinical isolates are indistinguishable from environmental isolates, and they do not exhibit a specific (disease) habitat selection. However, some important issues, such as the worldwide emergence of highly transmissible P. aeruginosa clones among cystic fibrosis (CF) patients and the spread and persistence of multidrug resistant (MDR) strains in hospital wards with high antibiotic pressure, remain contentious. To further investigate the population structure of P. aeruginosa, eight parameters were analyzed and combined for 328 unrelated isolates, collected over the last 125 years from 69 localities in 30 countries on five continents, from diverse clinical (human and animal) and environmental habitats. The analysed parameters were: i) O serotype, ii) Fluorescent Amplified-Fragment Length Polymorphism (FALFP) pattern, nucleotide sequences of outer membrane protein genes, iii) oprI, iv) oprL, v) oprD, vi) pyoverdine receptor gene profile (fpvA type and fpvB prevalence), and prevalence of vii) exoenzyme genes exoS and exoU and viii) group I pilin glycosyltransferase gene tfpO. These traits were combined and analysed using biological data analysis software and visualized in the form of a minimum spanning tree (MST). We revealed a network of relationships between all analyzed parameters and non-congruence between experiments. At the same time we observed several conserved clones, characterized by an almost identical data set. These observations confirm the nonclonal epidemic population structure of P. aeruginosa, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise. One of these clones is the renown and widespread MDR serotype O12 clone. On the other hand, we found no evidence for a widespread CF transmissible clone. All but one of the 43 analysed CF strains belonged to a ubiquitous P. aeruginosa “core lineage” and typically exhibited the exoS +/exoU − genotype and group B oprL and oprD alleles. This is to our knowledge the first report of an MST analysis conducted on a polyphasic data set. PMID:19936230
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Aronow, Bruce J.
2002-04-19
This project was based on preliminary data that was published by Dr. Boothman (Yang et al. 2000) which indicated a strong induction of apoJ gene expression, increased secretion of the protein, and accumulation of an apparently somewhat different form of the apoJ protein in the nucleus of MCF-7 breast carcinoma cells undergoing response to DNA damage. A clone expressing apoJ protein was isolated that was capable of interacting with Ku80, a component of the double strand break repair complex that is essential for the successful repair of rearranging immunoglobulin and T-cell receptor genes as evidenced by failure to produce maturemore » B and T cells in the absence of Ku70. ApoJ clones isolated and characterized by Dr. Boothman bound strongly to a Ku-70 ''bait'' protein. Over-expression of these same clones in a cell line was capable of killing the cell. ApoJ is very strongly induced in many instances of programmed cell death and has been proposed repeatedly to play some sort of effector role in the process. Our principle hypothesis for this study was that the strong induction of the apoJ gene and the particular expression of a nuclear form of the protein was potentially a causal factor in the decision point made by the cell as it attempts to repair double-strand breakage based DNA damage. The hypothesis was that if sufficiently high damage occurred, it would be deleterious to maintain the cell's viability through continued DNA repair. One method to inhibit DNA repair might be by inhibiting proteins such as Ku-70 that are necessary for double-strand break repair. If apoJ does play a critical role in tipping the decision balance over to cell death, we reasoned that deficiency of apoJ would cause increased accumulation of cells with DNA damage and that this might decrease cell death in response to DNA damage and increase tumor occurrence rates. To test this hypothesis and its potential implications, we exposed wildtype and apoJ deficient animals that we constructed through gene targeting to increasing levels of ionizing radiation from a Cesium source. Data gathered under the support of this grant application initially indicated that apoJ deficient animals were more resistant to radiation, but as we accumulated more and more data points and covered a tighter exposure range, the genotype-based differences became insignificant. However, the possibility existed that because mortality based radiation-resistance could be attributable to mechanism for which nuclear apoJ was not rate determining, we maintained a very large of colony of apoJ knockout and wildtype animals in both the C57/B16 and Cv129 strain backgrounds that were exposed to sub-lethal levels of ionizing radiation to monitor for the occurrence of tumors. These animals were allowed to fully recover and age normally in either germ free or normal animal housing. Our results demonstrated no significant differences between wildtype and apoJ knockout animals over a period that extended up to 30 months for individual animals. We recorded similar weight gain, a relatively low mortality rate, and a similar mixture and rate of sarcoma and adenocarcinomas after surviving the initial ionizing radiation exposures. Thus we conclude that apoJ gene function, which was totally eliminated by our gene targeting, did not influence radiation sensitivity or serve as a tumor suppressor in response to DNA damage.« less
-
Cherney, D J R
2004-03-01
There are scientists who believe that science is value-free and that social and ethical issues are not their concern. The birth of Dolly, the cloned lamb, greatly increased public and scientific awareness of ethical issues raised by molecular biology as they intersect with human experience. There are many other issues involving animal production systems, including animal welfare, rural community issues, and environmental concerns. Last year Germany became the first European nation to grant animals a constitutional right. Several European nations ban the use of traditional battery cages for laying hens and gestation crates for sows. In the US, 37 states have recently passed animal anticruelty laws. Times are changing, and if animal production systems are to be part of the future, animal scientists must join with society to solve these ethical issues. The Western Coordinating Committee-204 (WCC-204), Animal Bioethics, has as its goals to 1) create a forum in which animal scientists and nonanimal scientists may work together to examine and discuss contentious social issues, 2) provide a means of encouraging the development of research projects dealing with bioethics of the animal sciences, 3) develop mechanisms of outreach that would allow animal scientists to respond directly to consumers and critics, and 4) provide the means for ongoing critical analysis of the animal science professions in the context of their ability to address moral and sociopolitical issues. Animal scientists can no longer ignore social ethics, and by realizing the goals of Western Coordinating Committee-204, we can help maintain the future of animal production systems.
-
Lei, Tao; Tian, Wei; He, Liu; Huang, Xian-Hui; Sun, Yong-Xue; Deng, Yu-Ting; Sun, Yan; Lv, Dian-Hong; Wu, Cong-Ming; Huang, Liang-Zong; Shen, Jian-Zhong; Liu, Jian-Hua
2010-11-20
One thousand and thirty Escherichia coli isolates from food animals, animals-derived foods, and companion animals between 2007 and 2008 in Southern China were used to investigate their antimicrobial susceptibility to 14 different antimicrobials by the standard agar dilution method. More than 70% of isolates showed resistance to tetracycline, trimethoprim-sulphamethoxazole, nalidixic acid, and ampicillin. In general, resistance was less frequent in companion animal isolates vs food animals isolates, but cephalosporin and amikacin resistance was more frequent in companion animal isolates, 42.6% to 56.2% vs 14.1% to 24.3%, and 28.5% vs 18.8%, respectively, which was most likely due to the common use of these antimicrobials as treatment in pet animals. Fluoroquinolones resistance was high in all animal isolates (>50%). Food products showed lowest resistance among isolates from these three resources. PFGE analysis indicated that a majority of multidrug-resistant E. coli isolates showed unique, unrelated PFGE profiles and were unlikely to be the spread of a specific clone. This study provides useful information about the prevalence of antimicrobial resistance in E. coli isolated from animals and food products in China and provided evidence of the linkage of the use of antimicrobials in animals and its selection of antimicrobial resistance in bacterial isolates. The data from this study further warns the prudent use of antimicrobials in food and pet animals to reduce the risks of transmission of antimicrobial resistance zoonotic pathogen to humans. Copyright © 2010 Elsevier B.V. All rights reserved.
-
Chi, Xiangyang; Li, Jianmin; Liu, Weicen; Wang, Xiaolin; Yin, Kexin; Liu, Ju; Zai, Xiaodong; Li, Liangliang; Song, Xiaohong; Zhang, Jun; Zhang, Xiaopeng; Yin, Ying; Fu, Ling; Xu, Junjie; Yu, Changming; Chen, Wei
2015-05-01
The anthrax protective antigen (PA) is the central component of the three-part anthrax toxin, and it is the primary immunogenic component in the approved AVA anthrax vaccine and the "next-generation" recombinant PA (rPA) anthrax vaccines. Animal models have indicated that PA-specific antibodies (AB) are sufficient to protect against infection with Bacillus anthracis. In this study, we investigated the PA domain specificity, affinity, mechanisms of neutralization, and synergistic effects of PA-specific antibodies from a single donor following vaccination with the rPA vaccine. Antibody-secreting cells were isolated 7 days after the donor received a boost vaccination, and 34 fully human monoclonal antibodies (hMAb) were identified. Clones 8H6, 4A3, and 22F1 were able to neutralize lethal toxin (LeTx) both in vitro and in vivo. Clone 8H6 neutralized LeTx by preventing furin cleavage of PA in a dose-dependent manner. Clone 4A3 enhanced degradation of nicked PA, thereby interfering with PA oligomerization. The mechanism of 22F1 is still unclear. A fourth clone, 2A6, that was protective only in vitro was found to be neutralizing in vivo in combination with a toxin-enhancing antibody, 8A7, which binds to domain 3 of PA and PA oligomers. These results provide novel insights into the antibody response elicited by the rPA vaccine and may be useful for PA-based vaccine and immunotherapeutic cocktail design. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
-
A physical map of the bovine genome
Snelling, Warren M; Chiu, Readman; Schein, Jacqueline E; Hobbs, Matthew; Abbey, Colette A; Adelson, David L; Aerts, Jan; Bennett, Gary L; Bosdet, Ian E; Boussaha, Mekki; Brauning, Rudiger; Caetano, Alexandre R; Costa, Marcos M; Crawford, Allan M; Dalrymple, Brian P; Eggen, André; Everts-van der Wind, Annelie; Floriot, Sandrine; Gautier, Mathieu; Gill, Clare A; Green, Ronnie D; Holt, Robert; Jann, Oliver; Jones, Steven JM; Kappes, Steven M; Keele, John W; de Jong, Pieter J; Larkin, Denis M; Lewin, Harris A; McEwan, John C; McKay, Stephanie; Marra, Marco A; Mathewson, Carrie A; Matukumalli, Lakshmi K; Moore, Stephen S; Murdoch, Brenda; Nicholas, Frank W; Osoegawa, Kazutoyo; Roy, Alice; Salih, Hanni; Schibler, Laurent; Schnabel, Robert D; Silveri, Licia; Skow, Loren C; Smith, Timothy PL; Sonstegard, Tad S; Taylor, Jeremy F; Tellam, Ross; Van Tassell, Curtis P; Williams, John L; Womack, James E; Wye, Natasja H; Yang, George; Zhao, Shaying
2007-01-01
Background Cattle are important agriculturally and relevant as a model organism. Previously described genetic and radiation hybrid (RH) maps of the bovine genome have been used to identify genomic regions and genes affecting specific traits. Application of these maps to identify influential genetic polymorphisms will be enhanced by integration with each other and with bacterial artificial chromosome (BAC) libraries. The BAC libraries and clone maps are essential for the hybrid clone-by-clone/whole-genome shotgun sequencing approach taken by the bovine genome sequencing project. Results A bovine BAC map was constructed with HindIII restriction digest fragments of 290,797 BAC clones from animals of three different breeds. Comparative mapping of 422,522 BAC end sequences assisted with BAC map ordering and assembly. Genotypes and pedigree from two genetic maps and marker scores from three whole-genome RH panels were consolidated on a 17,254-marker composite map. Sequence similarity allowed integrating the BAC and composite maps with the bovine draft assembly (Btau3.1), establishing a comprehensive resource describing the bovine genome. Agreement between the marker and BAC maps and the draft assembly is high, although discrepancies exist. The composite and BAC maps are more similar than either is to the draft assembly. Conclusion Further refinement of the maps and greater integration into the genome assembly process may contribute to a high quality assembly. The maps provide resources to associate phenotypic variation with underlying genomic variation, and are crucial resources for understanding the biology underpinning this important ruminant species so closely associated with humans. PMID:17697342
-
Uchiyama, Jumpei; Suzuki, Masato; Nishifuji, Koji; Kato, Shin-Ichiro; Miyata, Reina; Nasukawa, Tadahiro; Yamaguchi, Kotoe; Takemura-Uchiyama, Iyo; Ujihara, Takako; Shimakura, Hidekatsu; Murakami, Hironobu; Okamoto, Noriaki; Sakaguchi, Yoshihiko; Shibayama, Keigo; Sakaguchi, Masahiro; Matsuzaki, Shigenobu
2016-08-01
Pseudomonas aeruginosa causes serious intractable infections in humans and animals. Bacteriophage (phage) therapy has been applied to treat P. aeruginosa infections, and phages belonging to the PB1-like virus genus in the Myoviridae family have been used as therapeutic phages. To achieve safer and more effective phage therapy, the use of preadapted phages is proposed. To understand in detail such phage preadaptation, the short-term antagonistic evolution of bacteria and phages should be studied. In this study, the short-term antagonistic evolution of bacteria and PB1-like phage was examined by studying phage-resistant clones of P. aeruginosa strain PAO1 and mutant PB1-like phages that had recovered their infectivity. First, phage KPP22 was isolated and characterized; it was classified as belonging to the PB1-like virus genus in the Myoviridae family. Subsequently, three KPP22-resistant PAO1 clones and three KPP22 mutant phages capable of infecting these clones were isolated in three sets of in vitro experiments. It was shown that the bacterial resistance to phage KPP22 was caused by significant decreases in phage adsorption and that the improved infectivity of KPP22 mutant phages was caused by significant increases in phage adsorption. The KPP22-resistant PAO1 clones and the KPP22 mutant phages were then analyzed genetically. All three KPP22-resistant PAO1 clones, which were deficient for the O5 antigen, had a common nonsense mutation in the wzy gene. All the KPP22 mutant phage genomes showed the same four missense mutations in the open reading frames orf060, orf065, and orf086 The information obtained in this study should be useful for further development of safe and efficient phage therapy. Pseudomonas aeruginosa causes serious intractable infections in humans and animals; bacteriophage (phage) therapy has been utilized to treat P. aeruginosa infections, and phages that belong to the PB1-like virus genus in the family Myoviridae have been used as therapeutic phages. The preadapted phage is trained in advance through the antagonistic evolution of bacteria and phage and is proposed to be used to achieve safer and more effective phage therapy. In this study, to understand the phage preadaptation, the in vitro short-term antagonistic evolution was studied using P. aeruginosa strain PAO1 and the newly isolated PB1-like phage KPP22. Phage KPP22 was characterized, and the molecular framework regarding the phage preadaptation of KPP22 was elucidated. The importance of study of antagonistic evolution of bacteria and phage in phage therapy is discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
-
Molecular analysis of methanogenic archaea in the forestomach of the alpaca (Vicugna pacos)
2012-01-01
Background Methanogens that populate the gastrointestinal tract of livestock ruminants contribute significantly to methane emissions from the agriculture industry. There is a great need to analyze archaeal microbiomes from a broad range of host species in order to establish causal relationships between the structure of methanogen communities and their potential for methane emission. In this report, we present an investigation of methanogenic archaeal populations in the foregut of alpacas. Results We constructed individual 16S rRNA gene clone libraries from five sampled animals and recovered a total of 947 sequences which were assigned to 51 species-level OTUs. Individuals were found to each have between 21 and 27 OTUs, of which two to six OTUs were unique. As reported in other host species, Methanobrevibacter was the dominant genus in the alpaca, representing 88.3% of clones. However, the alpaca archaeal microbiome was different from other reported host species, as clones showing species-level identity to Methanobrevibacter millerae were the most abundant. Conclusion From our analysis, we propose a model to describe the population structure of Methanobrevibacter-related methanogens in the alpaca and in previously reported host species, which may contribute in unraveling the complexity of symbiotic archaeal communities in herbivores. PMID:22221383
-
Kin Discrimination in Protists: From Many Cells to Single Cells and Backwards.
Paz-Y-Miño-C, Guillermo; Espinosa, Avelina
2016-05-01
During four decades (1960-1990s), the conceptualization and experimental design of studies in kin recognition relied on work with multicellular eukaryotes, particularly Unikonta (including invertebrates and vertebrates) and some Bikonta (including plants). This pioneering research had an animal behavior approach. During the 2000s, work on taxa-, clone- and kin-discrimination and recognition in protists produced genetic and molecular evidence that unicellular organisms (e.g. Saccharomyces, Dictyostelium, Polysphondylium, Tetrahymena, Entamoeba and Plasmodium) could distinguish between same (self or clone) and different (diverse clones), as well as among conspecifics of close or distant genetic relatedness. Here, we discuss some of the research on the genetics of kin discrimination/recognition and highlight the scientific progress made by switching emphasis from investigating multicellular to unicellular systems (and backwards). We document how studies with protists are helping us to understand the microscopic, cellular origins and evolution of the mechanisms of kin discrimination/recognition and their significance for the advent of multicellularity. We emphasize that because protists are among the most ancient organisms on Earth, belong to multiple taxonomic groups and occupy all environments, they can be central to reexamining traditional hypotheses in the field of kin recognition, reformulating concepts, and generating new knowledge. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.
-
Kin Discrimination in Protists: From Many Cells to Single Cells and Backwards1
Paz-y-Miño-C, Guillermo; Espinosa, Avelina
2016-01-01
During four decades (1960s to 1990s), the conceptualization and experimental design of studies in kin recognition relied on work with multicellular eukaryotes, particularly Unikonta (including invertebrates and vertebrates) and some Bikonta (including plants). This pioneering research had an animal behavior approach. During the 2000s, work on taxa-, clone- and kin-discrimination and recognition in protists produced genetic and molecular evidence that unicellular organisms (e.g. Saccharomyces, Dictyostelium, Polysphondylium, Tetrahymena, Entamoeba and Plasmodium) could distinguish between same (self or clone) and different (diverse clones), as well as among conspecifics of close or distant genetic relatedness. Here we discuss some of the research on the genetics of kin discrimination/recognition and highlight the scientific progress made by switching emphasis from investigating multicellular to unicellular systems (and backwards). We document how studies with protists are helping us to understand the microscopic, cellular origins and evolution of the mechanisms of kin discrimination/recognition and their significance for the advent of multicellularity. We emphasize that because protists are among the most ancient organisms on Earth, belong to multiple taxonomic groups and occupy all environments, they can be central to reexamining traditional hypotheses in the field of kin recognition, reformulating concepts, and generating new knowledge. PMID:26873616
-
Oliveira, R.J.; Mantovani, M.S.; da Silva, A.F.; Pesarini, J.R.; Mauro, M.O.; Ribeiro, L.R.
2014-01-01
The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero. PMID:24714812
-
Oliveira, R J; Mantovani, M S; Silva, A F da; Pesarini, J R; Mauro, M O; Ribeiro, L R
2014-04-01
The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.
-
Himaki, Takehiro; Mizobe, Yamato; Tsuda, Kenichirou; Suetomo, Masashi; Yamakuchi, Hiroshi; Miyoshi, Kazuchika; Takao, Sonshin; Yoshida, Mitsutoshi
2012-01-01
The objective of this study was to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerization inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from kidney fibroblasts of an aged Clawn miniature boar (12 years old). After electric activation, SCNT embryos were treated with 0, 0.5 or 1 μM LatA and cultured in vitro. The rate of blastocyst formation was significantly higher (P<0.05) in SCNT embryos treated with 0.5 μM LatA (38%) than those in control (14%). When cloned embryos treated with 0.5 μM LatA were transferred into the oviducts of two recipient miniature gilts to assess their development in vivo, both recipients became pregnant; one maintained pregnancy to term, and a live piglet (weighing 220 g) was delivered by Caesarean section. The results of this study indicated that the postactivation treatment with LatA was effective in improving in vitro developmental capacity of SCNT miniature pig embryos derived from kidney fibroblasts of an aged animal and that miniature pig cloned embryos treated with LatA had the ability to develop to term.
-
Cho, Su-Jin; Lee, Young S; Lee, Jae-Ik; Bang, Jae-Il; Yang, Jing; Klassen, Henry; Kong, Il-Keun
2010-12-01
The production of transgenic animals is highly desirable for biotechnology and basic research. We investigated the reproductive ability of a red fluorescence protein (RFP) transgenic cloned male cat (RFP TG cat) by natural mating with a domestic female cat. The RFP expression levels were examined in early embryogenesis, tissues from 45-day-old two fetuses, and four RFP TG cat offspring. The RFP gene was detected in tissue samples from one dead kitten, including several organs and the skin. Also, under a fluorescent light source, we were able to directly detect the RFP expression of in in vitro-produced blastocysts derived with sperm from the RFP TG cat. These results indicate that the RFP TG cat exhibits normal reproductive fertility, stable germ-line transmission of the RFP transgene, and characteristic RFP expression in its offspring. We isolated feline neural progenitor cells from a 45-day-old fetus derived from the natural mating of the RFP TG cat with a domestic female cat. Isolated brain and retinal progenitor cells were successfully passaged at least four times post isolation (day 23), and showed a high RFP expression level. This method of producing genetically modified cloned cats will be important for generating biomedical models of human diseases.
-
Stable long-term blood formation by stem cells in murine steady-state hematopoiesis.
Zavidij, Oksana; Ball, Claudia R; Herbst, Friederike; Oppel, Felix; Fessler, Sylvia; Schmidt, Manfred; von Kalle, Christof; Glimm, Hanno
2012-09-01
Hematopoietic stem cells (HSCs) generate all mature blood cells during the whole lifespan of an individual. However, the clonal contribution of individual HSC and progenitor cells in steady-state hematopoiesis is poorly understood. To investigate the activity of HSCs under steady-state conditions, murine HSC and progenitor cells were genetically marked in vivo by integrating lentiviral vectors (LVs) encoding green fluorescent protein (GFP). Hematopoietic contribution of individual marked clones was monitored by determination of lentiviral integration sites using highly sensitive linear amplification-mediated-polymerase chain reaction. A remarkably stable small proportion of hematopoietic cells expressed GFP in LV-injected animals for up to 24 months, indicating stable marking of murine steady-state hematopoiesis. Analysis of the lentiviral integration sites revealed that multiple hematopoietic clones with both myeloid and lymphoid differentiation potential contributed to long-term hematopoiesis. In contrast to intrafemoral vector injection, intravenous administration of LV preferentially targeted short-lived progenitor cells. Myelosuppressive treatment of mice prior to LV-injection did not affect the marking efficiency. Our study represents the first continuous analysis of clonal behavior of genetically marked hematopoietic cells in an unmanipulated system, providing evidence that multiple clones are simultaneously active in murine steady-state hematopoiesis. Copyright © 2012 AlphaMed Press.
-
Clone and genomic repositories at the American Type Culture Collection
DOE Office of Scientific and Technical Information (OSTI.GOV)
Maglott, D.R.; Nierman, W.C.
1990-01-01
The American Type Culture Collection (ATCC) has a long history of characterizing, preserving, and distributing biological resource materials for the scientific community. Starting in 1925 as a repository for standard bacterial and fungal strains, its collections have diversified with technologic advances and in response to the requirements of its users. To serve the needs of the human genetics community, the National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), established an international Repository of Human DNA Probes and Libraries at the ATCC in 1985. This repository expanded the existing collections of recombinant clones and librariesmore » at the ATCC, with the specific purposes of (1) obtaining, amplifying, and distribution probes detecting restriction fragment length polymorphisms (RFLPs); (2) obtaining, amplifying, and distributing genomic and cDNA clones from known genes independent of RFLP detection; (3) distributing the chromosome-specific libraries generated by the National Laboratory Gene Library Project at the Lawrence Livermore and Los Alamos National Laboratories and (4) maintaining a public, online database describing the repository materials. Because it was recognized that animal models and comparative mapping can be crucial to genomic characterization, the scope of the repository was broadened in February 1989 to include probes from the mouse genome.« less
-
Isolation and characterisation of mesenchymal stem/stromal cells in the ovine endometrium.
Letouzey, Vincent; Tan, Ker Sin; Deane, James A; Ulrich, Daniela; Gurung, Shanti; Ong, Y Rue; Gargett, Caroline E
2015-01-01
Mesenchymal stem/stromal cells (MSC) were recently discovered in the human endometrium. These cells possess key stem cell properties and show promising results in small animal models when used for preclinical tissue engineering studies. A small number of surface markers have been identified that enrich for MSC from bone marrow and human endometrium, including the Sushi Domain-containing 2 (SUSD2; W5C5) and CD271 markers. In preparation for developing a large animal preclinical model for urological and gynecological tissue engineering applications we aimed to identify and characterise MSC in ovine endometrium and determine surface markers to enable their prospective isolation. Ovine endometrium was obtained from hysterectomised ewes following progesterone synchronisation, dissociated into single cell suspensions and tested for MSC surface markers and key stem cell properties. Purified stromal cells were obtained by flow cytometry sorting with CD49f and CD45 to remove epithelial cells and leukocytes respectively, and MSC properties investigated. There was a small population CD271+ stromal cells (4.5 ± 2.3%) in the ovine endometrium. Double labelling with CD271 and CD49f showed that the sorted CD271+CD49f- stromal cell population possessed significantly higher cloning efficiency, serial cloning capacity and a qualitative increased ability to differentiate into 4 mesodermal lineages (adipocytic, smooth muscle, chondrocytic and osteoblastic) than CD271-CD49f- cells. Immunolabelling studies identified an adventitial perivascular location for ovine endometrial CD271+ cells. This is the first study to characterise MSC in the ovine endometrium and identify a surface marker profile identifying their location and enabling their prospective isolation. This knowledge will allow future preclinical studies with a large animal model that is well established for pelvic organ prolapse research.
-
Cloning and pharmacological characterization of the rabbit bradykinin B2 receptor.
Bachvarov, D R; Saint-Jacques, E; Larrivée, J F; Levesque, L; Rioux, F; Drapeau, G; Marceau, F
1995-12-01
Degenerate primers, corresponding to consensus sequences of third and sixth transmembrane domains of G protein-coupled receptor superfamily, were used for the polymerase chain reaction amplification and consecutive characterization of G protein-coupled receptors present in cultured rabbit aortic smooth muscle cells. One of the isolated resulting fragments was highly homologous to the corresponding region of the bradykinin (BK) B2 receptor cloned in other species. The polymerase chain reaction fragment was used to screen a rabbit genomic library, which allowed the identification of an intronless 1101-nucleotide open reading frame which codes for a 367-amino acid receptor protein. The rabbit B2 receptor sequence is more than 80% identical to the ones determined in three other species and retain putative glycosylation, palmitoylation and phosphorylation sites. In the rabbit genomic sequence, an acceptor splice sequence was found 8 base pairs upstream of the start codon. Northern blot analysis showed a high expression of a major transcript (4.2 kilobases) in the rabbit kidney and duodenum, and a less abundant expression in other tissues. Southern blot experiments suggest that a single copy of this gene exists in the rabbit genome. The cloned rabbit B2 receptor expressed in COS-1 cells binds [3H]BK in a saturable manner (KD 2.1 nM) and this ligand competes with a series of kinin agonists and antagonist with a rank order consistent with the B2 receptor identity. The insurmountable character of the antagonism exerted by Hoe 140 against BK on the rabbit B2 receptor, previously shown in pharmacological experiments, was confirmed in binding experiments with the cloned receptor expressed in a controlled manner. By contrast, Hoe 140 competed with [3H]BK in a surmountable manner for the human B2 receptor expressed in COS-1 cells. The cloning of the rabbit B2 receptor will be useful notably for the study of the structural basis of antagonist binding and for studies on receptor regulation in a relatively large animal.
-
Liang, Yuying; Lan, Shuiyun; Ly, Hinh
2009-09-01
Arenaviruses are enveloped single-strand RNA viruses that mostly have natural hosts in rodents. Upon infection of humans, several arenaviruses can cause severe hemorrhagic fever diseases, including Lassa fever that is endemic in West Africa. The virulence mechanism of these deadly arenaviruses can be studied in a safe and economical small animal model-guinea pigs infected by a nonpathogenic arenavirus Pichinde virus (PICV), a virulent strain of which can cause similar disease syndromes in guinea pigs as arenaviral hemorrhagic fevers in humans. We have recently developed molecular clones for both the virulent and avirulent strains of PICV. Using the available reverse genetics tools, we are characterizing the molecular determinants of virulent arenavirus infections in vivo.
-
Isolation of a peptide from Ph.D.-C7C phage display library for detection of Cry1Ab.
Wang, Yun; Wang, Qian; Wu, Ai-Hua; Hao, Zhen-Ping; Liu, Xian-Jin
2017-12-15
Traditional ELISA methods of using animal immunity yield antibodies for detection Cry toxin. Not only is this incredibly harmful to the animals, but is also time-intensive. Here we developed a simple method to yield the recognition element. Using a critical selection strategy and immunoassay we confirmed a clone from the Ph.D-C7C phage library, which has displayed the most interesting Cry1Ab-binding characteristics examined in this study (Fig. 1). The current study indicates that isolating peptide is an alternative method for the preparation of a recognition element, and that the developed assay is a potentially useful tool for detecting Cry1Ab. Copyright © 2017. Published by Elsevier Inc.
-
Methicillin-resistant Staphylococcus aureus in a neonatal alpaca
Stull, Jason W.; Kenney, Daniel G.; Slavić, Durda; Weese, J. Scott
2012-01-01
A 6-hour-old alpaca was presented for evaluation of respiratory difficulty. As part of routine surveillance, methicillin-resistant Staphylococcus aureus (MRSA) was identified from a nasal swab taken upon admission to the hospital. No signs of MRSA infection were noted. The MRSA strain recovered was a human epidemic clone that has been associated with horses. Methicillin-resistant S. aureus colonization can occur in camelids, and the potential animal and public health risks require consideration. PMID:23204589
-
Reddy, B A; Kloc, M; Etkin, L D
1992-12-01
We have cloned a cDNA (xlan4) from a Xenopus laevis oocyte cDNA library whose cognate mRNA is localized in the animal pole region of full grown oocytes. The cDNA can be translated in vitro to produce a predicted size protein of 35 kDa and, is also expressed in E. coli as a fusion protein. The conceptual protein encoded by the xlan4 cDNA is 17.5% proline rich and possesses several PEST sequences found in proteins with short half-lives. The xlan4 mRNA is 2.6 kb and during early development its titer decreases until the neurula stage after which it begins to reaccumulate. Northern blots on dissected embryos and in situ hybridization revealed that the zygotic expression is limited to the dorsal axial structures consisting primarily of the CNS. UV irradiation of the vegetal pole region immediately following fertilization that produces ventralized embryos results in a loss of zygotic xlan4 expression. In the adult, xlan4 mRNA is limited primarily to the brain. The presence of this mRNA in animal pole region which contributes to the future neural cell lineages suggests that this gene product may function either in the specification of neural cell types or in a neural specific function.
-
Islet-Derived CD4 T Cells Targeting Proinsulin in Human Autoimmune Diabetes
Michels, Aaron W.; Landry, Laurie G.; McDaniel, Kristen A.; Yu, Liping; Campbell-Thompson, Martha; Kwok, William W.; Jones, Kenneth L.; Gottlieb, Peter A.; Kappler, John W.; Tang, Qizhi; Roep, Bart O.; Atkinson, Mark A.; Mathews, Clayton E.
2017-01-01
Type 1 diabetes results from chronic autoimmune destruction of insulin-producing β-cells within pancreatic islets. Although insulin is a critical self-antigen in animal models of autoimmune diabetes, due to extremely limited access to pancreas samples, little is known about human antigenic targets for islet-infiltrating T cells. Here we show that proinsulin peptides are targeted by islet-infiltrating T cells from patients with type 1 diabetes. We identified hundreds of T cells from inflamed pancreatic islets of three young organ donors with type 1 diabetes with a short disease duration with high-risk HLA genes using a direct T-cell receptor (TCR) sequencing approach without long-term cell culture. Among 85 selected CD4 TCRs tested for reactivity to preproinsulin peptides presented by diabetes-susceptible HLA-DQ and HLA-DR molecules, one T cell recognized C-peptide amino acids 19–35, and two clones from separate donors responded to insulin B-chain amino acids 9–23 (B:9–23), which are known to be a critical self-antigen–driving disease progress in animal models of autoimmune diabetes. These B:9–23–specific T cells from islets responded to whole proinsulin and islets, whereas previously identified B:9–23 responsive clones from peripheral blood did not, highlighting the importance of proinsulin-specific T cells in the islet microenvironment. PMID:27920090
-
Kittelmann, Sandra; Janssen, Peter H
2011-03-01
The structure and variability of ciliate protozoal communities in the rumens of domestic New Zealand ruminants feeding on different diets was investigated. The relative abundance of ciliates compared with bacteria was similar across all samples. However, molecular fingerprinting of communities showed ruminant-specific differences in species composition. Community compositions of cattle were significantly influenced by diet. In contrast, diet effects in deer and sheep were weaker than the animal-to-animal variation. Cloning and sequencing of almost-full-length 18S rRNA genes from representative samples revealed that New Zealand ruminants were colonized by at least nine genera of ciliates and allowed the assignment of samples to two distinct community types. Cattle contained A-type communities, with most sequences closely related to those of the genera Polyplastron and Ostracodinium. Deer and sheep (with one exception) harboured B-type communities, with the majority of sequences belonging to the genera Epidinium and Eudiplodinium. It has been suggested that species composition of ciliate communities may impact methane formation in ruminants, with the B-type producing more methane. Therefore, manipulation of ciliate communities may be a means of mitigating methane emissions from grazing sheep and deer in New Zealand. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
-
After Dolly--ethical limits to the use of biotechnology on farm animals.
Lassen, Jesper; Gjerris, Mickey; Sandøe, Peter
2006-03-15
The cloning of Dolly the sheep gave rise to a widespread call for limits on interference with life. Until recently, the main limits were technical: what it is possible to do. Now scientists are faced with ethical limits as well: what it is acceptable to do. In this context, we take ethics to involve systematic and rational reflection on moral issues raised in the public sphere. The concerns of the general public are not necessarily valid, but they are the best point of departure if the discussion is to lead to a socially robust framework for setting limits to the use of animal biotechnology. To assess public understanding, we examine two sources of data: Eurobarometer surveys from 1991 to 2002 and a qualitative interview study carried out in Denmark in 2000. Based on these sources, we formulate, and then discuss closely, the following concerns: dangers to human health and the environment, animal welfare, animal integrity, and usefulness. In the final part of the article, it is proposed that a principle of proportionality should be the foundation for socially robust applications of animal biotechnology. Only in cases where the usefulness of the technology can be said to outweigh countervailing moral concerns, as in biomedical research, will applications of animal biotechnology stand up to scrutiny in the public sphere.
-
NASA Astrophysics Data System (ADS)
David, Peter H.; Hommel, Marcel; Miller, Louis H.; Udeinya, Iroka J.; Oligino, Lynette D.
1983-08-01
Sequestration, the adherence of infected erythrocytes containing late developmental stages of the parasite (trophozoites and schizonts) to the endothelium of capillaries and venules, is characteristic of Plasmodium falciparum infections. We have studied two host factors, the spleen and antibody, that influence sequestration of P. falciparum in the squirrel monkey. Sequestration of trophozoite/schizont-infected erythrocytes that occurs in intact animals is reduced in splenectomized animals; in vitro, when infected blood is incubated with monolayers of human melanoma cells, trophozoite/schizont-infected erythrocytes from intact animals but not from splenectomized animals bind to the melanoma cells. The switch in cytoadherence characteristics of the infected erythrocytes from nonbinding to binding occurs with a cloned parasite. Immune serum can inhibit and reverse in vitro binding to melanoma cells of infected erythrocytes from intact animals. Similarly, antibody can reverse in vivo sequestration as shown by the appearance of trophozoite/schizont-infected erythrocytes in the peripheral blood of an intact animal after inoculation with immune serum. These results indicate that the spleen modulates the expression of parasite alterations of the infected erythrocyte membrane responsible for sequestration and suggest that the prevention and reversal of sequestration could be one of the effector mechanisms involved in antibody-mediated protection against P. falciparum malaria.
-
Molecular identification of trypanosomatids in wild animals.
Tenório, M S; Oliveira e Sousa, L; Alves-Martin, M F; Paixão, M S; Rodrigues, M V; Starke-Buzetti, W A; Araújo Junior, J P; Lucheis, S B
2014-06-16
Diverse wild animal species can be reservoirs of zoonotic flagellate parasites, which can cause pathologic Chagas disease. The present study aimed to detect the natural occurrence of flagellate parasites through direct microscopic examination of the parasites in blood samples and through PCR of whole blood and blood culture (haemoculture) samples from 38 captive and 65 free-living wild animals in the Centre for Conservation of Wild Fauna (CCWF), an area endemic for leishmaniasis. For this study, PCR was accomplished using primers for the ribosomal region (ITS-1) of the flagellate parasites. The amplified fragments were cloned and sequenced to identify DNA of the Trypanosomatid parasite species, observed in blood cultures from 3.9% (04/103) of the animals. Through these techniques, Trypanosoma cruzi was identified in haemoculture samples of the following three free-living species: common agouti (Dasyprocta aguti), white-eared opossum (Didelphis albiventris), and nine-banded armadillo (Dasypus novemcinctus). Furthermore, Trypanosoma minasense was identified in whole blood samples from 01 (0.9%) captive animal (black howler monkey-Alouatta caraya). These results demonstrated the first report of T. cruzi isolation in wild species from the CCWF using blood culture, which can be applied in addition to molecular tools for epidemiological studies and to identify trypanosomatids in wild animals. Copyright © 2014 Elsevier B.V. All rights reserved.
-
Analysis of the Macaca mulatta transcriptome and the sequence divergence between Macaca and human.
Magness, Charles L; Fellin, P Campion; Thomas, Matthew J; Korth, Marcus J; Agy, Michael B; Proll, Sean C; Fitzgibbon, Matthew; Scherer, Christina A; Miner, Douglas G; Katze, Michael G; Iadonato, Shawn P
2005-01-01
We report the initial sequencing and comparative analysis of the Macaca mulatta transcriptome. Cloned sequences from 11 tissues, nine animals, and three species (M. mulatta, M. fascicularis, and M. nemestrina) were sampled, resulting in the generation of 48,642 sequence reads. These data represent an initial sampling of the putative rhesus orthologs for 6,216 human genes. Mean nucleotide diversity within M. mulatta and sequence divergence among M. fascicularis, M. nemestrina, and M. mulatta are also reported.
-
Ethical issues in transgenics.
Sherlock, R; Morrey, J D
2000-01-01
The arguments of critics and concerns of the public on generating transgenic cloned animals are analyzed for the absence or presence of logical structure. Critics' arguments are symbolically compared with "genetic trespassing," "genetic speeding," or "going the wrong way," and responses are provided to these arguments. Scientists will be empowered to participate in the public discussion and to engage the critics on these issues as they consider thoughtful, plausible responses to their concerns. Temporary moratoriums are recognized as a plausible approach to dealing with possible concerns of new scientific advancements.
-
2016-10-01
progress in subaim 1a, substantially improving the design of our proposed transgenic animal, the “deletion reporter mouse”, and are finalizing cloning...of necessary components. We expect to submit embryonic stem cells to the transgenic facility within the next few months. Furthermore, subaim 1b is...different mammary epithelial subpopulations. We will breed the reporter mouse created in aim 1 (or the CAG/UBC-GFP mouse) with BRCA1+/- and ATM+/- mutant
-
Satava, R M
2002-10-01
Numerous advanced technologies, both medical and nonmedical, are emerging faster than their social, behavioral, political, moral, and ethical implications can be understood. Some of these technologies will fundamentally challenge the practice of surgery: human cloning, genetic engineering, tissue engineering, intelligent robotics, nanotechnology, suspended animation, regeneration, and species prolongation. Because of the rapidity of change, the current status of these emerging technologies with their specific moral and ethical issues must be addressed at this time by the new generation of surgeons, or we must all face the consequences of an uncontrolled and unprepared future.
-
Turano, Helena; Gomes, Fernando; Medeiros, Micheli; Oliveira, Silvane; Fontes, Lívia C; Sato, Maria I Z; Lincopan, Nilton
2016-09-01
This study reports the presence of hospital-associated high-risk lineages of OXA-23-producing ST79 Acinetobacter baumannii and SPM-1-producing ST277 Pseudomonas aeruginosa in urban rivers in Brazil. These findings indicate that urban rivers can act as reservoirs of clinically important multidrug-resistant bacteria, which constitute a potential risk to human and animal health. Copyright © 2016 Elsevier Inc. All rights reserved.
-
1992-06-25
Michael See Bryzik, Walter I 205 Reid, Robert H. Oral, Microencapsulated, CFA/II III 35 Vaccine Against E. coli Diarrheal Disease: Preclinical Evaluation...Cloning, Characterization, and III 179 Expression of Animal Toxin Genes for Vaccine Development Smith, Stan See Juhasz, Arpad II 159 Soicher, Haim...modeling predictions that were supported by test data. FWD AND AFT SHELLS LONGERONS T300 GR/934 EPOXYAND T300 GR/934 EPOXY(BASELINE) CORK HS (BASELINE) GR
-
Molecular genetics of inherited eye disorders.
MacDonald, I M; Sasi, R
1994-10-01
In the past 10 y, there have been considerable advances in the mapping, isolation, and characterization of many genes for important ocular conditions: retinitis pigmentosa, Norrie disease, Waardenburg syndrome, choroideremia, aniridia, retinoblastoma, and others. The candidate gene approach has now supplemented classical linkage studies and positional cloning in the investigation of ocular disorders. Developmentally expressed genes and animal models have provided insights as to the etiology of other disorders. With this knowledge at hand, genetic counselling for heritable eye diseases has been greatly improved.
-
Prokof'ev, V V; Levakin, I A; Losev, E A; Zavirinskiĭ, Ia V; Galaktionov, K V
2011-01-01
The study was carried out on Himasthla elongata, a digenean common in the coastal ecosystems of the northern European seas. This species utilises intertidal prosobranchs Littorina spp. as the first intermediate host, bivalves (in the White Sea, Mytilus edulis) as the second intermediate host and gulls as the final host. The periwinkles Littorina littorea infected with H. elongata rediae (parthenogenetic generations) were sampled in the intertidal of the White Sea (66 degrees 20' N, 33 degrees 38' E) and used as the source of cercariae. Periwinkles were collected from the settlement with the low prevalence of H. elongata. As shown earlier with the use of AFLP (Amplified Fragment Length Polymorphisms) method, rediae groups in all the infected periwinkles of this settlement arise from the infection of a mollusc with a single miracidium. Therefore, the cercariae shed by an infected mollusc have the same genotype or, in other words, represent a clone. Photo- and geoorientation of cercariae originating from different clones and aged 1 h and 6 h were analysed separately. It was shown that in general the larvae of each clone followed the behavioural pattern characteristics of the species (positive geoorientation and negative photoorientation). However, the degree of expression of this typical behaviour was different in different clones. An especially high variability was observed in the manifestation of geoorientation (in several clones, most larvae demonstrated negative geoorientation). Differences in the distribution of cercariae in the illumination gradient were almost equally associated with the interclonal variability and the age of the larvae. On the whole, as the age of cercariae increased, the positive geoorientation became more prominent, whereas the ratio of cercariae with the typical (negative) photoorientation decreased. Statistical analysis revealed significant differences between the cercarial clones both in the initial manifestation of geo- and photoorientation and in the changes in the character of these reactions with the larval age. Taking into account that each cercarial clone investigated had the same genotype, it seems very likely that the interclonal differences noted in this study are hereditary. Maintenance of a rather high level of genetic polymorphism by the character "expression of orientation reaction" in trematode cercariae may enhance the chances for successful transmission of these larvae. Such variability increases the scale of cercarial dispersion in space and promotes the successful infection of the hosts, whose behaviour is also subject to intra- and inter-population variability. Besides, cercariae whose behaviour deviates from the basic behaviour of the species may play the role of the population's potential for colonisation of new species of animal hosts.
-
Yamamoto, Tatsuo; Nishiyama, Akihito; Takano, Tomomi; Yabe, Shizuka; Higuchi, Wataru; Razvina, Olga; Shi, Da
2010-08-01
Methicillin-resistant Staphylococcus aureus (MRSA) is able to persist not only in hospitals (with a high level of antimicrobial agent use) but also in the community (with a low level of antimicrobial agent use). The former is called hospital-acquired MRSA (HA-MRSA) and the latter community-acquired MRSA (CA-MRSA). It is believed MRSA clones are generated from S. aureus through insertion of the staphylococcal cassette chromosome mec (SCCmec), and outbreaks occur as they spread. Several worldwide and regional clones have been identified, and their epidemiological, clinical, and genetic characteristics have been described. CA-MRSA is likely able to survive in the community because of suitable SCCmec types (type IV or V), a clone-specific colonization/infection nature, toxin profiles (including Pantone-Valentine leucocidin, PVL), and narrow drug resistance patterns. CA-MRSA infections are generally seen in healthy children or young athletes, with unexpected cases of diseases, and also in elderly inpatients, occasionally surprising clinicians used to HA-MRSA infections. CA-MRSA spreads within families and close-contact groups or even through public transport, demonstrating transmission cores. Re-infection (including multifocal infection) frequently occurs, if the cores are not sought out and properly eradicated. Recently, attention has been given to CA-MRSA (USA300), which originated in the US, and is growing as HA-MRSA and also as a worldwide clone. CA-MRSA infection in influenza season has increasingly been noted as well. MRSA is also found in farm and companion animals, and has occasionally transferred to humans. As such, the epidemiological, clinical, and genetic behavior of CA-MRSA, a growing threat, is focused on in this study.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Reilly, D.S.; Nussbaum, R.L.
1994-09-01
The Lowe oculocerebrorenal syndrome (OCRL) is an X-linked disease characterized by congenital cataract, mental retardation, and renal tubular dysfunction. A candidate cDNA, OCRL-1, was identified by positional cloning and mutations in OCRL-1 have been detected in patients with Lowe syndrome. The OCRL-1 nucleotide sequence encodes a predicted protein of 968 amino acids and shares 51% amino acid identity with a human inositol polyphosphate-5-phosphatase. This suggests that the underlying defect in OCRL may be due to a defect in inositol phosphate metabolism. The isolation of OCRL-1 provides the opportunity to investigate its function through the use of animal model systems. Wemore » have isolated a partial cDNA clone encoding an OCRL-1 homologue, X-OCRL, from the South African clawed frog, Xenopus laevis. We used a portion of the human cDNA to screen a Xenopus laevis embryo cDNA library and isolated four positive clones. One clone, 42-5A, is a 650 bp insert with over 75% amino acid identity to the corresponding region of the human OCRL-1 sequence. 42-5A detects messenger RNA in adult Xenopus brain, stomach, small intestine, skin, muscle, lung, blood, and oviduct. X-OCRL messenger RNA is first detected during late gastrula and continues to be expressed throughout Xenopus development. In situ hybridization studies are underway to identify the cellular localization of X-OCRL expression in Xenopus embryos and adult tissues. We are especially interested in characterizing X-OCRL expression during formation of the amphibian lens since congenital cataracts are a constant feature of the human disease.« less
-
Michel, Marcus; Aliee, Maryam; Rudolf, Katrin; Bialas, Lisa; Jülicher, Frank; Dahmann, Christian
2016-01-01
The separation of cells with distinct fates and functions is important for tissue and organ formation during animal development. Regions of different fates within tissues are often separated from another along straight boundaries. These compartment boundaries play a crucial role in tissue patterning and growth by stably positioning organizers. In Drosophila, the wing imaginal disc is subdivided into a dorsal and a ventral compartment. Cells of the dorsal, but not ventral, compartment express the selector gene apterous. Apterous expression sets in motion a gene regulatory cascade that leads to the activation of Notch signaling in a few cell rows on either side of the dorsoventral compartment boundary. Both Notch and apterous mutant clones disturb the separation of dorsal and ventral cells. Maintenance of the straight shape of the dorsoventral boundary involves a local increase in mechanical tension at cell bonds along the boundary. The mechanisms by which cell bond tension is locally increased however remain unknown. Here we use a combination of laser ablation of cell bonds, quantitative image analysis, and genetic mutants to show that Notch and Apterous are required to increase cell bond tension along the dorsoventral compartment boundary. Moreover, clonal expression of the Apterous target gene capricious results in cell separation and increased cell bond tension at the clone borders. Finally, using a vertex model to simulate tissue growth, we find that an increase in cell bond tension at the borders of cell clones, but not throughout the cell clone, can lead to cell separation. We conclude that Apterous and Notch maintain the characteristic straight shape of the dorsoventral compartment boundary by locally increasing cell bond tension. PMID:27552097
-
NASA Astrophysics Data System (ADS)
Lider, Ofer; Karin, Nathan; Shinitzky, Meir; Cohen, Irun R.
1987-07-01
An ideal treatment for autoimmune diseases would be a nontoxic means of specifically neutralizing the autoreactive lymphocytes responsible for the disease. This goal has been realized in experimental autoimmunity models by immunizing rats or mice against their own autoimmune cells such that the animals generate an immune response specifically repressive to the disease-producing lymphocytes. This maneuver, termed lymphocyte vaccination, was demonstrated to be effective using some, but not all, autoimmune helper T-lymphocyte lines. We now report that T lymphocytes, otherwise incapable of triggering an immune response, can be transformed into effective immunogens by treating the cells in vitro with hydrostatic pressure. Clone A2b, as effector clone that recognized cartilage proteoglycan and caused adjuvant arthritis in Lewis rats, is such a cell. Untreated A2b could not trigger an immune response, but inoculating rats with pressure-treated A2b induced early remission of established adjuvant arthritis as well as resistance to subsequent disease. Specific resistance to arthritis was associated with anti-idiotypic T-cell reactivity to clone A2b and could be transferred from vaccinated rats to naive recipients using donor lymphoid cells. Aggregation of T-lymphocyte membrane components appeared to be important for an immune response because the effects of hydrostatic pressure could be reproduced by treatment of A2b with chemical cross-linkers or with agents disrupting the cytoskeleton. Populations of lymph node cells from antigen-primed rats, when treated with hydrostatic pressure, could also induce suppression of disease. Thus, effective vaccines can be developed without having to isolate the autoimmune T lymphocytes as lines or clones. These results demonstrate that effector T lymphocytes suitably treated may serve as agents for specifically controlling the immune system.
-
Kargar, Rohollah; Forouzanfar, Mohsen; Ghalamkari, Gholamreza; Nasr Esfahani, Mohammad Hossein
2017-02-01
Semen cryopreservation is affected by individual differences and use of clones animal from the same source is the main tool to eliminate genetic variation. Among many nutrients that are necessary for fertility, essential fatty acids and antioxidants are vital for production of healthy sperm by improving sperm membrane integrity and protecting sperm from oxidative stress. The goal of the current study was to investigate whether a flax seed oil or/and Vitamin E dietary supplementation could improve semen quality of cloned bucks following semen cryopreservation. Accordingly, eight adult cloned Bakhtiari bucks were divided randomly into four groups. Bucks were offered a base diet of hay and concentrate. The concentrate was enriched with flax seed oil, 30 gr/kg body weight/day (OIL), Vitamin E (VIT), 3 gr/kg body weight/day, or combined flax seed oil and the vitamin E (OIL-VIT). The concentrate with no supplements was considered as control group (CONT). Both flax seed oil and Vitamin E supplements were added to the total diet. The bucks were fed with their corresponding diets for a total of 9 weeks while sperm collection was carried out within 10-14 weeks. Ejaculates were diluted with Andromed ® and were frozen in liquid nitrogen. Sperm parameters and reactive oxygen species (ROS) contents were evaluated following freezing/thawing. According to the results of our study, dietary supplementation with flax seed oil, or/and Vitamin E can improve sperm motility, vitality and number of sperm with intact plasma membrane following freezing-thawing. But the degree of improvement in these parameters was significantly higher when Flax seed oil and vitamin E were co-supplemented. Copyright © 2016. Published by Elsevier Inc.
-
Zhao, Jianguo; Ross, Jason W.; Hao, Yanhong; Spate, Lee D.; Walters, Eric M.; Samuel, Melissa S.; Rieke, August; Murphy, Clifton N.; Prather, Randall S.
2009-01-01
The National Institutes of Health (NIH) miniature pig was developed specifically for xenotransplantation and has been extensively used as a large-animal model in many other biomedical experiments. However, the cloning efficiency of this pig is very low (<0.2%), and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying a histone deacetylase (HDAC) inhibitor such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However, some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Herein, we report that treatment with 500 nM 6-(1,3-dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDAC inhibitor, significantly enhanced the development of SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) were used as donors compared with the untreated group (21% vs. 9%, P < 0.05). Scriptaid treatment resulted in eight pregnancies from 10 embryo transfers (ETs) and 14 healthy NIH miniature pigs from eight litters, while no viable piglets (only three mummies) were obtained from nine ETs in the untreated group. Thus, scriptaid dramatically increased the cloning efficiency when using inbred genetics from 0.0% to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in in vitro fertilization embryos (from 37% to 26%, P < 0.05). In conclusion, the extremely low cloning efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns. PMID:19386991
-
Host age modulates parasite infectivity, virulence and reproduction.
Izhar, Rony; Ben-Ami, Frida
2015-07-01
Host age is one of the most striking differences among hosts within most populations, but there is very little data on how age-dependent effects impact ecological and evolutionary dynamics of both the host and the parasite. Here, we examined the influence of host age (juveniles, young and old adults) at parasite exposure on host susceptibility, fecundity and survival as well as parasite transmission, using two clones of the water flea Daphnia magna and two clones of its bacterial parasite Pasteuria ramosa. Younger D. magna were more susceptible to infection than older ones, regardless of host or parasite clone. Also, younger-infected D. magna became castrated faster than older hosts, but host and parasite clone effects contributed to this trait as well. Furthermore, the early-infected D. magna produced considerably more parasite transmission stages than late-infected ones, while host age at exposure did not affect virulence as it is defined in models (host mortality). When virulence is defined more broadly as the negative effects of infection on host fitness, by integrating the parasitic effects on host fecundity and mortality, then host age at exposure seems to slide along a negative relationship between host and parasite fitness. Thus, the virulence-transmission trade-off differs strongly among age classes, which in turn affects predictions of optimal virulence. Age-dependent effects on host susceptibility, virulence and parasite transmission could pose an important challenge for experimental and theoretical studies of infectious disease dynamics and disease ecology. Our results present a call for a more explicit stage-structured theory for disease, which will incorporate age-dependent epidemiological parameters. © 2015 The Authors. Journal of Animal Ecology © 2015 British Ecological Society.
-
Zhao, Jianguo; Ross, Jason W; Hao, Yanhong; Spate, Lee D; Walters, Eric M; Samuel, Melissa S; Rieke, August; Murphy, Clifton N; Prather, Randall S
2009-09-01
The National Institutes of Health (NIH) miniature pig was developed specifically for xenotransplantation and has been extensively used as a large-animal model in many other biomedical experiments. However, the cloning efficiency of this pig is very low (<0.2%), and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying a histone deacetylase (HDAC) inhibitor such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However, some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Herein, we report that treatment with 500 nM 6-(1,3-dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDAC inhibitor, significantly enhanced the development of SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) were used as donors compared with the untreated group (21% vs. 9%, P < 0.05). Scriptaid treatment resulted in eight pregnancies from 10 embryo transfers (ETs) and 14 healthy NIH miniature pigs from eight litters, while no viable piglets (only three mummies) were obtained from nine ETs in the untreated group. Thus, scriptaid dramatically increased the cloning efficiency when using inbred genetics from 0.0% to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in in vitro fertilization embryos (from 37% to 26%, P < 0.05). In conclusion, the extremely low cloning efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.
-
Houtman, Marien J C; Korte, Sanne M; Ji, Yuan; Kok, Bart; Vos, Marc A; Stary-Weinzinger, Anna; van der Heyden, Marcel A G
2014-10-03
Potassium inward rectifier KIR2.1 channels contribute to the stable resting membrane potential in a variety of muscle and neuronal cell-types. Mutations in the KIR2.1 gene KCNJ2 have been associated with human disease, such as cardiac arrhythmias and periodic paralysis. Crystal structure and homology modelling of KIR2.1 channels combined with functional current measurements provided valuable insights in mechanisms underlying channel function. KIR2.1 channels have been cloned and analyzed from all main vertebrate phyla, except reptilians. To address this lacuna, we set out to clone reptilian KIR2.1 channels. Using a degenerated primer set we cloned the KCNJ2 coding regions from muscle tissue of turtle, snake, bear, quail and bream, and compared their deduced amino acid sequences with those of KIR2.1 sequences from 26 different animal species obtained from Genbank. Furthermore, expression constructs were prepared for functional electrophysiological studies of ectopically expressed KIR2.1 ion channels. In general, KCNJ2 gene evolution followed normal phylogenetic patterns, however turtle KIR2.1 ion channel sequence is more homologues to avians than to snake. Alignment of all 31 KIR2.1 sequences showed that all disease causing KIR2.1 mutations, except V93I, V123G and N318S, are fully conserved. Homology models were built to provide structural insights into species specific amino acid substitutions. Snake KIR2.1 channels became expressed at the plasmamembrane and produced typical barium sensitive (IC50 ∼6μM) inward rectifier currents. Copyright © 2014 Elsevier Inc. All rights reserved.
-
Palomo, Gonzalo; Campos, Maria Jorge; Ugarte, María; Porrero, María Concepción; Alonso, Juan Manuel; Borge, Carmen; Vadillo, Santiago; Domínguez, Lucas; Quesada, Alberto; Píriz, Segundo
2013-02-01
Non-typhoidal salmonellosis is an important zoonotic disease caused by Salmonella enterica. This work focuses on the identification of Salmonella enterica clonal strains which, presenting a wide distribution potential, express resistance determinants that compromise effectiveness of the antimicrobial therapy. The screening was performed on 506 Salmonella enterica isolates from animals and humans, which were characterized by serovar and phage typing, genome macrorestriction and pulsed-field gel electrophoresis, and detection of phenotypic and genotypic traits for antimicrobial resistance. A Salmonella Enteritidis strain with strong quinolone resistance is spread on three host environments carrying one of the four variants found for the GyrA protein: (1) Asp87Tyr, the major polymorphism found in 39 Salmonella isolates from human origin and six from poultry; (2) Ser83Phe, with four isolates from human origin and one from white stork (Ciconia ciconia); and (3) Asp87Asn or (4) Asp87Gly, with two isolates each from human origins. Several Salmonella Typhimurium strains that presented int1 elements and the classically associated pentaresistance (ACSSuT) phenotype were found distributed between two host environments: domestic animals and humans, domestics and wild animals, or wild fauna plus humans. This study points out the importance of monitoring gut microbiota and its antimicrobial resistance from wildlife, in parallel to livestock animals and humans, especially for animal species that are in close contact with people.
-
Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line.
Dehler, Carola E; Boudinot, Pierre; Martin, Samuel A M; Collet, Bertrand
2016-08-01
CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening.
-
Faust, M; Ebensperger, C; Schulz, A S; Schleithoff, L; Hameister, H; Bartram, C R; Janssen, J W
1992-07-01
We have cloned the mouse homologue of the ufo oncogene. It encodes a novel tyrosine kinase receptor characterized by a unique extracellular domain containing two immunoglobulin-like and two fibronectin type III repeats. Comparison of the predicted ufo amino acid sequences of mouse and man revealed an overall identity of 87.6%. The ufo locus maps to mouse chromosome 7A3-B1 and thereby extends the known conserved linkage group between mouse chromosome 7 and human chromosome 19. RNA in situ hybridization analysis established the onset of specific ufo expression in the late embryogenesis at day 12.5 post coitum (p.c.) and localized ufo transcription to distinct substructures of a broad spectrum of developing tissues (e.g. subepidermal cells of the skin, mesenchymal cells of the periosteum). In adult animals ufo is expressed in cells forming organ capsules as well as in connective tissue structures. ufo may function as a signal transducer between specific cell types of mesodermal origin.
-
Verification of a canine PSMA (FolH1) antibody.
Wagner, Siegfried; Maibaum, Denise; Pich, Andreas; Nolte, Ingo; Murua Escobar, Hugo
2015-01-01
Canine prostate cancer (PC) is a highly aggressive malignancy. However, in contrast to man, neither standard screening strategies nor curative therapeutic options exist for the companion animal. A prostate-specific membrane antigen (PSMA) screening as molecular marker akin to human PC is currently not available for dogs as data on specific canine PSMA detection are contradictory. To evaluate an antibody for specific canine PSMA detection by western blotting (WB), lysates of three canine prostatic cell lines (CT1258, DT08/40, DT08/46) were comparatively analyzed by WB and mass spectrometry (MS) to the human cell lines VCaP, LnCaP and PC-3. MS analyses of the detected canine proteins confirmed cross reactivity of the antibody clone YPSMA-1 with canine PSMA. The MS analyses of the extracted canine protein bands proved that the YPSMA-1 clone is as well specific for canine PSMA in WB and, thus, represents a reliable tool for comparative PSMA studies. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
-
Cloning, monoclonal antibody production, and bodily distribution pattern of a bovine lipocalin.
Japaridze, Tamar; Senda, Akitsugu; Nozaki, Hirofumi; Yanagida, Mayumi; Suzuki, Takumi; Ganzorig, Khuukhenbaatar; Kushi, Yasunori; Kida, Katsuya; Urashima, Tadasu; Bruckmaier, Rupert M; Fukuda, Kenji
2012-01-01
A bovine lipocalin, previously identified as a putative odorant-binding protein in bovine colostrum (bcOBP), was cloned and expressed, and its monoclonal antibody was established. bcOBP was constantly secreted into milk on day of parturition until at least 10 d postpartum at a concentration of 181±39 µg/L. Besides milk, bcOBP occurred in the nasal mucus, saliva, amniotic fluid, vaginal discharge, and blood plasma. Despite its low concentration, the distribution pattern and the finding that bcOBP harbored a characteristic sequence motif, CxxxC, which is conserved among insect and mammal pheromone binding proteins, suggest that bcOBP functions as a pheromone carrier. The presence of bcOBP in the plasma at varied concentrations depending on the lactation period does not exclude the possibility that bcOBP is secreted into milk from the blood. Cross-reactivity of the monoclonal antibody indicated presence of proteins homologous to bcOBP in the colostrum of farm animals of Cetartiodactyla.
-
Cloning and functional expression of a plant voltage-dependent chloride channel.
Lurin, C; Geelen, D; Barbier-Brygoo, H; Guern, J; Maurel, C
1996-01-01
Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants. PMID:8624442
-
Cloning and High-Level Expression of α-Galactosidase cDNA from Penicillium purpurogenum
Shibuya, Hajime; Nagasaki, Hiroaki; Kaneko, Satoshi; Yoshida, Shigeki; Park, Gwi Gun; Kusakabe, Isao; Kobayashi, Hideyuki
1998-01-01
The cDNA coding for Penicillium purpurogenum α-galactosidase (αGal) was cloned and sequenced. The deduced amino acid sequence of the α-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic αGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was to Trichoderma reesei AGLI. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P. purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides. PMID:9797312
-
Churchill, A E
1987-04-04
Canine parvovirus isolated from a case of haemorrhagic enteritis in a breeding kennel in England was passaged and cloned in cultured feline and canine cells. No significant evidence of pathogenicity was found during six serial passages of the modified virus back through young dogs. The attenuated virus was excreted by inoculated animals and spread rapidly to uninoculated animals held in contact. When high titre attenuated virus was given to the six-week-old offspring of a seropositive dam a prompt seroconversion was observed. When the attenuated virus was used as an experimental vaccine in 108 pups in an infected breeding colony a highly significant improvement was obtained in the accumulated morbidity and mortality compared with a parallel group vaccinated with modified live feline panleucopenia virus.
-
Emerging Technologies to Create Inducible and Genetically Defined Porcine Cancer Models.
Schook, Lawrence B; Rund, Laurie; Begnini, Karine R; Remião, Mariana H; Seixas, Fabiana K; Collares, Tiago
2016-01-01
There is an emerging need for new animal models that address unmet translational cancer research requirements. Transgenic porcine models provide an exceptional opportunity due to their genetic, anatomic, and physiological similarities with humans. Due to recent advances in the sequencing of domestic animal genomes and the development of new organism cloning technologies, it is now very feasible to utilize pigs as a malleable species, with similar anatomic and physiological features with humans, in which to develop cancer models. In this review, we discuss genetic modification technologies successfully used to produce porcine biomedical models, in particular the Cre-loxP System as well as major advances and perspectives the CRISPR/Cas9 System. Recent advancements in porcine tumor modeling and genome editing will bring porcine models to the forefront of translational cancer research.
-
Characterisation of recombinant immunoreactive antigens of the scab mite Sarcoptes scabiei.
Kuhn, C; Lucius, R; Matthes, H F; Meusel, G; Reich, B; Kalinna, B H
2008-05-31
Sarcoptic mange (or scabies) is an important skin disease which can affect a variety of species including humans, cattle, goats, sheep, horses, pigs, rabbits, and dogs. Approximately 300 million people are affected worldwide and in lifestock animals the infestation may lead to substantial economic losses caused by depression in growth and feed conversion rates. Diagnosis of Sarcoptes infestation is difficult and only a few serological tests have been developed using whole mite antigen for diagnosis of mange in animals. Here we describe the isolation and characterisation of cDNAs of several immunoreactive clones and their recombinant expression in Escherichia coli. Three of the proteins contain repetitive sequences which suggests that they might be involved in immune evasion. The application of these antigens in serodiagnosis and the suitability for diagnosis is discussed.
-
New polymorphic markers in the vicinity of the pearl locus on mouse chromosome 13.
Xu, H P; Yanak, B L; Wigler, M H; Gorin, M B
1996-01-01
We have used a Mus domesticus/-Mus spretus congenic animal that was selected for retention of Mus spretus DNA around the pearl locus to create a highly polymorphic region suitable for screening new markers. Representation difference analysis (RDA) was performed with either DNA from the congenic animal or C57BL/6J as the driver for subtraction. Four clones were identified, characterized, and converted to PCR-based polymorphic markers. Three of the four markers equally subdivide a 10-cM interval containing the pearl locus, with the fourth located centromeric to it. These markers have been placed on the mouse genetic map by use of an interspecific backcross panel between Mus domesticus (C57BL/6J) and Mus spretus generated by The Jackson Laboratory.
-
2012-01-01
Background Mutualistic interactions are wide-spread but the mechanisms underlying their evolutionary stability and ecological dynamics remain poorly understood. Cultivation mutualisms in which hosts consume symbionts occur in phylogenetically diverse groups, but often have symbiont monocultures for each host. This is consistent with the prediction that symbionts should avoid coexistence with other strains so that host services continue to benefit relatives, but it is less clear whether hosts should always favor monocultures and what mechanisms they might have to manipulate symbiont diversity. Few mutualisms have been studied in sufficient genetic detail to address these issues, so we decided to characterize symbiont diversity in the complex mutualism between multiple root aphid species and Lasius flavus ants. After showing elsewhere that three of these aphid species have low dispersal and mostly if not exclusively asexual reproduction, we here investigate aphid diversity within and between ant nest mounds. Results The three focal species (Geoica utricularia, Forda marginata and Tetraneura ulmi) had considerable clonal diversity at the population level. Yet more than half of the ant mounds contained just a single aphid species, a significantly higher percentage than expected from a random distribution. Over 60% of these single-species mounds had a single aphid clone, and clones tended to persist across subsequent years. Whenever multiple species/clones co-occurred in the same mound, they were spatially separated with more than 95% of the aphid chambers containing individuals of a single clone. Conclusions L. flavus “husbandry” is characterized by low aphid “livestock” diversity per colony, especially at the nest-chamber level, but it lacks the exclusive monocultures known from other cultivation mutualisms. The ants appear to eat most of the early instar aphids, so that adult aphids are unlikely to face limited phloem resources and scramble competition with other aphids. We suggest that such culling of carbohydrate-providing symbionts for protein ingestion may maintain maximal host yield per aphid while also benefitting the domesticated aphids as long as their clone-mates reproduce successfully. The cost-benefit logic of this type of polyculture husbandry has striking analogies with human farming practices based on slaughtering young animals for meat to maximize milk-production by a carefully regulated adult livestock population. PMID:22747564
-
Song, Hui; Li, Hui; Huang, Mingrui; Xu, Dan; Wang, Ziyu; Wang, Feng
2016-02-01
Using of embryonic stem cells (ESCs) could improve production traits and disease resistance by improving the efficiency of somatic cell nuclear transfer (SCNT) technology. However, robust ESCs have not been established from domestic ungulates. In the present study, we generated goat induced pluripotent stem cells (giPSCs) and transgenic cloned dairy goat induced pluripotent stem cells (tgiPSCs) from dairy goat fibroblasts (gFs) and transgenic cloned dairy goat fibroblasts (tgFs), respectively, using lentiviruses that contained hOCT4, hSOX2, hMYC, and hKLF4 without chemical compounds. The giPSCs and tgiPSCs expressed endogenous pluripotent markers, including OCT4, SOX2, MYC, KLF4, and NANOG. Moreover, they were able to maintain a normal karyotype and differentiate into derivatives from all three germ layers in vitro and in vivo. Using SCNT, tgFs and tgiPSCs were used as donor cells to produce embryos, which were named tgF-Embryos and tgiPSC-Embryos. The fusion rates and cleavage rates had no significant differences between tgF-Embryos and tgiPSC-Embryos. However, the expression of IGF-2, which is an important gene associated with embryonic development, was significantly lower in tgiPSC-Embryos than in tgF-Embryos and was not significantly different from vivo-Embryos.
-
Abuqarn, Mehtap; Allmeling, Christina; Amshoff, Inga; Menger, Bjoern; Nasser, Inas; Vogt, Peter M; Reimers, Kerstin
2011-07-01
Urodele amphibians are exceptional in their ability to regenerate complex body structures such as limbs. Limb regeneration depends on a process called dedifferentiation. Under an inductive wound epidermis terminally differentiated cells transform to pluripotent progenitor cells that coordinately proliferate and eventually redifferentiate to form the new appendage. Recent studies have developed molecular models integrating a set of genes that might have important functions in the control of regenerative cellular plasticity. Among them is Msx1, which induced dedifferentiation in mammalian myotubes in vitro. Herein, we screened for interaction partners of axolotl Msx1 using a yeast two hybrid system. A two hybrid cDNA library of 5-day-old wound epidermis and underlying tissue containing more than 2×10⁶ cDNAs was constructed and used in the screen. 34 resulting cDNA clones were isolated and sequenced. We then compared sequences of the isolated clones to annotated EST contigs of the Salamander EST database (BLASTn) to identify presumptive orthologs. We subsequently searched all no-hit clone sequences against non redundant NCBI sequence databases using BLASTx. It is the first time, that the yeast two hybrid system was adapted to the axolotl animal model and successfully used in a screen for proteins interacting with Msx1 in the context of amphibian limb regeneration. 2011 Elsevier B.V. All rights reserved.
-
Yu, Haining; Gao, Jiuxiang; Lu, Yiling; Guang, Huijuan; Cai, Shasha; Zhang, Songyan; Wang, Yipeng
2013-11-01
Lysozymes are key proteins that play important roles in innate immune defense in many animal phyla by breaking down the bacterial cell-walls. In this study, we report the molecular cloning, sequence analysis and phylogeny of the first caudate amphibian g-lysozyme: a full-length spleen cDNA library from axolotl (Ambystoma mexicanum). A goose-type (g-lysozyme) EST was identified and the full-length cDNA was obtained using RACE-PCR. The axolotl g-lysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 184 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein are 21523.0 Da and 4.37, respectively. Expression of g-lysozyme mRNA is predominantly found in skin, with lower levels in spleen, liver, muscle, and lung. Phylogenetic analysis revealed that caudate amphibian g-lysozyme had distinct evolution pattern for being juxtaposed with not only anura amphibian, but also with the fish, bird and mammal. Although the first complete cDNA sequence for caudate amphibian g-lysozyme is reported in the present study, clones encoding axolotl's other functional immune molecules in the full-length cDNA library will have to be further sequenced to gain insight into the fundamental aspects of antibacterial mechanisms in caudate.
-
König, Enrico; Clark, Valerie C; Shaw, Chris; Bininda-Emonds, Olaf R P
2012-12-01
The skins of phyllomedusine frogs have long been considered as being tremendously rich sources of bioactive peptides. Previous studies of both peptides and cloning of their precursor encoding cDNAs have relied upon methanolic skin extracts or the dissected skins of recently deceased specimens and have not considered the different glands in isolation. We therefore focused our attention on the tibial gland of the Giant Monkey Frog, Phyllomedusa bicolor and constructed a cDNA library from the skin secretion that was obtained via mechanical stimulation of this macrogland. Using shotgun cloning, four precursors encoding host-defense peptides were identified: two archetypal dermaseptins, a phyllokinin and a phylloseptin that is new for this species but has been recently described from the Waxy Monkey Leaf Frog, Phyllomedusa sauvagii. Our study is the first to report defensive peptides specifically isolated from anuran tibial glands, confirming the hypothesis that these glands also contribute to chemical defense. Moreover, the discovery of novel compounds for this otherwise very well characterized species suggests that this largely neglected gland might possess a different cocktail of secretions from glands elsewhere in the same animal. We will also discuss some evolutionary implications of our findings with respect to the adaptive plasticity of secretory glands. Copyright © 2012 Elsevier Inc. All rights reserved.
-
Zhang, Yan-Li; Zhang, Guo-Min; Jia, Ruo-Xin; Wan, Yong-Jie; Yang, Hua; Sun, Ling-Wei; Han, Le; Wang, Feng
2018-01-01
Pre-implantation embryo metabolism demonstrates distinctive characteristics associated with the development potential of embryos. We aim to determine if metabolic differences correlate with embryo morphology. In this study, gas chromatography - mass spectroscopy (GC-MS)-based metabolomics was used to assess the culture media of goat cloned embryos collected from high-quality (HQ) and low-quality (LQ) groups based on morphology. Expression levels of amino acid transport genes were further examined by quantitative real-time PCR. Results showed that the HQ group presented higher percentages of blastocysts compared with the LQ counterparts (P < 0.05). Metabolic differences were also present between HQ and LQ groups. The culture media of the HQ group showed lower levels of valin, lysine, glutamine, mannose and acetol, and higher levels of glucose, phytosphingosine and phosphate than those of the LQ group. Additionally, expression levels of amino acid transport genes SLC1A5 and SLC3A2 were significantly lower in the HQ group than the LQ group (P < 0.05, respectively). To our knowledge, this is the first report which uses GC-MS to detect metabolic differences in goat cloned embryo culture media. The biochemical profiles may help to select the most in vitro viable embryos. © 2017 Japanese Society of Animal Science.
-
Effects of chemical smokes on flora and fauna under field and laboratory exposures
DOE Office of Scientific and Technical Information (OSTI.GOV)
Schaeffer, D.J.; Novak, E.W.; Lower, W.R.
1987-06-01
Various types of obscurant smokes are used routinely in training by the U.S. Army. Because continued routine use of the smokes could be detrimental to the native flora and fauna at training sites, a preliminary biological and chemical field study of fogoil, hexachloroethane, and tank diesel smokes was conducted. Smoke plumes were sampled and chemically analyzed at distances of 15-150 m from the smoke source where Tradescantia clones 4430 and 03 and the native plant Ambrosia dumosa and the native rodent Dipodomys merriami were exposed for 30 min. In addition, Tradescantia clone 4430 was exposed to tank diesel in themore » laboratory at concentration levels equivalent to exposure at 15 and 50 m. Tradescantia clones were examined for mutagenic effects indicated by micronuclei induction in developing pollen and pink somatic mutations in stamen hairs. Photosynthetic perturbations were measured in Tradescantia and A. dumosa using variable fluorescence induction. Animals were examined for sister chromatid exchanges and chromosome aberrations. It was found that all of the smokes tested exerted varying degrees of physiological and mutagenic effects in one or more assay system at one or more exposure distance. The studies reported here indicate that exposed ecological systems, or at least components of these systems, are at a higher risk than are unexposed components (e.g., organisms) for several types of damage attributed to obscurant smoke exposure.« less
-
Construction of High-Quality Camel Immune Antibody Libraries.
Romão, Ema; Poignavent, Vianney; Vincke, Cécile; Ritzenthaler, Christophe; Muyldermans, Serge; Monsion, Baptiste
2018-01-01
Single-domain antibodies libraries of heavy-chain only immunoglobulins from camelids or shark are enriched for high-affinity antigen-specific binders by a short in vivo immunization. Thus, potent binders are readily retrieved from relatively small-sized libraries of 10 7 -10 8 individual transformants, mostly after phage display and panning on a purified target. However, the remaining drawback of this strategy arises from the need to generate a dedicated library, for nearly every envisaged target. Therefore, all the procedures that shorten and facilitate the construction of an immune library of best possible quality are definitely a step forward. In this chapter, we provide the protocol to generate a high-quality immune VHH library using the Golden Gate Cloning strategy employing an adapted phage display vector where a lethal ccdB gene has to be substituted by the VHH gene. With this procedure, the construction of the library can be shortened to less than a week starting from bleeding the animal. Our libraries exceed 10 8 individual transformants and close to 100% of the clones harbor a phage display vector having an insert with the length of a VHH gene. These libraries are also more economic to make than previous standard approaches using classical restriction enzymes and ligations. The quality of the Nanobodies that are retrieved from immune libraries obtained by Golden Gate Cloning is identical to those from immune libraries made according to the classical procedure.
-
[Embryonic stem cells and therapeutic cloning].
Sunde, A; Eftedal, I
2001-08-30
Increased interest in the therapeutic use of human stem cells has emerged following significant progress in ongoing research. The cloning of a sheep, the isolation of human embryonic stem cells, and the discovery that adult stem cells may be reprogrammed taken together give substance to hopes that novel principles of treatment may be developed for a variety of serious conditions. Embryonic stem cells are derived from pre-embryos at the blastocyst stage and may give rise to all bodily tissues and cells. Animal models have demonstrated that embryonic stem cells when transplanted into adult hosts may differentiate and develop into cells and tissues applicable for treatment of a variety of conditions, including Parkinson's disease, multiple sclerosis, spinal injuries, cardiac stroke and cancer. Transplanted embryonic stem cells are exposed to immune reactions similar to those acting on organ transplants, hence immunosuppression of the recipient is generally required. It is, however, possible to obtain embryonic stem cells that are genetically identical to the patient's own cells by means of therapeutic cloning techniques. The nucleus from a somatic cell is transferred into an egg after removal of the egg's own genetic material. Under specific condition the egg will use genetic information from the somatic cell in organising the formation of a blastocyst which in turn generates embryonic stem cells. These cells have a genetic composition identical to that of the patient and are suitable for stem cell therapy.
-
Batchelder, Cynthia A; Hoffert, Kara A; Bertolini, Marcelo; Moyer, Alice L; Mason, Jeffery B; Petkov, Stoyan G; Famula, Thomas R; Anderson, Gary B
2005-01-01
Potential applications of somatic cell nuclear transfer to agriculture and medicine are currently constrained by low efficiency and high rates of embryonic, fetal, and neonatal loss. Nuclear transfer efficiency in cattle was compared between three donor-cell treatments from a single animal, between four donor-cell treatments in sequential stages of differentiation from a single cell lineage and genotype, and between the same cell type in two donors. Cumulus and granulosa donor cells resulted in a greater proportion of viable day-7 embryos than ear-skin cells; pregnancy rate and losses were not different among treatments. The least differentiated cell type in the follicular cell lineage, preantral follicle cells, resulted in fewer cloned blastocysts (11%) than cumulus (30%), granulosa (23%), and luteal (25%) donor cells. Cloned blastocysts that did develop from preantral follicle cells (75%) were more likely to progress through implantation into later stages of pregnancy than cloned blastocysts from cumulus (10%), granulosa (9%), and luteal (11%) donor cells (p < 0.05). Day-7 embryo development from granulosa cells was similar between two donors (19 vs. 24%) and proved to be a poor indicator of further development as day-30 pregnancy rates varied threefold between donors (48 vs. 15%, p < 0.05). Results reported here emphasize the crucial role of the nuclear donor cell in the outcome of the nuclear-transfer process.
-
Jiang, Chunyan; Wang, Hougen; Kang, Qianjin; Liu, Jing
2012-01-01
Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity. PMID:22156425
-
Pang, Jen-Chieh; Lin, Jer-Sheng; Tsai, Cheng-Chih; Tsen, Hau-Yang
2006-10-01
Seventy-seven animal isolates of Salmonella enterica serovar Enteritidis (S. Enteritidis) obtained from the United States were analyzed by phage typing and pulsed field gel electrophoresis (PFGE). Thirty-nine strains were found with phage types (PT) 4, 8, and 13a. When the chromosomal DNA of these 39 isolated strains with PT4, 8, and 13a were digested with XbaI, SpeI and NotI, followed by PFGE analysis, 28 strains were found with a pattern combination of X4S4N4, which was the major subtype. When PFGE patterns of the US isolates with PT 4 and 8 were compared with those of the Taiwanese and German isolates, pattern X3S3N3 was confirmed to be the world-wide subtype shared by PT 4 isolates, as previously reported, while pattern X4S4N4 was newly found to be the most common subtype shared by PT 8 strains. The presence of such major world-wide clones, however, does not necessarily mean that these clones are highly virulent, at least not according to the results of invasiveness assays using cultured human intestinal epithelium cell line Int-407 and living BALB/mice.
-
Effects of chemical smokes on flora and fauna under field and laboratory exposures.
Schaeffer, D J; Novak, E W; Lower, W R; Yanders, A; Kapila, S; Wang, R
1987-06-01
Various types of obscurant smokes are used routinely in training by the U.S. Army. Because continued routine use of the smokes could be detrimental to the native flora and fauna at training sites, a preliminary biological and chemical field study of fogoil, hexachloroethane, and tank diesel smokes was conducted. Smoke plumes were sampled and chemically analyzed at distances of 15-150 m from the smoke source where Tradescantia clones 4430 and 03 and the native plant Ambrosia dumosa and the native rodent Dipodomys merriami were exposed for 30 min. In addition, Tradescantia clone 4430 was exposed to tank diesel in the laboratory at concentration levels equivalent to exposure at 15 and 50 m. Tradescantia clones were examined for mutagenic effects indicated by micronuclei induction in developing pollen and pink somatic mutations in stamen hairs. Photosynthetic perturbations were measured in Tradescantia and A. dumosa using variable fluorescence induction. Animals were examined for sister chromatid exchanges and chromosome aberrations. It was found that all of the smokes tested exerted varying degrees of physiological and mutagenic effects in one or more assay system at one or more exposure distance. The studies reported here indicate that exposed ecological systems, or at least components of these systems, are at a higher risk than are unexposed components (e.g., organisms) for several types of damage attributed to obscurant smoke exposure.
-
Human Staphylococcus aureus lineages among Zoological Park residents in Greece
Drougka, E.; Foka, A.; Posantzis, D.; Giormezis, N.; Anastassiou, E.D.; Petinaki, E.; Spiliopoulou, I.
2015-01-01
Staphylococcus aureus is a part of the microbiota flora in many animal species. The clonal spread of S. aureus among animals and personnel in a Zoological Park was investigated. Samples were collected from colonized and infected sites among 32 mammals, 11 birds and eight humans. The genes mecA, mecC, lukF/lukS-PV (encoding Panton-Valentine leukocidin, PVL) and tst (toxic shock syndrome toxin-1) were investigated by PCR. Clones were defined by Multilocus Sequence Typing (MLST), spa type and Pulsed-Field Gel Electrophoresis (PFGE). Seven S. aureus isolates were recovered from four animals and one from an employee. All were mecA, mecC and tst–negative, whereas, one carried the PVL genes and was isolated from an infected Squirrel monkey. Clonal analysis revealed the occurrence of seven STs, eight PFGE and five spa types including ones of human origin. Even though a variety of genotypes were identified among S. aureus strains colonizing zoo park residents, our results indicate that colonization with human lineages has indeed occurred. PMID:26623381
-
Myelnikov, Dmitriy
2017-12-01
The Animal Breeding Research Organisation in Edinburgh (ABRO, founded in 1945) was a direct ancestor of the Roslin Institute, celebrated for the cloning of Dolly the sheep. After a period of sustained growth as an institute of the Agricultural Research Council (ARC), ABRO was to lose most of its funding in 1981. This decision has been absorbed into the narrative of the Thatcherite attack on science, but in this article I show that the choice to restructure ABRO pre-dated major government cuts to agricultural research, and stemmed from the ARC's wish to prioritize biotechnology in its portfolio. ABRO's management embraced this wish and campaigned against the cuts based on a promise of biotechnological innovation, shifting its focus from farm animal genetics to the production of recombinant pharmaceuticals in sheep milk. By tracing interaction between government policies, research council agendas and local strategies, I show how novel research programmes such as genetic modification could act as a lifeline for struggling institutions.
-
NASA Astrophysics Data System (ADS)
Ward, N.; Page, S.; Heidelberg, J.; Eisen, J. A.; Fraser, C. M.
2002-12-01
The composition of microbial communities associated with deep-sea hydrothermal vent animals is of interest because of the key role of bacterial symbionts in driving the chemosynthetic food chain of the vent system, and also because bacterial biofilms attached to animal exterior surfaces may play a part in settlement of larval forms. Sequence analysis of 16S ribosomal RNA (rRNA) genes from such communities provides a snapshot of community structure, as this gene is present in all Bacteria and Archaea, and a useful phylogenetic marker for both cultivated microbial species, and uncultivated species such as many of those found in the deep-sea environment. Specimens of giant tube worms (Riftia pachyptila), mussels (Bathymodiolus thermophilus), and clams (Calyptogena magnifica) were collected during the 2002 R/V Atlantis research cruises to the East Pacific Rise (9N) and Galápagos Rift. Microbial biofilms attached to the exterior surfaces of individual animals were sampled, as were tissues known to harbor chemosynthetic bacterial endosymbionts. Genomic DNA was extracted from the samples using a commercially available kit, and 16S rRNA genes amplified from the mixed bacterial communities using the polymerase chain reaction (PCR) and oligonucleotide primers targeting conserved terminal regions of the 16S rRNA gene. The PCR products obtained were cloned into a plasmid vector and the recombinant plasmids transformed into cells of Escherichia coli. Individual cloned 16S rRNA genes were sequenced at the 5' end of the gene (the most phylogenetically informative region in most taxa) and the sequence data compared to publicly available gene sequence databases, to allow a preliminary assignment of clones to taxonomic groups within the Bacteria and Archaea, and to determine the overall composition and phylogenetic diversity of the animal-associated microbial communities. Analysis of Riftia pachyptila exterior biofilm samples revealed the presence of members of the delta and epsilon proteobacteria, low GC Gram positive bacteria (firmicutes), spirochetes, CFB (Cytophaga-Flavobacterium-Bacteroides) group, green nonsulfur bacteria, acidobacteria, verrucomicrobia, and planctomycetes. The presence of the latter three taxonomic groups is of special interest, as they represent phylogenetically distinct groups within the Bacteria for which specific ecological functions have not yet been identified, but which have been found to be widely distributed and often numerically significant in diverse terrestrial and aquatic habitats. Although further sequencing is required to demonstrate the presence of a Riftia-associated microbial population distinct from that of the surrounding seawater, results available from three Riftia individuals from the East Pacific Rise suggest this to be the case. Analysis of microbial communities associated with the gill tissue of the mussel Bathymodiolus thermophilus shows a population dominated by gamma-Proteobacterial chemoautotrophic symbionts, although lower frequency novel phylotypes have been detected. Representatives of specific taxonomic groups have been selected for sequencing of the complete 16S rRNA gene, and the sequences used to reconstruct phylogenetic trees to more accurately determine the evolutionary relationships between the novel sequences, and available sequences for both cultured and non-cultured bacteria.
-
Golla, K; Selokar, N L; Saini, M; Chauhan, M S; Manik, R S; Palta, P; Singla, S K
2012-10-01
Somatic cells in milk are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important in animals that are susceptible to risks of bacterial infection on biopsy collection. In this study, a minimum of 10 milk samples were collected from each of the three buffaloes representing Murrah breed. All the samples were processed immediately and cell colonies were obtained. Cell colonies from one buffalo (MU-442) survived beyond 10 passages and were evaluated by fluorescence microscopy and used in nuclear transfer experiments. In culture, these cells expressed vimentin, indicating they were of fibroblast origin similar to ear cells. We compared the effectiveness of cloning using those milk-derived fibroblast (MDF) cells and fibroblast cells derived from the ear derived fibroblast (EDF). Fusion and cleavage rates of MDF-NT and EDF-NT embryos were found to be similar (92.43 ± 1.28% vs 94.98 ± 1.24%, and 80.27 ± 1.75% vs 84.56 ± 3.73%, respectively; p > 0.01); however, development to blastocyst stage and total cell number was higher for EDF-NT embryos (50.24 ± 2.54%, 227.14 ± 13.04, respectively, p < 0.01), than for MDF-NT embryos (16.44 ± 0.75%, 170.57 ± 4.50 respectively). We conclude that somatic cells from milk can be cultured effectively and used as nucleus donor to produce cloned blastocyst-stage embryos. © 2012 Blackwell Verlag GmbH.
-
Arii, Jun; Hushur, Orkash; Kato, Kentaro; Kawaguchi, Yasushi; Tohya, Yukinobu; Akashi, Hiroomi
2006-04-01
Canine herpesvirus (CHV) is an attractive candidate not only for use as a recombinant vaccine to protect dogs from a variety of canine pathogens but also as a viral vector for gene therapy in domestic animals. However, developments in this area have been impeded by the complicated techniques used for eukaryotic homologous recombination. To overcome these problems, we used bacterial artificial chromosomes (BACs) to generate infectious BACs. Our findings may be summarized as follows: (i) the CHV genome (pCHV/BAC), in which a BAC flanked by loxP sites was inserted into the thymidine kinase gene, was maintained in Escherichia coli; (ii) transfection of pCHV/BAC into A-72 cells resulted in the production of infectious virus; (iii) the BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus CHV/BAC by co-infection with CHV/BAC and a recombinant adenovirus that expressed the Cre recombinase; and (iv) a recombinant virus in which the glycoprotein C gene was deleted was generated by lambda recombination followed by Flp recombination, which resulted in a reduction in viral titer compared with that of the wild-type virus. The infectious clone pCHV/BAC is useful for the modification of the CHV genome using bacterial genetics, and CHV/BAC should have multiple applications in the rapid generation of genetically engineered CHV recombinants and the development of CHV vectors for vaccination and gene therapy in domestic animals.
-
Continuous nucleus extraction by optically-induced cell lysis on a batch-type microfluidic platform.
Huang, Shih-Hsuan; Hung, Lien-Yu; Lee, Gwo-Bin
2016-04-21
The extraction of a cell's nucleus is an essential technique required for a number of procedures, such as disease diagnosis, genetic replication, and animal cloning. However, existing nucleus extraction techniques are relatively inefficient and labor-intensive. Therefore, this study presents an innovative, microfluidics-based approach featuring optically-induced cell lysis (OICL) for nucleus extraction and collection in an automatic format. In comparison to previous micro-devices designed for nucleus extraction, the new OICL device designed herein is superior in terms of flexibility, selectivity, and efficiency. To facilitate this OICL module for continuous nucleus extraction, we further integrated an optically-induced dielectrophoresis (ODEP) module with the OICL device within the microfluidic chip. This on-chip integration circumvents the need for highly trained personnel and expensive, cumbersome equipment. Specifically, this microfluidic system automates four steps by 1) automatically focusing and transporting cells, 2) releasing the nuclei on the OICL module, 3) isolating the nuclei on the ODEP module, and 4) collecting the nuclei in the outlet chamber. The efficiency of cell membrane lysis and the ODEP nucleus separation was measured to be 78.04 ± 5.70% and 80.90 ± 5.98%, respectively, leading to an overall nucleus extraction efficiency of 58.21 ± 2.21%. These results demonstrate that this microfluidics-based system can successfully perform nucleus extraction, and the integrated platform is therefore promising in cell fusion technology with the goal of achieving genetic replication, or even animal cloning, in the near future.
-
Zischka, Melanie; Künne, Carsten T; Blom, Jochen; Wobser, Dominique; Sakιnç, Türkân; Schmidt-Hohagen, Kerstin; Dabrowski, P Wojtek; Nitsche, Andreas; Hübner, Johannes; Hain, Torsten; Chakraborty, Trinad; Linke, Burkhard; Goesmann, Alexander; Voget, Sonja; Daniel, Rolf; Schomburg, Dietmar; Hauck, Rüdiger; Hafez, Hafez M; Tielen, Petra; Jahn, Dieter; Solheim, Margrete; Sadowy, Ewa; Larsen, Jesper; Jensen, Lars B; Ruiz-Garbajosa, Patricia; Quiñones Pérez, Dianelys; Mikalsen, Theresa; Bender, Jennifer; Steglich, Matthias; Nübel, Ulrich; Witte, Wolfgang; Werner, Guido
2015-03-12
Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type. We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro). Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.
-
Donate Home Cloning What is Cloning What is Cloning Clones are organisms that are exact genetic copies. ... clones made through modern cloning technologies. How Is Cloning Done? Many people first heard of cloning when ...
-
Deuterostome evolution: early development in the enteropneust hemichordate, Ptychodera flava
NASA Technical Reports Server (NTRS)
Henry, J. Q.; Tagawa, K.; Martindale, M. Q.
2001-01-01
Molecular and morphological comparisons indicate that the Echinodermata and Hemichordata represent closely related sister-phyla within the Deuterostomia. Much less is known about the development of the hemichordates compared to other deuterostomes. For the first time, cell lineage analyses have been carried out for an indirect-developing representative of the enteropneust hemichordates, Ptychodera flava. Single blastomeres were iontophoretically labeled with Dil at the 2- through 16-cell stages, and their fates followed through development to the tornaria larval stage. The early cleavage pattern of P. flava is similar to that of the direct-developing hemichordate, Saccoglossus kowalevskii, as well as that displayed by indirect-developing echinoids. The 16-celled embryo contains eight animal "mesomeres," four slightly larger "macromeres," and four somewhat smaller vegetal "micromeres." The first cleavage plane was not found to bear one specific relationship relative to the larval dorsoventral axis. Although individual blastomeres generate discrete clones of cells, the appearance and exact locations of these clones are variable with respect to the embryonic dorsoventral and bilateral axes. The eight animal mesomeres generate anterior (animal) ectoderm of the larva, which includes the apical organ; however, contributions to the apical organ were found to be variable as only a subset of the animal blastomeres end up contributing to its formation and this varies from embryo to embryo. The macromeres generate posterior larval ectoderm, and the vegetal micromeres form all the internal, endomesodermal tissues. These blastomere contributions are similar to those found during development of the only other hemichordate studied, the direct-developing enteropneust, S. kowalevskii. Finally, isolated blastomeres prepared at either the two- or the four-cell stage are capable of forming normal-appearing, miniature tornaria larvae. These findings indicate that the fates of these cells and embryonic dorsoventral axial properties are not committed at these early stages of development. Comparisons with the developmental programs of other deuterostome phyla allow one to speculate on the conservation of some key developmental events/mechanisms and propose basal character states shared by the ancestor of echinoderms and hemichordates.
-
McCrackin, M A; Helke, Kristi L; Galloway, Ashley M; Poole, Ann Z; Salgado, Cassandra D; Marriott, Bernadette P
2016-10-02
Controversy continues concerning antimicrobial use in food animals and its relationship to drug-resistant infections in humans. We systematically reviewed published literature for evidence of a relationship between antimicrobial use in agricultural animals and drug-resistant foodborne campylobacteriosis in humans. Based on publications from the United States (U.S.), Canada and Denmark from 2010 to July 2014, 195 articles were retained for abstract review, 50 met study criteria for full article review with 36 retained for which data are presented. Two publications reported increase in macrolide resistance of Campylobacter coli isolated from feces of swine receiving macrolides in feed, and one of these described similar findings for tetracyclines and fluoroquinolones. A study in growing turkeys demonstrated increased macrolide resistance associated with therapeutic dosing with Tylan® in drinking water. One publication linked tetracycline-resistant C. jejuni clone SA in raw cow's milk to a foodborne outbreak in humans. No studies that identified farm antimicrobial use also traced antimicrobial-resistant Campylobacter from farm to fork. Recent literature confirms that on farm antibiotic selection pressure can increase colonization of animals with drug-resistant Campylobacter spp. but is inadequately detailed to establish a causal relationship between use of antimicrobials in agricultural animals and prevalence of drug-resistant foodborne campylobacteriosis in humans.
-
Marusina, Alina I; Ono, Yoko; Merleev, Alexander A; Shimoda, Michiko; Ogawa, Hiromi; Wang, Elizabeth A; Kondo, Kayo; Olney, Laura; Luxardi, Guillaume; Miyamura, Yoshinori; Yilma, Tilahun D; Villalobos, Itzel Bustos; Bergstrom, Jennifer W; Kronenberg, Daniel G; Soulika, Athena M; Adamopoulos, Iannis E; Maverakis, Emanual
2017-02-01
It is widely accepted that central and effector memory CD4 + T cells originate from naïve T cells after they have encountered their cognate antigen in the setting of appropriate co-stimulation. However, if this were true the diversity of T cell receptor (TCR) sequences within the naïve T cell compartment should be far greater than that of the memory T cell compartment, which is not supported by TCR sequencing data. Here we demonstrate that aged mice with far fewer naïve T cells, respond to the model antigen, hen eggwhite lysozyme (HEL), by utilizing the same TCR sequence as their younger counterparts. CD4 + T cell repertoire analysis of highly purified T cell populations from naive animals revealed that the HEL-specific clones displayed effector and central "memory" cell surface phenotypes even prior to having encountered their cognate antigen. Furthermore, HEL-inexperienced CD4 + T cells were found to reside within the naïve, regulatory, central memory, and effector memory T cell populations at similar frequencies and the majority of the CD4 + T cells within the regulatory and memory populations were unexpanded. These findings support a new paradigm for CD4 + T cell maturation in which a specific clone can undergo a differentiation process to exhibit a "memory" or regulatory phenotype without having undergone a clonal expansion event. It also demonstrates that a foreign-specific T cell is just as likely to reside within the regulatory T cell compartment as it would the naïve compartment, arguing against the specificity of the regulatory T cell compartment being skewed towards self-reactive T cell clones. Finally, we demonstrate that the same set of foreign and autoreactive CD4 + T cell clones are repetitively generated throughout adulthood. The latter observation argues against T cell-depleting strategies or autologous stem cell transplantation as therapies for autoimmunity-as the immune system has the ability to regenerate pathogenic clones. Published by Elsevier Ltd.
-
Liu, Jun; Luo, Yan; Liu, Qingqing; Zheng, Liming; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong
2013-03-01
Transgenic animals that express antimicrobial agents in their milk can inhibit bacterial pathogens that cause mastitis. Our objective was to produce human β-defensin-3 (HBD3) transgenic embryos by nuclear transfer using goat mammary epithelial cells (GMECs) as donor cells. Three GMEC lines (GMEC1, GMEC2, and GMEC3) were transfected with a HBD3 mammary-specific expression vector by electroporation. There was a difference (P < 0.05) in the rate of geneticin-resistant colony formation among cell lines GMEC1, GMEC2, and GMEC3 (39 and 47 vs. 19 colonies per 3 × 10(6) cells, respectively). After inducing expression, the mRNA and protein of HBD3 were detected by reverse transcription polymerase chain reaction and Western blot analysis in transgenic cells. Transgenic clonal cells expressing HBD3 were used as donor cells to investigate development of cloned embryos. There were no significant differences in rates of cleavage or blastocyst formation of cloned embryos from transgenic (GMEC1T2 and GMEC2T3) and nontransgenic (GMEC1 and GMEC2) GMECs (72.3 ± 5.0%, 69.5 ± 2.3%, 61.8 ± 4.8%, and 70.0 ± 2%; and 16.8 ± 0.5%, 17.5 ± 0.7%, 16.7 ± 0.9%, and 17.5 ± 0.6%, respectively). However, the fusion rate, cleavage rate, and blastocyst formation rate of cloned embryos from a transgenic clonal cell line (GMEC2T6, 50.7 ± 2.1%, 55.5 ± 2.0%, and 11.1 ± 0.6%) were lower than those of other groups (P < 0.05). We concluded that genetic modification of GMECs might not influence the in vitro development of cloned embryos, but that some of the transgenic clonal cells were not suitable for nuclear transfer to produce transgenic goats, because of low developmental rates. However, transgenic GMECs expressing HBD3 might be used as donor cells for producing transgenic goats that express increased concentrations of β-defensins in their milk. Copyright © 2013 Elsevier Inc. All rights reserved.
-
Comparative analysis of charged particle-induced autosomal mutations in murine cells and tissues
NASA Astrophysics Data System (ADS)
Kronenberg, Amy; Gauny, Stacey; Turker, Mitchell; Dan, Cristian; Kwoh, Ely
Carcinogenesis requires the accumulation of mutations and most of these mutations of occur on autosomes. This study seeks to determine the effect of the tissue microenvironment on the frequency and types of autosomal mutations in epithelial cells exposed to the types of charged particles in space radiation environments. Epithelial cells are the principal cells at risk for the development of solid cancers in humans. Aprt heterozygous mice from a cross between C57BL/6 and DBA/2 mice (B6D2F1) are used for these studies. The tissue of interest here is the kidney. We evaluated the effects of Fe ion on cytotoxicity, mutant frequency, and mutant spectra in kidney epithelium exposed in vivo. In vitro studies use primary kidney clones from B6D2F1 mice. Animals or cells were exposed to graded doses (0-2 Gy) of 1 GeV/amu Fe ions at the NASA Space Radiation Laboratories at Brookhaven National Laboratory. Animals were given whole body exposure in plexiglas holders. Cells were irradiated in T-75 flasks as monolayers. Cytotoxicity for cells exposed as monolayers were performed immediately post-irradiation. In vitro mutation assays were performed after a 5-6 day expression period post-irradiation, at which time cells were seeded in standard medium supplemented with 2,6 diaminopurine to screen for Aprt mutants. Colony formation was assessed in parallel in standard medium. In contrast, mice were euthanized after 2-4 months post-irradiation (early) or 8-10 months post-irradiation (late) to determine the cytotoxic and mutagenic response to Fe ion irradiation. Once the kidneys were digested, the cytotoxicity and mutation assays were performed using the same methodology employed for cells in vitro. Individual Apr t mutant colonies were collected from separate flasks exposed in vitro to 2 Gy of Fe ions. A similar group of Aprt mutants were collected from separate, un-irradiated flasks Aprt mutant colonies were also collected from individual kidneys for un-irradiated mice and for mice exposed to 2 Gy of Fe ions. Mutant spectra were analyzed via PCR using a series of heterozygous markers along mouse chromosome 8. Cytotoxicity assays were performed immediately after Fe ion exposure of cells from two primary clones. Cells irradiated in vitro demonstrated a dose-dependent decrement in cloning efficiency with no evidence of a shoulder. The results demonstrate the two clones behave similarly (unpaired t-test, p>0.3) with a D0 of 84.3 cGy for the combined data set. Mutation data were obtained using cells from one of the primary clones. In three experiments, we observed a linear dose-response for Aprt mutation with an induced mutant frzction of 1.06 x 10-3 /Gy. Kidney epithelial cells irradiated in vivo and incubated for 2-4 months in situ prior to harvest also showed an exponential reduction of cloning efficiency. Cells harvested 8-10 months postirradiation showed evidence of recovery for doses up to 1.5 Gy, but there was no improvement in cloning efficiency for kidney cells exposed to 2 Gy Fe ions in vivo evaluated at the late time point. Results for Aprt mutation induction in vivo indicated considerable inter-animal variation within each dose group (0, 1.0, 1.5 and 2 Gy). Fe ion exposures were mutagenic to the kidney, even at the lowest dose (p<0.01). A comparison of the mutant frequency results at the two harvest times indicates that the dose response did not vary with incubation time in vivo. Analysis of the pooled data from the 2-4 months harvests and the 8-10 month harvests indicated an increase in mutant frequency of 1.49 fold per Gy (p=0.01, CI 1.11-2.01). Molecular analysis of Aprtdeficient cells collected after a 2 Gy exposure to Fe ions in vitro showed an increased proportion of mutants arising via interstitial deletion or mitotic recombination, with an indication of an increase in chromosome loss. Similar results have been obtained for Aprt mutants isolated from mice exposed to 2 Gy of Fe ions, as compared with mutants collected from sham-irradiated mice. Taken together, the results to date demonstrate that Fe ions are mutagenic to mouse kidney epithelium exposed in vitro assayed at short times post-irradiation and they are also mutagenic to kidney epithelium exposed in vivo. While cytotoxicity is somewhat ameliorated in vivo, toxicity was evident at the highest dose up to 8-10 months post-exposure. A comparison of the Aprt mutant frequency analyses (in vitro vs. in vivo) and mutation spectra analyses (in vitro vs. in vivo) reflect similar trends. Supported by NASA grant T-403X to A. Kronenberg.
-
Madec, J-Y; Haenni, M; Nordmann, P; Poirel, L
2017-11-01
There has been a great and long-term concern that extended-spectrum β-lactamase (ESBL)/AmpC- and carbapenemase-producing Enterobacteriaceae occurring in animals may constitute a public-health issue. A large number of factors with complex interrelations contribute to the spread of those bacteria among animals and humans. ESBL/AmpC- or carbapenemase-encoding genes are most often located on mobile genetic elements favouring their dissemination. Some shared reservoirs of ESBL/AmpC or carbapenemase genes, plasmids or clones have been identified and suggest cross-transmissions. Even though exposure to animals is regarded as a risk factor, evidence for a direct transfer of ESBL/AmpC-producing bacteria from animals to humans through close contacts is limited. Nonetheless, the size of the commensal ESBL/AmpC reservoir in non-human sources is dramatically rising. This may constitute an indirect risk to public health by increasing the gene pool from which pathogenic bacteria can pick up ESBL/AmpC/carbapenemase genes. The extent to which food contributes to potential transmission of ESBL/AmpC producers to humans is also not well established. Overall, events leading to the occurrence of ESBL/AmpC- and carbapenemase-encoding genes in animals seem very much multifactorial. The impact of animal reservoirs on human health still remains debatable and unclear; nonetheless, there are some examples of direct links that have been identified. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
-
Emerging Technologies to Create Inducible and Genetically Defined Porcine Cancer Models
Schook, Lawrence B.; Rund, Laurie; Begnini, Karine R.; Remião, Mariana H.; Seixas, Fabiana K.; Collares, Tiago
2016-01-01
There is an emerging need for new animal models that address unmet translational cancer research requirements. Transgenic porcine models provide an exceptional opportunity due to their genetic, anatomic, and physiological similarities with humans. Due to recent advances in the sequencing of domestic animal genomes and the development of new organism cloning technologies, it is now very feasible to utilize pigs as a malleable species, with similar anatomic and physiological features with humans, in which to develop cancer models. In this review, we discuss genetic modification technologies successfully used to produce porcine biomedical models, in particular the Cre-loxP System as well as major advances and perspectives the CRISPR/Cas9 System. Recent advancements in porcine tumor modeling and genome editing will bring porcine models to the forefront of translational cancer research. PMID:26973698
-
Adams, Mark; Foster, Ralph; Hutchinson, Mark N; Hutchinson, Rhonda G; Donnellan, Steve C
2003-11-01
Molecular data derived from allozymes and mitochondrial nucleotide sequences, in combination with karyotypes, sex ratios, and inheritance data, have revealed the widespread Australian lizard Menetia greyii to be a complex of sexual and triploid unisexual taxa. Three sexual species, three presumed parthenogenetic lineages, and one animal of uncertain status were detected amongst 145 animals examined from south-central Australia, an area representing less than one-seventh of the total distribution of the complex. Parthenogenesis appears to have originated via interspecific hybridization, although presumed sexual ancestors could only be identified in two cases. The allozyme and mtDNA data reveal the presence of many distinct clones within the presumed parthenogenetic lineages. This new instance of vertebrate parthenogenesis is a first for the Scincidae and only the second definitive case of unisexuality in an indigenous Australian vertebrate.
-
NASA Astrophysics Data System (ADS)
Olsen, David; Chang, Robert; Williams, Kim E.; Polarek, James W.
We have developed a recombinant expression system to produce a series of novel recombinant human gelatins that can substitute for animal sourced gelatin preparations currently used in pharmaceutical and nutraceutical applications. This system allows the production of human sequence gelatins, or, if desired, gelatins from any other species depending on the availability of the cloned gene. The gelatins produced with this recombinant system are of defined molecular weight, unlike the animal-sourced gelatins, which consist of numerous polypeptides of varying size. The fermentation and purification process used to prepare these recombinant gelatins does not use any human- or animal-derived components and thus this recombinant material should be free from viruses and agents that cause transmissible spongiform encephalopathies. The recombinant gelatins exhibit lot-to-lot reproducibility and we have performed extensive analytical testing on them. We have demonstrated the utility of these novel gelatins as biological stabilizers and plasma expanders, and we have shown they possess qualities that are important in applications where gel formation is critical. Finally, we provide examples of how our system allows the engineering of these recombinant gelatins to optimize the production process.
-
Anticonvulsant Effect of Diazoxide against Dichlorvos-Induced Seizures in Mice
Jazayeri, Amin; Zolfaghari, Samira; Ostadhadi, Sattar
2013-01-01
Dichlorvos, a synthetic organophosphate toxin, is used as pesticides. These toxins can be used as pesticides in farming and medicine for the devastation and/or elimination of ectoparasites of animals. Reports have shown that Dichlorvos generate seizure effects in various animals. Potassium channel opener is extensively used for medication of cardiovascular and other diseases. Studies have shown that potassium channel opener has anticonvulsant effects in different animal models. The goal of this study was to evaluate the effect of dizoxide on Dichlorvos-induced seizures in mice. In this research, the animals received different doses of Diazoxide (1, 2.5, 5, 10, and 20 mg/kg b.wt.) intraperitoneally 30 min before intraperitoneal injection of Dichlorvos (50 mg/kg b.w.t). After Dichlorvos injection, latency of clones, severity of seizure, and finally death as the fate were investigated. Results showed that Diazoxide dose-dependently decreased the severity of Dichlorvos-induced seizures, so that Diazoxide at a dose of 5 mg (the lowest, P < 0.05) and 20 mg/kg b.wt. (the highest, P < 0.001) has anticonvulsant effects. Thus, our data suggest that diazoxide as ATP-sensitive potassium channels opener has anticonvulsant activity against dichlorvas-induced seizure. PMID:24453891
-
Li, Yang; Cang, Ming; Lee, Andrew Stephen; Zhang, Kehua; Liu, Dongjun
2011-01-01
Animal embryonic stem cells (ESCs) provide powerful tool for studies of early embryonic development, gene targeting, cloning, and regenerative medicine. However, the majority of attempts to establish ESC lines from large animals, especially ungulate mammals have failed. Recently, another type of pluripotent stem cells, known as induced pluripotent stem cells (iPSCs), have been successfully generated from mouse, human, monkey, rat and pig. In this study we show sheep fibroblasts can be reprogrammed to pluripotency by defined factors using a drug-inducible system. Sheep iPSCs derived in this fashion have a normal karyotype, exhibit morphological features similar to those of human ESCs and express AP, Oct4, Sox2, Nanog and the cell surface marker SSEA-4. Pluripotency of these cells was further confirmed by embryoid body (EB) and teratoma formation assays which generated derivatives of all three germ layers. Our results also show that the substitution of knockout serum replacement (KSR) with fetal bovine serum in culture improves the reprogramming efficiency of sheep iPSCs. Generation of sheep iPSCs places sheep on the front lines of large animal preclinical trials and experiments involving modification of animal genomes. PMID:21253598
-
Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers
Haylock, Anna-Karin; Nilvebrant, Johan; Mortensen, Anja; Velikyan, Irina; Nestor, Marika; Falk, Ronny
2017-01-01
Aim The aim of this study was to generate and characterize scFv antibodies directed to human CD44v6, as well as to radiolabel and evaluate top candidates in vitro and in vivo for their potential use in CD44v6-targeted molecular imaging in cancer patients. Materials and methods Phage display selections were used to isolate CD44v6-specific scFvs. A chain shuffling strategy was employed for affinity maturation based on a set of CD44v6-specific first-generation clones. Two second-generation scFv clones were then chosen for labeling with 111In or 125I and assessed for CD44v6-specific binding on cultured tumor cells. In vivo uptake and distribution was evaluated in tumor-bearing mice using a dual tumor model. Finally, a proof-of-concept small animal PET-CT study was performed on one of the candidates labeled with 124I. Results Two affinity-matured clones, CD44v6-scFv-A11 and CD44v6-scFv-H12, displayed promising binding kinetics. Seven out of eight radiolabeled conjugates demonstrated CD44v6-specific binding. In vivo studies on selected candidates demonstrated very advantageous tumor-to-organ ratios, in particular for iodinated conjugates, where 125I-labeled scFvs exhibited favorable kinetics and tumor-to-blood ratios above five already at 24 hours p.i.. The small animal PET-CT study using 124I-labeled CD44v6-scFv-H12 was in line with the biodistribution data, clearly visualizing the high CD44v6-expressing tumor. Conclusion The single chain fragments, CD44v6-scFv-A11 and CD44v6-scFv-H12 specifically bind to CD44v6, and the radiolabeled counterparts provide high tumor-to-blood ratios and fast clearance from organs and blood. We conclude that radioiodinated CD44v6-scFv-A11 and CD44v6-scFv-H12 possess features highly suitable for stringent molecular imaging. PMID:29029420
-
NASA Astrophysics Data System (ADS)
Huang, Xiaoyan; Yan, Yan; Wang, Sha; Wang, Qinying; Shi, Jian; Shao, Zhanshe; Dai, Jiejie
2017-11-01
CD28 is one of the most important co-stimulatory molecules expressed by naive and primed T cells. The tree shrews (Tupaia belangeri), as an ideal animal model for analyzing mechanism of human diseases receiving extensive attentions, demands essential research tools, in particular in the study of cellular markers and monoclonal antibodies for immunological studies. However, little is known about tree shrew CD28 (tsCD28) until now. In this study, a 663 bp of the full-length CD28 cDNA, encoding a polypeptide of 220 amino acids was cloned from tree shrew spleen lymphocytes. The nucleotide sequence of the tsCD28 showed 85%, 76%, and 75% similarities with human, rat, and mouse, respectively, which showed the affinity relationship between tree shrew and human is much closer than between human and rodents. The open reading frame (ORF) sequence of tsCD28 gene was predicted to be in correspondence with the signal sequence, immunoglobulin variable-like (IgV) domain, transmembrane domain and cytoplasmic tail, respectively.We also analyzed its molecular characteristics with other mammals by using biology software such as Clustal W 2.0 and so forth. Our results showed that tsCD28 contained many features conserved in CD28 genes from other mammals, including conserved signal peptide and glycosylation sites, and several residues responsible for binding to the CD28R, and the tsCD28 amino acid sequence were found a close genetic relationship with human and monkey. The crystal structure and surface charge revealed most regions of tree shrew CD28 molecule surface charges are similar as human. However, compared with human CD28 (hCD28) regions, in some areas, the surface positive charge of tsCD28 was less than hCD28, which may affect antibody binding. The present study is the first report of cloning and characterization of CD28 in tree shrew. This study provides a theoretical basis for the further study the structure and function of tree shrew CD28 and utilize tree shrew as an effective animal model of human disease.
-
2012-01-01
Background Foot-and-mouth disease (FMD) is the most economically important and highly contagious disease of cloven-hoofed animals worldwide. Control of the disease has been mainly based on large-scale vaccinations with whole-virus inactivated vaccines. In recent years, a series of outbreaks of type O FMD occurred in China (including Chinese Taipei, Chinese Hong Kong) posed a tremendous threat to Chinese animal husbandry. Its causative agent, type O FMDV, has evolved into three topotypes (East–South Asia (ME-SA), Southeast Asia (SEA), Cathay (CHY)) in these regions, which represents an important obstacle to disease control. The available FMD vaccine in China shows generally good protection against ME-SA and SEA topotype viruses infection, but affords insufficient protection against some variants of the CHY topotype. Therefore, the choice of a new vaccine strain is of fundamental importance. Results The present study describes the generation of a full-length infectious cDNA clone of FMDV vaccine strain and a genetically modified virus with some amino acid substitutions in antigenic sites 1, 3, and 4, based on the established infectious clone. The recombinant viruses had similar growth properties to the wild O/HN/CHA/93 virus. All swine immunized with inactivated vaccine prepared from the O/HN/CHA/93 were fully protected from challenge with the viruses of ME-SA and SEA topotypes and partially protected against challenge with the virus of CHY topotype at 28 days post-immunization. In contrast, the swine inoculated with the genetically modified vaccine were completely protected from the infection of viruses of the three topotypes. Conclusions Some amino acid substitutions in the FMDV vaccine strain genome did not have an effect on the ability of viral replication in vitro. The vaccine prepared from genetically modified FMDV by reverse genetics significantly improved the protective efficacy to the variant of the CHY topotype, compared with the wild O/HN/CHA/93 virus. Thus, the full-length cDNA clone of FMDV can be a useful tool to develop genetically engineered FMDV vaccine candidates to help control porcinophilic FMD epidemics in China. PMID:22591597
-
Ufnar, Jennifer A; Ufnar, David F; Wang, Shiao Y; Ellender, R D
2007-08-01
The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10(-6) g of wet pig feces in 500 ml of phosphate-buffered saline and 10(-4) g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker.
-
Harada, Kazuki; Shimizu, Takae; Mukai, Yujiro; Kuwajima, Ken; Sato, Tomomi; Kajino, Akari; Usui, Masaru; Tamura, Yutaka; Kimura, Yui; Miyamoto, Tadashi; Tsuyuki, Yuzo; Ohki, Asami; Kataoka, Yasushi
2017-01-01
The emergence of antimicrobial resistance among Enterobacter spp., including resistance to extended-spectrum cephalosporins (ESC), is of great concern in both human and veterinary medicine. In this study, we investigated the prevalence of antimicrobial resistance among 60 isolates of Enterobacter spp., including E. cloacae (n = 44), E. aerogenes (n = 10), and E. asburiae (n = 6), from clinical specimens of dogs and cats from 15 prefectures in Japan. Furthermore, we characterized the resistance mechanisms harbored by these isolates, including extended-spectrum β-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR); and assessed the genetic relatedness of ESC-resistant Enterobacter spp. strains by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Antimicrobial susceptibility testing demonstrated the resistance rates to ampicillin (93.3%), amoxicillin-clavulanic acid (93.3%), cefmetazole (93.3%), chloramphenicol (46.7%), ciprofloxacin (43.3%), tetracycline (40.0%), ceftazidime (33.3%), cefotaxime (33.3%), trimethoprim/sulfamethoxazole (28.3%), gentamicin (23.3%), and meropenem (0%). Phenotypic testing detected ESBLs in 16 of 18 ESC-resistant E. cloacae isolates but not in the other species. The most frequent ESBL was CTX-M-15 (n = 8), followed by SHV-12 (n = 7), and CTX-M-3 (n = 1). As for AmpC β-lactamases, CMY-2 (n = 2) and DHA-1 (n = 2) were identified in ESC-resistant E. cloacae strains with or without ESBLs. All of the ESC-resistant E. cloacae strains also harbored one or two PMQRs, including qnrB (n = 15), aac(6')-Ib-cr (n = 8), and qnrS (n = 2). Based on MLST and PFGE analysis, E. cloacae clones of ST591-SHV-12, ST171-CTX-M-15, and ST121-CTX-M-15 were detected in one or several hospitals. These results suggested intra- and inter-hospital dissemination of E. cloacae clones co-harboring ESBLs and PMQRs among companion animals. This is the first report on the large-scale monitoring of antimicrobial-resistant isolates of Enterobacter spp. from companion animals in Japan.
-
Ufnar, Jennifer A.; Ufnar, David F.; Wang, Shiao Y.; Ellender, R. D.
2007-01-01
The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10−6 g of wet pig feces in 500 ml of phosphate-buffered saline and 10−4 g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker. PMID:17586669
-
Linninge, Caroline; Ahrné, Siv; Molin, Göran
2015-01-01
The composition of the gut microbiota can vary widely between individual mice of the same batch and thereby affect the resulting outcome in experimental studies. Therefore, an efficient method is needed to equalize the gut microbiota prior to the start of critical experiments. In order to minimize variations in gut microbiota between animals and provide the animals with a Gram-negative flora exposing lipopolysaccharides in the cell-walls, C57BL/6 mice were given a mixture of ampicillin, metronidazole and clindamycin in the drinking water for 3 days and then Escherichia coli for two additional days. Treatment with antibiotics alone or with antibiotics in combination with E. coli was well tolerated by all animals. Body weight and liver weight were not affected, although higher hepatic fat content was found in treated animals (p < 0.05). The diversity of the gut microbiota was strongly reduced in animals treated with antibiotics and antibiotics in combination with E. coli (p < 0.01), without affecting the total amount of bacteria. Cloned and sequenced 16S rRNA genes showed high presence of Enterobacteriaceae and Porphymonadaceae in the treated animals. Analysis with Principal Component Analysis gave a clear separation of the composition in microbiota between different treatment groups. The described treatment efficiently equalized the gut microbiota and provided the animals with a strong abundance of Enterobacteriaceae without changing the total load of bacteria. This is a straightforward, lenient and efficient method of pre-treatment to equalize the gut microbiota of mice as a starting procedure of animal studies.
-
Three concepts of cloning in human beings.
Cui, Ke-Hui
2005-07-01
Human cloning, organ cloning and tissue cloning are various types of cloning that occur at different levels with different methodologies. According to three standards of terminology for an embryo (fertilization through germ cells, development in the uterus and having the potential to produce a human life), tissue cloning and type I organ cloning will not produce an embryo. In contrast, human cloning and type II organ cloning will produce an embryo. Thus, only non-germinal tissue cloning and type I organ cloning are beyond the ethical question and will not change human beings as a species. Using cloned tissues to make new tissues or organs is promising for the future of medicine.
-
Muzaffar, Musharifa; Selokar, Naresh L.; Singh, Karn P.; Zandi, Mohammad; Singh, Manoj K.; Shah, Riaz A.; Chauhan, Manmohan S.; Singla, Suresh K.; Palta, Prabhat
2012-01-01
Abstract This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production. PMID:22582863
-
Progress in gene targeting and gene therapy for retinitis pigmentosa
DOE Office of Scientific and Technical Information (OSTI.GOV)
Farrar, G.J.; Humphries, M.M.; Erven, A.
1994-09-01
Previously, we localized disease genes involved in retinitis pigmentosa (RP), an inherited retinal degeneration, close to the rhodopsin and peripherin genes on 3q and 6p. Subsequently, we and others identified mutations in these genes in RP patients. Currently animal models for human retinopathies are being generated using gene targeting by homologous recombination in embryonic stem (ES) cells. Genomic clones for retinal genes including rhodopsin and peripherin have been obtained from a phage library carrying mouse DNA isogenic with the ES cell line (CC1.2). The peripherin clone has been sequenced to establish the genomic structure of the mouse gene. Targeting vectorsmore » for rhodopsin and peripherin including a neomycin cassette for positive selection and thymidine kinase genes enabling selection against random intergrants are under construction. Progress in vector construction will be presented. Simultaneously we are developing systems for delivery of gene therapies to retinal tissues utilizing replication-deficient adenovirus (Ad5). Efficacy of infection subsequent to various methods of intraocular injection and with varying viral titers is being assayed using an adenovirus construct containing a CMV promoter LacZ fusion as reporter and the range of tissues infected and the level of duration of LacZ expression monitored. Viral constructs with the LacZ reporter gene under the control of retinal specific promoters such as rhodopsin and IRBP cloned into pXCJL.1 are under construction. An update on developments in photoreceptor cell-directed expression of virally delivered genes will be presented.« less
-
Cloning, sequencing, and expression of interferon-γ from elk in North America
Sweeney, Steven J.; Emerson, Carlene; Eriks, Inge S.
2001-01-01
Eradication of Mycobacterium bovis relies on accurate detection of infected animals, including potential domestic and wildlife reservoirs. Available diagnostic tests lack the sensitivity and specificity necessary for accurate detection, particularly in infected wildlife populations. Recently, an in vitro diagnostic test for cattle which measures plasma interferon-gamma (IFN-γ) levels in blood following in vitro incubation with M. bovis purified protein derivative has been enveloped. This test appears to have increased sensitivity over traditional testing. Unfortunately, it does not detect IFN-γ from Cervidae. To begin to address this problem, the IFN-γ gene from elk (Cervus elaphus) was cloned, sequenced, expressed, and characterized. cDNA was cloned from mitogen stimulated peripheral blood mononuclear cells. The predicted amino acid (aa) sequence was compared to known sequences from cattle, sheep, goats, red deer (Cervus elaphus), humans, and mice. Biological activity of the recombinant elk IFN-γ (rElkIFN-γ) was confirmed in a vesicular stomatitis virus cytopathic effect reduction assay. Production of monoclonal antibodies to IFN-γ epitopes conserved between ruminant species could provide an important tool for the development of reliable, practical diagnostic assays for detection of a delayed type hypersensitivity response to a variety of persistent infectious agents in ruminants, including M. bovis and Brucella abortus. Moreover, development of these reagents will aid investigators in studies to explore immunological responses of elk that are associated with resistance to infectious diseases.
-
Muzaffar, Musharifa; Selokar, Naresh L; Singh, Karn P; Zandi, Mohammad; Singh, Manoj K; Shah, Riaz A; Chauhan, Manmohan S; Singla, Suresh K; Palta, Prabhat; Manik, Radheysham
2012-06-01
This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production.
-
Silverman, Lee R.; Phipps, Andrew J.; Montgomery, Andy; Fernandez, Soledad; Tsukahara, Tomonori; Ratner, Lee; Lairmore, Michael D.
2005-01-01
Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell lymphoma/leukemia (ATL). The HTLV-1 envelope gene exhibits limited variability when examined from infected individuals, but has not been tested using infectious clones of the virus in animal models. In vitro assays indicate that HTLV-1 envelope (Env) Ser75Ile, Asn95Asp, and Asn195Asp surface unit (SU) mutants are able to replicate in and immortalize lymphocytes. Herein, we examined the effects of these Env mutants in rabbits inoculated with HTLV-1 immortalized ACH.75, ACH.95, or ACH.195 cell lines (expressing full-length molecular clones with the SU mutations) or the ACH.1 cell line (expressing wild-type SU). All rabbits became infected, and the fidelity of the mutations was maintained throughout the 8-week study. However, SU point mutations resulted in decreased antibody responses to viral group-associated antigen (Gag) and Env antigens. ACH.195 rabbits had a selective decreased antibody response to SU, and one ACH.195 rabbit had an antibody response to both HTLV-1 and HTLV-2 SUs. Some mutant inoculation groups had altered proviral loads. However, peripheral-blood mononuclear cell (PBMC) proviral loads did not correlate with antibody responses. Our data are the first to demonstrate that mutations in critical determinants of HTLV-1 Env SU altered antibody responses and proviral loads, but do not prevent viral replication in vivo. PMID:16046523
-
Suzuki, Saori; Mori, Ken-Ichi; Higashino, Atsunori; Iwasaki, Yuki; Yasutomi, Yasuhiro; Maki, Noboru; Akari, Hirofumi
2016-01-01
The development of effective hepatitis C virus (HCV) vaccines is essential for the prevention of further HCV dissemination, especially in developing countries. Therefore the aim of this study is to establish a feasible and immunocompetent surrogate animal model of HCV infection that will help in evaluation of the protective efficacy of newly developing HCV vaccine candidates. To circumvent the narrow host range of HCV, an HCV genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B (GBV-B), which is closely related to HCV, was generated. The chimera between HCV and GBV-B, named HCV/G, replicated more efficiently as compared with the HCV clone in primary marmoset hepatocytes. Furthermore, it was found that the chimera persistently replicated in a tamarin for more than 2 years after intrahepatic inoculation of the chimeric RNA. Although relatively low (<200 copies/mL), the viral RNA loads in plasma were detectable intermittently during the observation period. Of note, the chimeric RNA was found in the pellet fraction obtained by ultracentrifugation of the plasma at 73 weeks, indicating production of the chimeric virus. Our results will help establish a novel non-human primate model for HCV infection on the basis of the HCV/G chimera in the major framework of the HCV genome. © 2015 The Societies and John Wiley & Sons Australia, Ltd.
-
McCarthy, D
1999-01-01
Is cloning human beings morally wrong? The basis for the one serious objection to cloning is that, because of what a clone is, clones would have much worse lives than non-clones. I sketch a fragment of moral theory to make sense of the objection. I then outline several ways in which it might be claimed that, because of what a clone is, clones would have much worse lives than non-clones. In particular, I look at various ideas connected with autonomy. I conclude that there is no basis to the claim that, because of what a clone is, clones would have much worse lives than non-clones. I therefore reject the claim that cloning human beings is morally wrong. PMID:10226912
-
Transmitter responsiveness in two newly isolated clones of neuroblastoma X glioma hybrid.
Ogura, A; Amano, T
1983-01-10
Mouse neuroblastoma clone N1E-115 cells and rat glioma clone C6 cells were hybridized and two new clones were isolated. One clone, designated NG115-301, possessed weak electric excitability to an applied current pulse, while another clone, NG115-401, generated an action potential in response to the pulse. The former clone responded to serotonin and catecholamines with slow hyperpolarizations, while the latter clone responded to catecholamines with transient depolarizations. Both clones did not respond to acetylcholine. These types of responses have not been reported in any available clones. These clones may enrich the repertoire of cell clones useful for the characterization of transmitter reception mechanisms in the nervous system.
-
Response of mouse epidermal cells to single doses of heavy-particles
NASA Technical Reports Server (NTRS)
Leith, J. T.; Schilling, W. A.; Welch, G. P.
1972-01-01
The survival of mouse epidermal cells to heavy-particles has been studied In Vivo by the Withers clone technique. Experiments with accelerated helium, lithium and carbon ions were performed. The survival curve for the helium ion irradiations used a modified Bragg curve method with a maximum tissue penetration of 465 microns, and indicated that the dose needed to reduce the original cell number to 1 surviving cell/square centimeters was 1525 rads with a D sub o of 95 rads. The LET at the basal cell layer was 28.6 keV per micron. Preliminary experiments with lithium and carbon used treatment doses of 1250 rads with LET's at the surface of the skin of 56 and 193 keV per micron respectively. Penetration depths in skin were 350 and 530 microns for the carbon and lithium ions whose Bragg curves were unmodified. Results indicate a maximum RBE for skin of about 2 using the skin cloning technique. An attempt has been made to relate the epidermal cell survival curve to mortality of the whole animal for helium ions.
-
Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A.; Shen, Li; Inoue, Azusa; Zhang, Yi
2014-01-01
SUMMARY Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major epigenetic barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by IVF but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells, and its removal by ectopic expression of the H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency. PMID:25417163
-
Johnson, S C; Ewart, K V; Osborne, J A; Delage, D; Ross, N W; Murray, H M
2002-09-01
The salmon louse, Lepeophtheirus salmonis, is a marine ectoparasitic copepod that infects salmonid fishes. We are studying the interactions between this parasite and its salmonid hosts, as it is a common cause of disease in both wild and farmed stocks of salmon. In this paper, we report on the cloning and sequencing of seven trypsin-like enzymes from a cDNA library prepared from whole body preadult female and male L. salmonis. The predicted trypsin activation peptides are 23 or 24 residues in length, considerably longer than previously reported activation peptides of other animals. Differences in the putative signal and activation peptide sequences of the trypsin isoforms suggest that these forms differ in their regulation and function. The calculated molecular weights of the trypsins range from 23.6 to 23.7 kDa. There are eight cysteine residues, which suggest the presence of four disulfide bridges. These trypsins are very similar (>or=46% aa identity) to other crustacean trypsins and insect hypodermins. Using in situ hybridization techniques trypsinogen expression could be identified in all three cell types of the midgut.
-
T, Perrone; P M, Aso; A, Mijares; P, Holzmuller; M, Gonzatti; N, Parra
2018-04-15
Livestock trypanosomoses, caused by three species of the Trypanozoon subgenus, Trypanosoma brucei brucei, T. evansi and T. equiperdum are widely distributed and limit animal production throughout the world. The infectivity and virulence of clones derived from Trypanosoma evansi and Trypanosoma equiperdum Venezuelan strains were compared in an in vivo mouse model. Primary infectivity and virulence determinants such as survival rates, parasitemia levels, PCV, and changes in body weight and survival rates were monitored for up to 32 days. The T. equiperdum strain was the most virulent, with 100% mortality in mice, with the highest parasitemia levels (7.0 × 10 7 Tryps/ml) and loss of physical condition. The T. evansi strains induced 100% and 20% fatality in mice. Our results show that the homogeneous parasite populations maintain the virulent phenotype of the original T. equiperdum and T. evansi stocks. This is the first comparative study of infectivity and virulence determinants among clonal populations of T. equiperdum and T. evansi. Copyright © 2018 Elsevier B.V. All rights reserved.
-
In Vivo Imaging of Chikungunya Virus in Mice and Aedes Mosquitoes Using a Renilla Luciferase Clone
Ziegler, Sarah A.; Nuckols, John; McGee, Charles E.; Huang, Yan-Jang Scott; Vanlandingham, Dana L.; Tesh, Robert B.
2011-01-01
Abstract Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that in humans causes an acute febrile illness characterized by fever, arthralgia, and rash. It is currently associated with large outbreaks in Asia, Africa, and islands of the Indian Ocean and has been introduced from these tropical regions into Europe, where local transmission has been recorded on two occasions. The underlying basis of the pathogenesis of CHIKV and related alphaviruses that produce similar symptoms remains unclear. By applying new techniques, for example, in vivo imaging in live animals and arthropods, we may improve our understanding of viral pathogenesis in vertebrates and viral replication in mosquitoes. This technical report describes the evaluation of a CHIKV–luciferase clone to visualize infection and dissemination in both Aedes aegypti and Aedes albopictus mosquitoes and mice. In mosquitoes, luciferase activity was seen at 3 and 7 days post-infection in both head and abdomens. In vivo imaging of CHIKV–luciferase was detected in mice for up to 5 days post-infection at the site of inoculation with limited dissemination to the skeletal muscle. PMID:21668347
-
In vivo imaging of chikungunya virus in mice and Aedes mosquitoes using a Renilla luciferase clone.
Ziegler, Sarah A; Nuckols, John; McGee, Charles E; Huang, Yan-Jang Scott; Vanlandingham, Dana L; Tesh, Robert B; Higgs, Stephen
2011-11-01
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that in humans causes an acute febrile illness characterized by fever, arthralgia, and rash. It is currently associated with large outbreaks in Asia, Africa, and islands of the Indian Ocean and has been introduced from these tropical regions into Europe, where local transmission has been recorded on two occasions. The underlying basis of the pathogenesis of CHIKV and related alphaviruses that produce similar symptoms remains unclear. By applying new techniques, for example, in vivo imaging in live animals and arthropods, we may improve our understanding of viral pathogenesis in vertebrates and viral replication in mosquitoes. This technical report describes the evaluation of a CHIKV?luciferase clone to visualize infection and dissemination in both Aedes aegypti and Aedes albopictus mosquitoes and mice. In mosquitoes, luciferase activity was seen at 3 and 7 days post-infection in both head and abdomens. In vivo imaging of CHIKV-luciferase was detected in mice for up to 5 days post-infection at the site of inoculation with limited dissemination to the skeletal muscle.
-
The production of recombinant human erythropoietin.
Inoue, N; Takeuchi, M; Ohashi, H; Suzuki, T
1995-01-01
Erythropoietin (EPO) is the glycoprotein hormone that promotes differentiation of erythroid progenitor cells in bone marrow. The normal kidney produces EPO to maintain erythrocyte for oxygen supply. This hormone activity was found in the serum of anemic animals in the 1890s. Renal failure results in severe anemia because of reduced EPO production, therefore anemia patients expected EPO treatment for long time. However, this was difficult due to the limited amount of EPO. Many researchers have tried to isolate EPO since the 1950s. Finally Miyake and Goldwasser purified highly active EPO from the urine of aplastic anemia patients. Since then, the characteristics and structural information from the purified material accelerated the cloning of the EPO gene. Mammalian cells were essential to produce EPO, because EPO contains 40% carbohydrate that plays some important roles in its activity, stability and biosynthesis. In 1984, two groups succeeded in cloning the EPO gene and expressing this gene in mammalian cells. Recombinant human EPO is currently available for anemia treatment. In this paper, we review production in mammalian cells, molecular characterization, especially carbohydrate moieties, and clinical applications of recombinant EPO.
-
Generation of a reliable full-length cDNA of infectiousTembusu virus using a PCR-based protocol.
Liang, Te; Liu, Xiaoxiao; Cui, Shulin; Qu, Shenghua; Wang, Dan; Liu, Ning; Wang, Fumin; Ning, Kang; Zhang, Bing; Zhang, Dabing
2016-02-02
Full-length cDNA of Tembusu virus (TMUV) cloned in a plasmid has been found instable in bacterial hosts. Using a PCR-based protocol, we generated a stable full-length cDNA of TMUV. Different cDNA fragments of TMUV were amplified by reverse transcription (RT)-PCR, and cloned into plasmids. Fragmented cDNAs were amplified and assembled by fusion PCR to produce a full-length cDNA using the recombinant plasmids as templates. Subsequently, a full-length RNA was transcribed from the full-length cDNA in vitro and transfected into BHK-21 cells; infectious viral particles were rescued successfully. Following several passages in BKH-21 cells, the rescued virus was compared with the parental virus by genetic marker checks, growth curve determinations and animal experiments. These assays clearly demonstrated the genetic and biological stabilities of the rescued virus. The present work will be useful for future investigations on the molecular mechanisms involved in replication and pathogenesis of TMUV. Copyright © 2015 Elsevier B.V. All rights reserved.
-
Schütz, Ekkehard; Urnovitz, Howard B; Iakoubov, Leonid; Schulz-Schaeffer, Walter; Wemheuer, Wilhelm; Brenig, Bertram
2005-07-01
Circulating nucleic acids (CNA) are known to be enriched in repetitive DNA sequences in humans. Here, bovine sera CNA were analyzed to determine if cell stress-related short interspersed nucleotide elements (SINEs) could be detected in sera from cattle associated with bovine spongiform encephalopathy (BSE). Nucleic acids were extracted, amplified, cloned, and sequenced from the sera of protease-resistant prion protein (PrP(res))-positive cattle (n = 2) and sera from BSE-cohort cows (n = 6); 150 out of 163 clones revealed the presence of, on average, an 80-bp sequence from the 3' region of Bov-tA SINE. A PCR protocol was developed that differentially identified SINE-associated CNA in BSE-exposed versus normal cattle. CNA were extracted from a serum vesicular fraction after controlled blood collection and processing procedures. Sera from four confirmed cases of BSE, 137 BSE-exposed cohort animals associated with eight confirmed BSE cases, and 845 healthy, PrP(res)-negative control cows were tested. All four sera from confirmed BSE cases were repeatedly reactive in the assay. BSE-exposed cohorts had a 100-fold higher occurrence of repeatedly reactive individuals per cohort (average = 63%; range = 33% to 91%), compared to healthy controls (average = 0.6%; P < 0.001). This study shows that BSE-confirmed and cohort animals possess a unique profile of SINE-associated serum CNA that can be utilized as a marker that highly correlates to BSE exposure.
-
Schütz, Ekkehard; Urnovitz, Howard B.; Iakoubov, Leonid; Schulz-Schaeffer, Walter; Wemheuer, Wilhelm; Brenig, Bertram
2005-01-01
Circulating nucleic acids (CNA) are known to be enriched in repetitive DNA sequences in humans. Here, bovine sera CNA were analyzed to determine if cell stress-related short interspersed nucleotide elements (SINEs) could be detected in sera from cattle associated with bovine spongiform encephalopathy (BSE). Nucleic acids were extracted, amplified, cloned, and sequenced from the sera of protease-resistant prion protein (PrPres)-positive cattle (n = 2) and sera from BSE-cohort cows (n = 6); 150 out of 163 clones revealed the presence of, on average, an 80-bp sequence from the 3′ region of Bov-tA SINE. A PCR protocol was developed that differentially identified SINE-associated CNA in BSE-exposed versus normal cattle. CNA were extracted from a serum vesicular fraction after controlled blood collection and processing procedures. Sera from four confirmed cases of BSE, 137 BSE-exposed cohort animals associated with eight confirmed BSE cases, and 845 healthy, PrPres-negative control cows were tested. All four sera from confirmed BSE cases were repeatedly reactive in the assay. BSE-exposed cohorts had a 100-fold higher occurrence of repeatedly reactive individuals per cohort (average = 63%; range = 33% to 91%), compared to healthy controls (average = 0.6%; P < 0.001). This study shows that BSE-confirmed and cohort animals possess a unique profile of SINE-associated serum CNA that can be utilized as a marker that highly correlates to BSE exposure. PMID:16002628
-
Primitive ATP-activated P2X receptors: discovery, function and pharmacology
Fountain, Samuel J.
2013-01-01
Adenosine 5-triphosphate (ATP) is omnipresent in biology. It is therefore no surprise that organisms have evolved multifaceted roles for ATP, exploiting its abundance and restriction of passive diffusion across biological membranes. A striking role is the emergence of ATP as a bona fide transmitter molecule, whereby the movement of ATP across membranes serves as a chemical message through a direct ligand-receptor interaction. P2X receptors are ligand-gated ion channels that mediate fast responses to the transmitter ATP in mammalian cells including central and sensory neurons, vascular smooth muscle, endothelium, and leukocytes. Molecular cloning of P2X receptors and our understanding of structure-function relationships has provided sequence information with which to query an exponentially expanding wealth of genome sequence information including protist, early animal and human pathogen genomes. P2X receptors have now been cloned and characterized from a number of simple organisms. Such work has led to surprising new cellular roles for the P2X receptors family and an unusual phylogeny, with organisms such as Drosophila and C. elegans notably lacking P2X receptors despite retaining ionotropic receptors for other common transmitters that are present in mammals. This review will summarize current work on the evolutionary biology of P2X receptors and ATP as a signaling molecule, discuss what can be drawn from such studies when considering the action of ATP in higher animals and plants, and outline how simple organisms may be exploited experimentally to inform P2X receptor function in a wider context. PMID:24367292
-
Holt, Kathryn E.; Wertheim, Heiman; Zadoks, Ruth N.; Baker, Stephen; Whitehouse, Chris A.; Dance, David; Jenney, Adam; Connor, Thomas R.; Hsu, Li Yang; Severin, Juliëtte; Brisse, Sylvain; Cao, Hanwei; Wilksch, Jonathan; Gorrie, Claire; Schultz, Mark B.; Edwards, David J.; Nguyen, Kinh Van; Nguyen, Trung Vu; Dao, Trinh Tuyet; Mensink, Martijn; Minh, Vien Le; Nhu, Nguyen Thi Khanh; Schultsz, Constance; Kuntaman, Kuntaman; Newton, Paul N.; Moore, Catrin E.; Strugnell, Richard A.; Thomson, Nicholas R.
2015-01-01
Klebsiella pneumoniae is now recognized as an urgent threat to human health because of the emergence of multidrug-resistant strains associated with hospital outbreaks and hypervirulent strains associated with severe community-acquired infections. K. pneumoniae is ubiquitous in the environment and can colonize and infect both plants and animals. However, little is known about the population structure of K. pneumoniae, so it is difficult to recognize or understand the emergence of clinically important clones within this highly genetically diverse species. Here we present a detailed genomic framework for K. pneumoniae based on whole-genome sequencing of more than 300 human and animal isolates spanning four continents. Our data provide genome-wide support for the splitting of K. pneumoniae into three distinct species, KpI (K. pneumoniae), KpII (K. quasipneumoniae), and KpIII (K. variicola). Further, for K. pneumoniae (KpI), the entity most frequently associated with human infection, we show the existence of >150 deeply branching lineages including numerous multidrug-resistant or hypervirulent clones. We show K. pneumoniae has a large accessory genome approaching 30,000 protein-coding genes, including a number of virulence functions that are significantly associated with invasive community-acquired disease in humans. In our dataset, antimicrobial resistance genes were common among human carriage isolates and hospital-acquired infections, which generally lacked the genes associated with invasive disease. The convergence of virulence and resistance genes potentially could lead to the emergence of untreatable invasive K. pneumoniae infections; our data provide the whole-genome framework against which to track the emergence of such threats. PMID:26100894
-
Effects of cattle husbandry on abundance and activity of methanogenic archaea in upland soils.
Radl, Viviane; Gattinger, Andreas; Chronáková, Alica; Nemcová, Anna; Cuhel, Jiri; Simek, Miloslav; Munch, Jean Charles; Schloter, Michael; Elhottová, Dana
2007-09-01
In the present study, we tested the hypothesis that animal treading associated with a high input of organic matter would favour methanogenesis in soils used as overwintering pasture. Hence, methane emissions and methanogen populations were examined at sections with different degree of cattle impact in a Farm in South Bohemia, Czech Republic. In spring, methane emission positively corresponded to the gradient of animal impact. Applying phospholipid etherlipid analysis, the highest archaeal biomass was found in section severe impact (SI), followed by moderate impact (MI) and no impact. The same trend was observed for the methanogens as showed by real-time quantitative PCR analyses of methyl coenzyme M reductase (mcrA) genes. The detection of monounsaturated isoprenoid side chain hydrocarbons (i20:1) indicated the presence of acetoclastic methanogens in the cattle-impacted sites. This result was corroborated by the phylogenetic analysis of mcrA gene sequences obtained from section SI, which showed that 33% of the analysed clones belonged to the genus Methanosarcina. The majority of the sequenced clones (41%) showed close affiliations with uncultured rumen archaeons. This leads to the assumption that a substantial part of the methanogenic community in plot SI derived from the grazing cattle itself. Compared to the spring sampling, in autumn, a significant reduction in archaeal biomass and number of copies of mcrA genes was observed mainly for section MI. It can be concluded that after 5 months without cattle impact, the severely impact section maintained its methane production potential, whereas the methane production potential under moderate impact returned to background values.
-
Experimental embryology of mammals at the Jastrzebiec Institute of Genetics and Animal Breeding.
Karasiewicz, Jolanta; Andrzej-Modlinski, Jacek
2008-01-01
Our Department of Experimental Embryology originated from The Laboratory of Embryo Biotechnology, which was organized and directed by Dr. Maria Czlonkowska until her premature death in 1991. Proving successful international transfer of frozen equine embryos and generation of an embryonic sheep-goat chimaera surviving ten years were outstanding achievements of her term. In the 1990s, we produced advanced fetuses of mice after reconstructing enucleated oocytes with embryonic stem (ES) cells, as well as mice originating entirely from ES cells by substitution of the inner cell mass with ES cells. Attempts at obtaining ES cells in sheep resulted in the establishment of embryo-derived epithelioid cell lines from Polish Heatherhead and Polish Merino breeds, producing overt chimaeras upon blastocyst injection. Successful re-cloning was achieved from 8-cell rabbit embryos, and healthy animals were born from the third generation of cloned embryos. Recently mice were born after transfer of 8-cell embryonic nuclei into selectively enucleated zygotes, and mouse blastocysts were produced from selectively enucleated germinal vesicle oocytes surrounded by follicular cells, upon their reconstruction with 2-cell nuclei and subsequent activation. Embryonic-somatic chimaeras were born after transfer of foetal fibroblasts into 8-cell embryos (mouse) and into morulae and blastocysts (sheep). We also regularly perform the following applications: in vitro production of bovine embryos from slaughterhouse oocytes or those recovered by ovum pick up; cryopreservation of oocytes and embryos (freezing: mouse, rabbit, sheep, goat; vitrification: rabbit, cow); and banking of somatic cells from endangered wild mammalian species (mainly Cervidae).
-
iPBS: a universal method for DNA fingerprinting and retrotransposon isolation.
Kalendar, Ruslan; Antonius, Kristiina; Smýkal, Petr; Schulman, Alan H
2010-11-01
Molecular markers are essential in plant and animal breeding and biodiversity applications, in human forensics, and for map-based cloning of genes. The long terminal repeat (LTR) retrotransposons are well suited as molecular markers. As dispersed and ubiquitous transposable elements, their "copy and paste" life cycle of replicative transposition leads to new genome insertions without excision of the original element. Both the overall structure of retrotransposons and the domains responsible for the various phases of their replication are highly conserved in all eukaryotes. Nevertheless, up to a year has been required to develop a retrotransposon marker system in a new species, involving cloning and sequencing steps as well as the development of custom primers. Here, we describe a novel PCR-based method useful both as a marker system in its own right and for the rapid isolation of retrotransposon termini and full-length elements, making it ideal for "orphan crops" and other species with underdeveloped marker systems. The method, iPBS amplification, is based on the virtually universal presence of a tRNA complement as a reverse transcriptase primer binding site (PBS) in LTR retrotransposons. The method differs from earlier retrotransposon isolation methods because it is applicable not only to endogenous retroviruses and retroviruses, but also to both Gypsy and Copia LTR retrotransposons, as well as to non-autonomous LARD and TRIM elements, throughout the plant kingdom and to animals. Furthermore, the inter-PBS amplification technique as such has proved to be a powerful DNA fingerprinting technology without the need for prior sequence knowledge.
-
Engineering of EPA/DHA omega-3 fatty acid production by Lactococcus lactis subsp. cremoris MG1363.
Amiri-Jami, Mitra; Lapointe, Gisele; Griffiths, Mansel W
2014-04-01
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to be of major importance in human health. Therefore, these essential polyunsaturated fatty acids have received considerable attention in both human and farm animal nutrition. Currently, fish and fish oils are the main dietary sources of EPA/DHA. To generate sustainable novel sources for EPA and DHA, the 35-kb EPA/DHA synthesis gene cluster was isolated from a marine bacterium, Shewanella baltica MAC1. To streamline the introduction of the genes into food-grade microorganisms such as lactic acid bacteria, unnecessary genes located upstream and downstream of the EPA/DHA gene cluster were deleted. Recombinant Escherichia coli harboring the 20-kb gene cluster produced 3.5- to 6.1-fold more EPA than those carrying the 35-kb DNA fragment coding for EPA/DHA synthesis. The 20-kb EPA/DHA gene cluster was cloned into a modified broad-host-range low copy number vector, pIL252m (4.7 kb, Ery) and expressed in Lactococcus lactis subsp. cremoris MG1363. Recombinant L. lactis produced DHA (1.35 ± 0.5 mg g(-1) cell dry weight) and EPA (0.12 ± 0.04 mg g(-1) cell dry weight). This is believed to be the first successful cloning and expression of EPA/DHA synthesis gene cluster in lactic acid bacteria. Our findings advance the future use of EPA/DHA-producing lactic acid bacteria in such applications as dairy starters, silage adjuncts, and animal feed supplements.
-
Morach, Marina; Stephan, Roger; Schmitt, Sarah; Ewers, Christa; Zschöck, Michael; Reyes-Velez, Julian; Gilli, Urs; Del Pilar Crespo-Ortiz, María; Crumlish, Margaret; Gunturu, Revathi; Daubenberger, Claudia A; Ip, Margaret; Regli, Walter; Johler, Sophia
2018-03-01
Streptococcus agalactiae is a leading cause of morbidity and mortality among neonates and causes severe infections in pregnant women and nonpregnant predisposed adults, in addition to various animal species worldwide. Still, information on the population structure of S. agalactiae and the geographical distribution of different clones is limited. Further data are urgently needed to identify particularly successful clones and obtain insights into possible routes of transmission within one host species and across species borders. We aimed to determine the population structure and virulence gene profiles of S. agalactiae strains from a diverse set of sources and geographical origins. To this end, 373 S. agalactiae isolates obtained from humans and animals from five different continents were typed by DNA microarray profiling. A total of 242 different S. agalactiae strains were identified and further analyzed. Particularly successful clonal lineages, hybridization patterns, and strains were identified that were spread across different continents and/or were present in more than one host species. In particular, several strains were detected in both humans and cattle, and several canine strains were also detected in samples from human, bovine, and porcine hosts. The findings of our study suggest that although S. agalactiae is well adapted to various hosts including humans, cattle, dogs, rodents, and fish, interspecies transmission is possible and occurs between humans and cows, dogs, and rabbits. The virulence and resistance gene profiles presented enable new insights into interspecies transmission and make a crucial contribution to the identification of suitable targets for therapeutic agents and vaccines.
-
Physical mapping of complex genomes
Evans, G.A.
1993-06-15
A method for the simultaneous identification of overlapping cosmid clones among multiple cosmid clones and the use of the method for mapping complex genomes are provided. A library of cosmid clones that contains the DNA to be mapped is constructed and arranged in a manner such that individual clones can be identified and replicas of the arranged clones prepared. In preferred embodiments, the clones are arranged in a two dimensional matrix. In such embodiments, the cosmid clones in a row are pooled, mixed probes complementary to the ends of the DNA inserts in the pooled clones are synthesized, hybridized to a first replica of the library. Hybridizing clones, which include the pooled row, are identified. A second portion of clones is prepared by pooling cosmid clones that correspond to a column in the matrix. The second pool thereby includes one clone from the first portion pooled clones. This common clone is located on the replica at the intersection of the column and row. Mixed probes complementary to the ends of the DNA inserts in the second pooled portion of clones are prepared and hybridized to a second replica of the library. The hybridization pattern on the first and second replicas of the library are compared and cross-hybridizing clones, other than the clones in the pooled column and row, that hybridize to identical clones in the first and second replicas are identified. These clones necessarily include DNA inserts that overlap with the DNA insert in the common clone located at the intersection of the pooled row and pooled column. The DNA in the entire library may be mapped by pooling the clones in each of the rows and columns of the matrix, preparing mixed end-specific probes and hybridizing the probes from each row or column to a replica of the library. Since all clones in the library are located at the intersection of a column and a row, the overlapping clones for all clones in the library may be identified and a physical map constructed.
-
Miller, G W; Wang, Y M; Gainetdinov, R R; Caron, M G
2001-01-01
One of the most valuable methods for understanding the function of a particular protein is the generation of animals that have had the gene encoding for the protein of interest disrupted, commonly known as a "quo;knockout"quo; or null mutant. By incorporating a sequence of DNA (typically encoding antibiotic resistance to aid in the selection of the mutant gene) into embryonic stem cells by homologous recombination, the normal transcription of the gene is effectively blocked (Fig. 1). Since a particular protein is encoded by two copies of a gene, it is necessary to have the gene on both alleles "quo;knocked out."quo; This is performed by cross-breeding animals with one affected allele (heterozygote) to generate offspring that have inherited two mutant alleles (homozygote). This procedure has been used to generate animals lacking either the plasma membrane dopamine transporter (DAT; Fig. 2) or the vesicular monoamine transporter (VMAT2; Fig. 3). Both DAT and VMAT2 are essential for dopamine homeostasis and are thought to participate in the pathogenesis of Parkinson's disease (1-5). Fig. 1. Maps of the targeting vector and the mock construct. The mouse genomic fragment (clone 11) was isolated from a Stratagene 129 SvJ library by standard colony hybridization using a PCR probe from the 5' end of rat cDNA. The restriction site abbreviations are as follows: H, HindIII; N, NotI; Sc, SacI; Sn, SnaI; X, XbaI; and Xh, XhoI. The region between HindIII and SnaI on clone 11 containing the coding sequence from transmembrane domains 3 and 4 of VMAT2 was deleted and replaced with PGK-neo. The 3' fragment of clone 11 was reserved as an external probe for Southern analysis. To facilitate PCR screening of embryonic stem cell clones, a mock construct containing the SnaI/XbaI fragment and part of the Neo cassette was generated as a positive control. pPNT and pGEM4Z were used to construct knockout and mock vectors, respectively. (Reproduced with permission from ref. 1). Fig. 2. DAT and VMAT2 expression in wild-type and DAT knockout midbrain. DAT immunoreactivity in wild-type (A) and DAT knockout midbrain (B). VMAT2 immunoreactivity in wild-type (C) and DAT knockout midbrain (D). Robust immunoreactivity was observed in the ventral tegmental area and substantia nigra pars compacta and reticulata in the wild-type brain. Note absence of DAT immunoreactivity and modest reduction of VMAT2 immunoreactivity in the DAT knockout. Fig. 3. Characterization of VMAT2 gene disruption. (A) Southern blot analysis of mouse genomic DNA. The Southern blot was prepared with 15 μg of genomic DNA per lane and probed with a 1.4-kb 3' external genomic fragment. +/+, wild type littermates; +/-, heterozygote; -/-, homozygote. (B) RT-PCR analysis of mouse brain poly(A)+ RNA. For each reverse transcription assay, 0.5 μg of poly(A)+ RNA was used. Equal volumes of cDNA templates were used for each PCR assay. The PCR primers used flank the neomycin cassette for the purpose of detecting potential readthrough of the neomycin DNA. The heterozygote has a reduced amount of transcripts compared with the wild-type littermate; the homozygote is devoid of VMAT2 transcripts. G3PDH was used as internal control. (C) Western blot analysis of wholebrain synaptic vesicles. Samples (25 μg) of vesicles were solubilized and separated by SDS-PAGE, transferred to nitrocellulose, subjected to Western blot analysis with anti-VMAT2-Ct (top) or anti-a-tubulin (bottom) antibodies, and developed with chemiluminescence. Molecular mass markers (kDa) are shown to the left. To confirm equal loading and transfer of proteins, the blots were stripped and reprobed with an antibody to α-tubulin. (Reproduced with permission from ref. 1). The importance of DAT in neuronal function is highlighted in animals in which DAT has been genetically deleted (DAT KO) (3). In the homozygote DAT KO mice, released dopamine remains in the extracellular space up to 300 times longer than normal. As expected, these animals display behaviors consistent with persistent activation of dopamine receptors, such as hyperlocomotion. Genetic deletion of VMAT2 reveals the essential role of vesicular storage and release of monoamines. Homozygote VMAT2 knockout mice survive for only a few days, whereas heterozygotes appear normal. Studies performed in homozygote pups and heterozygote adults clearly show that the level of VMAT2 expression calibrates the level of vesicular filling (1,2,bi4). With only 50% of normal VMAT2, heterozygote animals have reduced vesicular filling and release. These alterations in presynaptic monoamine function in the heterozygotes are thought to be responsible for the observed sensitization to the psychostimulants cocaine and amphetamine and to ethanol (1). Knockout animals also appear to parallel the changes that occur in reserpinized animals, suggesting that the adverse actions of this drug are mediated by VMAT2.
-
Functional expression of a cattle MHC class II DR-like antigen on mouse L cells
DOE Office of Scientific and Technical Information (OSTI.GOV)
Fraser, D.C.; Craigmile, S.; Campbell, J.D.M.
1996-09-01
Cattle DRA and DRB genes, cloned by reverse-transcription polymerase chain reaction, were transfected into mouse L cells. The cattle DR-expressing L-cell transfectant generated was analyzed serologically, biochemically, and functionally. Sequence analysis of the transfected DRB gene clearly showed showed that it was DRB3 allele DRB3*0101, which corresponds to the 1D-IEF-determined allele DRBF3. 1D-IEF analysis of the tranfectant confirmed that the expressed DR product was DRBF3. Functional integrity of the transfected gene products was demonstrated by the ability of the transfectant cell line to present two antigens (the foot-and-mouth disease virus-derived peptide FMDV15, and ovalbumin) to antigen-specific CD4{sup +} T cellsmore » from both the original animal used to obtain the genes, and also from an unrelated DRBF3{sup +} heterozygous animal. Such transfectants will be invaluable tools, allowing us to dissect the precise contributions each locus product makes to the overall immune response in heterozygous animals, information essential for rational vaccine design. 45 refs., 5 figs., 1 tab.« less
-
Characterization of Pisrt1/Foxl2 in Ellobius lutescens and exclusion as sex-determining genes.
Baumstark, Annette; Hameister, Horst; Hakhverdyan, Mikhayil; Bakloushinskaya, Irina; Just, Walter
2005-04-01
The rodent Ellobius lutescens is an exceptional mammal which determines male sex constitutively without the SRY gene and, therefore, may serve as an animal model for human 46,XX female-to-male sex reversal. It was suggested that other factors of the network of sex-determining genes determine maleness in these animals. However, some sex-determining genes like SOX9 and SF1 have already been excluded by segregation analysis as primary sex-determining factors in E. lutescens. In this work, we have cloned and characterized two genes of the PIS (polled intersex syndrome) gene interval, which were reported as candidates in female-to-male sex reversal in hornless goats recently. The genes Foxl2 and Pisrt1 from that interval were identified in E. lutescens DNA and mapped to Chromosome 8. We have excluded linkage of Foxl2 and Pisrt1 loci with the sex of the animals. Hence, the involvement of this gene region in sex determination may be specific for goats and is not a general mechanism of XX sex reversal or XX male sex determination.
-
Billington, Stephen J; Songer, J Glenn; Jost, B Helen
2002-05-01
Tetracycline resistance is common among isolates of the animal commensal and opportunistic pathogen Arcanobacterium pyogenes. The tetracycline resistance determinant cloned from two bovine isolates of A. pyogenes was highly similar at the DNA level (92% identity) to the tet(W) gene, encoding a ribosomal protection tetracycline resistance protein, from the rumen bacterium Butyrivibrio fibrisolvens. The tet(W) gene was found in all 20 tetracycline-resistant isolates tested, indicating that it is a widely distributed determinant of tetracycline resistance in this organism. In 25% of tetracycline-resistant isolates, the tet(W) gene was associated with a mob gene, encoding a functional mobilization protein, and an origin of transfer, suggesting that the determinant may be transferable to other bacteria. In fact, low-frequency transfer of tet(W) was detected from mob+ A. pyogenes isolates to a tetracycline-sensitive A. pyogenes recipient. The mobile nature of this determinant and the presence of A. pyogenes in the gastrointestinal tract of cattle and pigs suggest that A. pyogenes may have inherited this determinant within the gastrointestinal tracts of these animals.
-
Džunková, Mária; D'Auria, Giuseppe; Pérez-Villarroya, David; Moya, Andrés
2012-01-01
Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb) cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs) with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.
-
Mwaengo, Dufton M.; Novembre, Francis J.
1998-01-01
We have previously described the development of AIDS in a chimpanzee (C499) infected with human immunodeficiency virus type 1 (HIV-1) and the subsequent pathogenic HIV-1 infection in another chimpanzee (C455) transfused with blood from C499 (F. J. Novembre et al., J. Virol. 71:4086–4091, 1997). In the present study, two virus isolates were derived from these animals: HIV-1JC from peripheral blood mononuclear cells (PBMC) of C499, and HIV-1NC from plasma of C455. These virus isolates were used to generate two infectious molecular clones, termed HIV-1JC16 and HIV-1NC7 (JC16 and NC7, respectively). Comparative analyses of the sequences of the two clones showed that they were highly interrelated but distinct. Based on heteroduplex mobility assays, JC16 and NC7 appear to represent dominant viruses in the uncloned stock population. Compared with amino acid sequences of the parental viruses HIV-1SF2, HIV-1LAV-1b, and HIV-1NDK, JC16 and NC7 showed a number of differences, including insertions, deletions, and point mutations spread throughout the genome. However, insertion/deletion footprints in several genes of both JC16 and NC7 suggested that recombination between SF2 and LAV-1b could have occurred, possibly contributing to the generation of a pathogenic virus. Comparative in vitro analyses of the molecular clones and the uncloned stocks of HIV-1JC and HIV-1NC revealed that these viruses had strikingly similar replicative abilities in mitogen-stimulated PBMC and in macrophages. Compared to the SF2 and LAV-1b isolates of HIV-1, HIV-1JC and HIV-1NC isolates were more similar to LAV-1b with respect to the ability to replicate in mitogen-stimulated PBMC and macrophages. These viruses should prove to be useful in mapping determinants of pathogenesis. PMID:9765443
-
Hargreaves, Melissa L.; Shaw, Kristin M.; Dobbins, Ginette; Snippes Vagnone, Paula M.; Harper, Jane E.; Boxrud, Dave; Lynfield, Ruth; Aziz, Maliha; Price, Lance B.; Silverstein, Kevin A. T.; Danzeisen, Jessica L.; Youmans, Bonnie; Case, Kyle; Sreevatsan, Srinand
2015-01-01
Carbapenemase-producing, carbapenem-resistant Enterobacteriaceae, or CP-CRE, are an emerging threat to human and animal health, because they are resistant to many of the last-line antimicrobials available for disease treatment. Carbapenemase-producing Enterobacter cloacae harboring blaKPC-3 recently was reported in the upper midwestern United States and implicated in a hospital outbreak in Fargo, North Dakota (L. M. Kiedrowski, D. M. Guerrero, F. Perez, R. A. Viau, L. J. Rojas, M. F. Mojica, S. D. Rudin, A. M. Hujer, S. H. Marshall, and R. A. Bonomo, Emerg Infect Dis 20:1583–1585, 2014, http://dx.doi.org/10.3201/eid2009.140344). In early 2009, the Minnesota Department of Health began collecting and screening CP-CRE from patients throughout Minnesota. Here, we analyzed a retrospective group of CP-E. cloacae isolates (n = 34) collected between 2009 and 2013. Whole-genome sequencing and analysis revealed that 32 of the strains were clonal, belonging to the ST171 clonal complex and differing collectively by 211 single-nucleotide polymorphisms, and it revealed a dynamic clone under positive selection. The phylogeography of these strains suggests that this clone existed in eastern North Dakota and western Minnesota prior to 2009 and subsequently was identified in the Minneapolis and St. Paul metropolitan area. All strains harbored identical IncFIA-like plasmids conferring a CP-CRE phenotype and an additional IncX3 plasmid. In a single patient with multiple isolates submitted over several months, we found evidence that these plasmids had transferred from the E. cloacae clone to an Escherichia coli ST131 bacterium, rendering it as a CP-CRE. The spread of this clone throughout the upper midwestern United States is unprecedented for E. cloacae and highlights the importance of continued surveillance to identify such threats to human health. PMID:26438492
-
Staunstrup, Nicklas Heine; Madsen, Johannes; Primo, Maria Nascimento; Li, Juan; Liu, Ying; Kragh, Peter M.; Li, Rong; Schmidt, Mette; Purup, Stig; Dagnæs-Hansen, Frederik; Svensson, Lars; Petersen, Thomas K.; Callesen, Henrik; Bolund, Lars; Mikkelsen, Jacob Giehm
2012-01-01
Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage-induced inflammation and should be of great potential in studies aiming at the development and refinement of topical therapies for cutaneous inflammation including psoriasis. PMID:22590584
-
Nakajima, Noriaki; Inomata, Tomo; Ito, Junya; Kashiwazaki, Naomi
2008-12-01
In several mammalian species including rats, successfully cloned animals have been generated using somatic cell nuclear transfer (SCNT). However, in the case of rats, additional treatment with MG132, a proteasome inhibitor, before enucleation of oocytes seems to be required for successful cloning because ovulated rat oocytes are spontaneously activated, and hence, their suppression is the key to successful cloning. A previous study on rats demonstrated that matured oocytes potentially possess lower cytostatic factor (CSF) activity compared to mouse oocytes, resulting in a low incidence of premature chromosome condensation in the reconstructed embryos after SCNT. It is known that mice having more than two pronuclei are generally observed in nuclear-transferred oocytes after induction of premature chromosome condensation, which implies successful reprogramming. This leads us to the hypothesis that MG132 treatment affects not only the inhibition of spontaneous activation but also the reprogramming and developmental ability of reconstructed rat embryos. If so, prolonged MG132 treatment during and/or after SCNT may further improve the survivability. However, the effect of MG132 treatment on reconstructed embryos after SCNT has been very limited in rats and other species. We show here that prolonged MG132 treatment during and after SCNT improves survival and the number of pronuclei in reconstructed rat embryos after activation. These reconstructed embryos treated before, during, and after SCNT showed significantly higher p34(cdc2) kinase activity involving CSF activity compared to that of the control embryos. On the other hand, p34(cdc2) kinase activity was not recovered in nuclear-transferred oocytes without MG132, which suggested that the enucleation had detrimental effects on the development of reconstructed oocytes. Taken together, MG132 treatment during SCNT increases survival and pronuclear numbers in reconstructed rat embryos via maintenance of high CSF activity. The data suggest that MG132 treatment is indispensable for at least rat SCNT.
-
Mohiuddin, Muhammad M; Singh, Avneesh K; Corcoran, Philip C; Hoyt, Robert F; Thomas, Marvin L; Ayares, David; Horvath, Keith A
2014-09-01
Cardiac transplantation and available mechanical alternatives are the only possible solutions for end-stage cardiac disease. Unfortunately, because of the limited supply of human organs, xenotransplantation may be the ideal method to overcome this shortage. We have recently seen significant prolongation of heterotopic cardiac xenograft survival from 3 to 12 months and beyond. Hearts from genetically engineered piglets that were alpha 1-3 galactosidase transferase knockout and expressed the human complement regulatory gene, CD46 (groups A-C), and the human thrombomodulin gene (group D) were heterotropically transplanted in baboons treated with antithymocyte globulin, cobra venom factor, anti-CD20 antibody, and costimulation blockade (anti-CD154 antibody [clone 5C8]) in group A, anti-CD40 antibody (clone 3A8; 20 mg/kg) in group B, clone 2C10R4 (25 mg/kg) in group C, or clone 2C10R4 (50 mg/kg) in group D, along with conventional nonspecific immunosuppressive agents. Group A grafts (n = 8) survived for an average of 70 days, with the longest survival of 236 days. Some animals in this group (n = 3) developed microvascular thrombosis due to platelet activation and consumption, which resulted in spontaneous hemorrhage. The median survival time was 21 days in group B (n = 3), 80 days in group C (n = 6), and more than 200 days in group D (n = 5). Three grafts in group D are still contracting well, with the longest ongoing graft survival surpassing the 1-year mark. Genetically engineered pig hearts (GTKOhTg.hCD46.hTBM) with modified targeted immunosuppression (anti-CD40 monoclonal antibody) achieved long-term cardiac xenograft survival. This potentially paves the way for clinical xenotransplantation if similar survival can be reproduced in an orthotopic transplantation model. Copyright © 2014 The American Association for Thoracic Surgery. All rights reserved.
-
Generation of CMAHKO/GTKO/shTNFRI-Fc/HO-1 quadruple gene modified pigs.
Kim, Geon A; Lee, Eun Mi; Jin, Jun-Xue; Lee, Sanghoon; Taweechaipaisankul, Anukul; Hwang, Jong Ik; Alam, Zahid; Ahn, Curie; Lee, Byeong Chun
2017-08-01
As an alternative source of organs for transplantation into humans, attention has been directed to pigs due to their similarities in biological features and organ size. However, severe immune rejection has prevented successful xenotransplantation using pig organs and tissues. To overcome immune rejection, recently developed genetic engineering systems such as TALEN coupled with somatic cell nuclear transfer (SCNT) to make embryos could be used to produce pigs compatible with xenotransplantation. We used the TALEN system to target the non-Gal antigen cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene in pigs that is naturally deleted in humans. Gal-deleted cells expressing both soluble human tumor necrosis factor receptor I IgG 1 -Fc (shTNFRI-Fc) and human hemagglutinin -tagged-human heme oxygenase-1 (hHO-1) were transfected with a TALEN target for CMAH. Cells lacking CMAH were negatively selected using N-glyconeuraminic acid (Neu5Gc)/magnetic beads and the level of Neu5Gc expression of isolated cells were analyzed by FACS and DNA sequencing. Cloned embryos using 3 different genetically modified cell clones were respectively transferred into 3 recipients, with 55.6% (5/9) becoming pregnant and three cloned pigs were produced. Successful genetic disruption of the CMAH gene was confirmed by sequencing, showing lack of expression of CMAH in tail-derived fibroblasts of the cloned piglets. Besides decreased expression of Neu5Gc in piglets produced by SCNT, antibody-mediated complement-dependent cytotoxicity assays and natural antibody binding for examining immuno-reactivity of the quadruple gene modified pigs derived from endothelial cells and fibroblasts were reduced significantly compared to those of wild type animals. We conclude that by combining the TALEN system and transgenic cells, targeting of multiple genes could be useful for generating organs for xenotransplantation. We produced miniature pigs with quadruple modified genes CMAHKO/GTKO/shTNFRI-Fc/hHO-1 that will be suitable for xenotransplantation by overcoming hyperacute, acute and anti-inflammatory rejection.
-
Jahanbakhsh, Seyedehameneh; Smith, Matthew G; Kohan-Ghadr, Hamid-Reza; Letellier, Ann; Abraham, Sam; Trott, Darren J; Fairbrother, John Morris
2016-08-01
The aim of this study was to investigate the evolution with time of ceftiofur-resistant Escherichia coli clinical isolates from pigs in Québec, Canada, between 1997 and 2012 with respect to pathotypes, clones and antimicrobial resistance. Eighty-five ceftiofur-resistant E. coli isolates were obtained from the OIE (World Organisation for Animal Health) Reference Laboratory for Escherichia coli. The most prevalent pathovirotypes were enterotoxigenic E. coli (ETEC):F4 (40%), extraintestinal pathogenic E. coli (ExPEC) (16.5%) and Shiga toxin-producing E. coli (STEC):F18 (8.2%). Susceptibility testing to 15 antimicrobial agents revealed a high prevalence of resistance to 13 antimicrobials, with all isolates being multidrug-resistant. blaCMY-2 (96.5%) was the most frequently detected β-lactamase gene, followed by blaTEM (49.4%) and blaCTX-M (3.5%). Pulsed-field gel electrophoresis (PFGE) applied to 45 representative E. coli isolates revealed that resistance to ceftiofur is spread both horizontally and clonally. In addition, the emergence of extended-spectrum β-lactamase-producing E. coli isolates carrying blaCTX-M was observed in 2011 and 2012 in distinct clones. The most predominant plasmid incompatibility (Inc) groups were IncFIB, IncI1, IncA/C and IncFIC. Resistance to gentamicin, kanamycin and chloramphenicol as well as the frequency of blaTEM and IncA/C significantly decreased over the study period, whereas the frequency of IncI1 and multidrug resistance to seven antimicrobial categories significantly increased. These findings reveal that extended-spectrum cephalosporin-resistant porcine E. coli isolates in Québec belong to several different clones with diverse antimicrobial resistance patterns and plasmids. Furthermore, blaCMY-2 was the major β-lactamase gene in these isolates. From 2011, we report the emergence of blaCTX-M in distinct clones. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.
-
Clone DB: an integrated NCBI resource for clone-associated data
Schneider, Valerie A.; Chen, Hsiu-Chuan; Clausen, Cliff; Meric, Peter A.; Zhou, Zhigang; Bouk, Nathan; Husain, Nora; Maglott, Donna R.; Church, Deanna M.
2013-01-01
The National Center for Biotechnology Information (NCBI) Clone DB (http://www.ncbi.nlm.nih.gov/clone/) is an integrated resource providing information about and facilitating access to clones, which serve as valuable research reagents in many fields, including genome sequencing and variation analysis. Clone DB represents an expansion and replacement of the former NCBI Clone Registry and has records for genomic and cell-based libraries and clones representing more than 100 different eukaryotic taxa. Records provide details of library construction, associated sequences, map positions and information about resource distribution. Clone DB is indexed in the NCBI Entrez system and can be queried by fields that include organism, clone name, gene name and sequence identifier. Whenever possible, genomic clones are mapped to reference assemblies and their map positions provided in clone records. Clones mapping to specific genomic regions can also be searched for using the NCBI Clone Finder tool, which accepts queries based on sequence coordinates or features such as gene or transcript names. Clone DB makes reports of library, clone and placement data on its FTP site available for download. With Clone DB, users now have available to them a centralized resource that provides them with the tools they will need to make use of these important research reagents. PMID:23193260
-
What Do We Know About NOD-Like Receptors in Plant Immunity?
Zhang, Xiaoxiao; Dodds, Peter N; Bernoux, Maud
2017-08-04
The first plant disease resistance (R) genes were identified and cloned more than two decades ago. Since then, many more R genes have been identified and characterized in numerous plant pathosystems. Most of these encode members of the large family of intracellular NLRs (NOD-like receptors), which also includes animal immune receptors. New discoveries in this expanding field of research provide new elements for our understanding of plant NLR function. But what do we know about plant NLR function today? Genetic, structural, and functional analyses have uncovered a number of commonalities and differences in pathogen recognition strategies as well as how NLRs are regulated and activate defense signaling, but many unknowns remain. This review gives an update on the latest discoveries and breakthroughs in this field, with an emphasis on structural findings and some comparison to animal NLRs, which can provide additional insights and paradigms in plant NLR function.
-
Prospects for new viral vaccines.
Marmion, B P
1980-08-11
Animal virology has made outstanding contributions to preventive medicine by the development of vaccines for the control of infectious disease in man and animals. Cost-benefit analysis indicates substantial savings in health care costs from the control of diseases such as smallpox, poliomyelitis, yellow fever and measels. Areas for further development include vaccines for influenza (living, attenuated virus), the herpes group (varicella: cytomegalovirus), respiratory syncytial virus, rotavirus and hepatitis A, B, and non A/non B. The general options for vaccine formulation are discussed with particular emphasis on approaches with the use of viral genetics to 'tailor make' vaccine viruses with defined growth potential in laboratory systems, low pathogenicity, and defined antigens. Current progress with the development of an inactivated hepatitis B vaccine is reviewed as a case study in vaccine development. The impact of recent experiments in cloning hepatitis B virus DNA in E. coli on the production of a purified viral polypeptide vaccine is assessed.
-
Biotechnology and DNA vaccines for aquatic animals
Kurath, G.
2008-01-01
Biotechnology has been used extensively in the development of vaccines for aquaculture. Modern molecular methods such as polymerase chain reaction (PCR), cloning and microarray analysis have facilitated antigen discovery, construction of novel candidate vaccines, and assessments of vaccine efficacy, mode of action, and host response. This review focuses on DNA vaccines for finfish to illustrate biotechnology applications in this field. Although DNA vaccines for fish rhabdoviruses continue to show the highest efficacy, DNA vaccines for several other viral and bacterial fish pathogens have now been proven to provide significant protection against pathogen challenge. Studies of the fish rhabdovirus DNA vaccines have elucidated factors that affect DNA vaccine efficacy as well as the nature of the fish innate and adaptive immune responses to DNA vaccines. As tools for managing aquatic animal disease emergencies, DNA vaccines have advantages in speed, flexibility, and safety, and one fish DNA vaccine has been licensed.
-
Physical mapping of complex genomes
Evans, Glen A.
1993-01-01
Method for simultaneous identification of overlapping cosmid clones among multiple cosmid clones and the use of the method for mapping complex genomes are provided. A library of cosmid clones that contains the DNA to be mapped is constructed and arranged in a manner such that individual clones can be identified and replicas of the arranged clones prepared. In preferred embodiments, the clones are arranged in a two dimensional matrix. In such embodiments, the cosmid clones in a row are pooled, mixed probes complementary to the ends of the DNA inserts int he pooled clones are synthesized, hybridized to a first replica of the library. Hybridizing clones, which include the pooled row, are identified. A second portion of clones is prepared by pooling cosmid clones that correspond to a column in the matrix. The second pool thereby includes one clone from the first portion pooled clones. This common clone is located on the replica at the intersection of the column and row. Mixed probes complementary to the ends of the DNA inserts in the second pooled portion of clones are prepared and hybridized to a second replica of the library. The hybridization pattern on the first and second replicas of the library are compared and cross-hybridizing clones, other than the clones in the pooled column and row, that hybridize to identical clones in the first and second replicas are identified. These clones necessarily include DNA inserts that overlap with the DNA insert int he common clone located at the intersection of the pooled row and pooled column. The DNA in the entire library may be mapped by pooling the clones in each of the rows and columns of the matrix, preparing mixed end-specific probes and hybridizing the probes from each row or column to a replica of the library. Since all clones in the library are located at the intersection of a column and a row, the overlapping clones for all clones in the library may be identified and a physical map constructed. In other preferred embodiments, the cosmid clones are arranged in a three dimensional matrix, pooled and compared in threes according to intersecting planes of the three dimensional matrix. Arrangements corresponding to geometries of higher dimensions may also be prepared and used to simultaneously identify overlapping clones in highly complex libraries with relatively few hybridization reactions.
-
Mshana, Stephen E; Matee, Mecky; Rweyemamu, Mark
2013-10-12
A review of the published and unpublished literature on bacterial resistance in human and animals was performed. Sixty-eight articles/reports from the Democratic Republic of Congo (DRC), Mozambique, Tanzania and Zambia were reviewed. The majority of these articles were from Tanzania. There is an increasing trend in the incidence of antibiotic resistance; of major concern is the increase in multidrug- resistant Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Vibrio cholera, non-typhoid Salmonella and other pathogens responsible for nosocomial infections. The increase in methicillin- resistant Staphylococcus aureus and extended-spectrum beta-lactamase (ESBL) producers in the countries under review confirms the spread of these clones worldwide. Clinical microbiology services in these countries need to be strengthened in order to allow a coordinated surveillance for antimicrobial resistance and provide data for local treatment guidelines and for national policies to control antimicrobial resistance. While the present study does not provide conclusive evidence to associate the increasing trend in antibiotic resistance in humans with the use of antibiotics in animals, either as feed additives or veterinary prescription, we strongly recommend a one-health approach of systematic surveillance across the public and animal health sectors, as well as the adherence to the FAO (Food and Agriculture Organization)-OIE (World Organization of animal Health) -WHO(World Health Organization) recommendations for non-human antimicrobial usage.
-
Menzies-Gow, N J; Young, N J
2011-09-28
Enterococcus faecium, a major cause of potentially life-threatening hospital-acquired human infections, can be resistant to several antimicrobials, such that streptogramin quinupristin-dalfopristin (Q/D) is one of the few antibiotics still effective. Consequently use of the streptogramin virginiamycin as an animal growth promoter was banned in the EU in 1999 as some believed this contributed to the emergence of Q/D resistant E. faecium. Virginiamycin is advocated for preventing equine pasture-associated laminitis, but its effect on equine faecal bacterial Q/D resistance has not been determined. Faecal samples were obtained from horses receiving virginiamycin, horses co-grazing and horses not exposed to virginiamycin. Streptogramin resistant E. faecium were cultured from 70% (21/30) of animals treated with virginiamycin, 75% (18/24) of co-grazing animals and 69% (11/16) of animals not exposed. ermB and vatD genes were detected using real time PCR in 63% and 66% of animals treated with virginiamycin, 75% and 71% of co-grazing animals and 63% and 69% of animals not exposed. Antimicrobial resistance genes were present only in samples which had cultured Q/D resistant E. faecium. There was no significant difference between groups with respect to antimicrobial resistance. The gene load of vatD was significantly (p=0.04) greater in unexposed animals compared to those treated with virginiamycin. The use of virginiamycin to prevent pasture-associated laminitis does not appear to be related to an increased Q/D resistance frequency. However, in view of the high frequency of resistance within all groups, the horse is a reservoir of Q/D resistant genes and clones that potentially could be transferred transiently to humans. Copyright © 2011 Elsevier B.V. All rights reserved.
-
Argudín, M Angeles; Vanderhaeghen, Wannes; Vandendriessche, Stien; Vandecandelaere, Ilse; André, François-Xavier; Denis, Olivier; Coenye, Tom; Butaye, Patrick
2015-07-09
While Staphylococcus epidermidis, as part of the commensal flora, is a well-known human opportunistic pathogen, only little is known about the genetic relatedness of S. epidermidis carriage isolates from animal and human origin. This study aimed to compare S. epidermidis recovered from livestock, livestock-farmers and humans associated with the hospital environment. A total of 193 S. epidermidis isolates from three populations [animals (n=33), farmers (n=86) and hospital-associated (n=74)] were characterized by broth microdilution antimicrobial susceptibility testing, staphylococcal cassette chromosome mec (SCCmec) typing, pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The overall S. epidermidis nasal colonization rate was low in animals (1-9%) but high among farmers (75%). High levels of multi-resistance were found in all populations. Tetracycline resistance was high in animal and farmer isolates; resistance to erythromycin, clindamycin and trimethoprim was high in animal and hospital-associated isolates. Methicillin-resistant S. epidermidis - MRSE isolates were found in all collections, with 22 (67%) MRSE in animals, 44 (51%) MRSE in farmers and 42 (57%) MRSE associated with the hospital-setting. Known SCCmec types and variants were detected in 79% of MRSE; the rest were non-typeable cassettes. In total 79 PFGE-types were found, of which 22 were shared between livestock, farmers and the hospital settings. Clonal complex 2 was predominant in all three populations and most STs corresponded to types previously observed in community and nosocomial S. epidermidis populations. S. epidermidis isolates from livestock, farmers and hospital-setting showed a high level of diversity, but some clones can be found in humans as well as in animals. Copyright © 2015 Elsevier B.V. All rights reserved.
-
Schwartz, Mark D.; Beaubien, Elisabeth G.; Crimmins, Theresa M.; Weltzin, Jake F.; Edited by Schwartz, Mark D.
2013-01-01
Plant phenological observations and networks in North America have been largely local and regional in extent until recent decades. In the USA, cloned plant monitoring networks were the exception to this pattern, with data collection spanning the late 1950s until approximately the early 1990s. Animal observation networks, especially for birds have been more extensive. The USA National Phenology Network (USA-NPN), established in the mid-2000s is a recent effort to operate a comprehensive national-scale network in the United States. In Canada, PlantWatch, as part of Nature Watch, is the current national-scale plant phenology program.
-
Germline Transformation of Drosophila Virilis Mediated by the Transposable Element Hobo
Lozovskaya, E. R.; Nurminsky, D. I.; Hartl, D. L.; Sullivan, D. T.
1996-01-01
A laboratory strain of Drosophila virilis was genetically transformed with a hobo vector carrying the miniwhite cassette using a helper plasmid with an hsp70-driven hobo transposase-coding sequence. The rate of transformation was 0.5% per fertile G0 animal. Three transgenic insertions were cloned and characterized and found to be authentic hobo insertions. These results, together with the known wide-spread distribution of hobo in diverse insect species, suggest that hobo and related transposable elements may be of considerable utility in the germline transformation of insects other than D. melanogaster. PMID:8770594
-
Isolation and characterization of Agouti: a diabetes/obesity related gene
Woychik, Richard P.
1998-01-01
The present invention relates to the cloning and expression of the Agouti gene and analogous genes in transformed, transfected and transgenic mice. The present invention provides an animal model for the study of diabetes, obesity and tumors for the testing of potential therapeutic agents. The present invention provides oligonucleotide probes for the detection of the Agouti gene and mutations in the gene. The present invention also relates to the isolation and recombinant production of the Agouti gene product, production of antibodies to the Agouti gene product and their use as diagnostic and therapeutic agents.
-
Isolation and characterization of Agouti: a diabetes/obesity related gene
Woychik, Richard P.
2000-06-27
The present invention relates to the cloning and expression of the Agouti gene and analogous genes in transformed, transfected and transgenic mice. The present invention provides an animal model for the study of diabetes, obesity and tumors for the testing of potential therapeutic agents. The present invention provides oligonucleotide probes for the detection of the Agouti gene and mutations in the gene. The present invention also relates to the isolation and recombinant production of the Agouti gene product, production of antibodies to the Agouti gene product and their use as diagnostic and therapeutic agents.
-
Harada, Kazuki; Shimizu, Takae; Mukai, Yujiro; Kuwajima, Ken; Sato, Tomomi; Usui, Masaru; Tamura, Yutaka; Kimura, Yui; Miyamoto, Tadashi; Tsuyuki, Yuzo; Ohki, Asami; Kataoka, Yasushi
2016-01-01
The emergence of antimicrobial resistance in Klebsiella spp., including resistance to extended-spectrum cephalosporins (ESC) and fluoroquinolones, is of great concern in both human and veterinary medicine. In this study, we investigated the prevalence of antimicrobial resistance in a total of 103 Klebsiella spp. isolates, consisting of Klebsiella pneumoniae complex (KP, n = 89) and K. oxytoca (KO, n = 14) from clinical specimens of dogs and cats in Japan. Furthermore, we characterized the resistance mechanisms, including extended-spectrum β-lactamase (ESBL), plasmid-mediated AmpC β-lactamase (PABL), and plasmid-mediated quinolone resistance (PMQR); and assessed genetic relatedness of ESC-resistant Klebsiella spp. strains by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Antimicrobial susceptibility testing demonstrated that resistance rates to ampicillin, cephalothin, enrofloxacin, ciprofloxacin, trimethoprim/sulfamethoxazole, cefotaxime, gentamicin, tetracycline, chloramphenicol, amoxicillin-clavulanic acid, and cefmetazole were 98.1, 37.9, 37.9, 35.9, 35.0, 34.0, 31.1, 30.1, 28.2, 14.6, and 6.8%, respectively. Phenotypic testing detected ESBLs and/or AmpC β-lactamases in 31 of 89 (34.8%) KP isolates, but not in KO isolates. Resistances to 5 of the 12 antimicrobials tested, as well as the three PMQRs [qnrB, qnrS, and aac(6′)-Ib-cr], were detected significantly more frequently in ESBL-producing KP, than in non-ESBL-producing KP and KO. The most frequent ESBL was CTX-M-15 (n = 13), followed by CTX-M-14 (n = 7), CTX-M-55 (n = 6), SHV-2 (n = 5), CTX-M-2 (n = 2), and CTX-M-3 (n = 2). Based on the rpoB phylogeny, all ESBL-producing strains were identified as K. pneumoniae, except for one CTX-M-14-producing strain, which was identified as K. quasipneumoniae. All of AmpC β-lactamase positive isolates (n = 6) harbored DHA-1, one of the PABLs. Based on MLST and PFGE analysis, ST15 KP clones producing CTX-M-2, CTX-M-15, CTX-M-55, and/or SHV-2, as well as KP clones of ST1844-CTX-M-55, ST655-CTX-M-14, and ST307-CTX-M-15, were detected in one or several hospitals. Surprisingly, specific clones were detected in different patients at an interval of many months. These results suggest that multidrug-resistant ESBL-producing KP were clonally disseminated among companion animals via not only direct but also indirect transmission. This is the first report on large-scale monitoring of antimicrobial-resistant Klebsiella spp. isolates from companion animals in Japan. PMID:27446056
-
How does embryo manipulation fit into present and future pig reproduction?
Polge, C
1985-01-01
Available techniques for the collection and direct transplantation of pig embryos are simple and efficient and could be used for the expansion of new lines, for increasing selection pressure in nucleus herds and for extracting healthy stock from a diseased source. However, the reduced viability of pig embryos during culture in vitro and the inability as yet to preserve them by deep-freezing impose limits to the use of embryo transplantation for the export or import of potential breeding stock. The efficiency of breeding schemes could be improved by the sexing of embryos and the possibility of producing genetically identical twins or quadruplets by micromanipulation of embryos should improve the efficiency of animal experimentation. Chimaerism may be used to rescue embryos of a non-viable genotype such as parthenotes or those derived by hybridization, but the greatest revolution in pig breeding may be brought about by the introduction of foreign cloned genes into eggs and the production of transgenic animals. Eggs at an appropriate stage for microinjection may be provided in the future by techniques for the maturation and fertilization of oocytes in vitro. Animal breeders should be aware of the potential impact of techniques for the manipulation of eggs and embryos on future developments in animal production.
-
Goff, Will L.; McElwain, Terry F.; Suarez, Carlos E.; Johnson, Wendell C.; Brown, Wendy C.; Norimine, Junzo; Knowles, Donald P.
2003-01-01
The competitive enzyme-linked immunosorbent assay (cELISA) format has proven to be an accurate, reliable, easily standardized, and high-throughput method for detecting hemoparasite infections. In the present study, a species-specific, broadly conserved, and tandemly repeated B-cell epitope within the C terminus of the rhoptry-associated protein 1 of the hemoparasite Babesia bovis was cloned and expressed as a histidine-tagged thioredoxin fusion peptide and used as antigen in a cELISA. The assay was optimized with defined negative and positive bovine sera, where positive sera inhibited the binding of the epitope-specific monoclonal antibody BABB75A4. The cELISA accurately differentiated animals with B. bovis-specific antibodies from uninfected animals and from animals with antibodies against other tick-borne hemoparasites (98.7% specificity). In addition, B. bovis-specific sera from Australia, Argentina, Bolivia, Puerto Rico, and Morocco inhibited the binding of BABB75A4, confirming conservation of the epitope. The assay first detected experimentally infected animals between 13 and 17 days postinfection, and with sera from naturally infected carrier cattle, was comparable to indirect immunofluorescence (98.3% concordance). The assay appears to have the characteristics necessary for an epidemiologic and disease surveillance tool. PMID:12522037
-
O'Sullivan, M G; Anderson, D C; Fikes, J D; Bain, F T; Carlson, C S; Green, S W; Young, N S; Brown, K E
1994-01-01
Although human B19 parvovirus infection has been clearly associated with a number of distinct syndromes (including severe anemia, abortion, and arthritis), detailed knowledge of its pathogenesis has been hindered by the lack of a suitable animal model. We have identified a novel simian parvovirus in cynomolgus monkeys with severe anemia. Sequencing of a 723-bp fragment of cloned viral DNA extracted from serum revealed that the simian parvovirus has 65% homology at the DNA level with the human B19 parvovirus but little homology with other known parvoviruses. Light microscopic examination of bone marrow from infected animals showed intranuclear inclusion bodies, and ultrastructural studies showed viral arrays characteristic of parvoviruses. Another striking feature was the presence of marked dyserythropoiesis in cells of the erythroid lineage, raising the possibility that B19 parvovirus infection may underlie related dyserythropoietic syndromes in human beings. Affected animals had concurrent infection with the immunosuppressive type D simian retrovirus, analogous to HIV patients who develop severe anemia because of infection with B19 parvovirus. The remarkable similarities between the simian and B19 parvoviruses suggest that experimentally infected cynomolgus monkeys may serve as a useful animal model of human B19 infection. Images PMID:8163659
-
Lichens: Unexpected anti-prion agents?
Rodriguez, Cynthia M.; Bennett, James P.; Johnson, Christopher J.
2012-01-01
The prion diseases sheep scrapie and cervid chronic wasting disease are transmitted, in part, via an environmental reservoir of infectivity; prions released from infected animals persist in the environment and can cause disease years later. Central to controlling disease transmission is the identification of methods capable of inactivating these agents on the landscape. We have found that certain lichens, common, ubiquitous, symbiotic organisms, possess a serine protease capable of degrading prion protein (PrP) from prion-infected animals. The protease functions against a range of prion strains from various hosts and reduces levels of abnormal PrP by at least two logs. We have now tested more than 20 lichen species from several geographical locations and from various taxa and found that approximately half of these species degrade PrP. Critical next steps include examining the effect of lichens on prion infectivity and cloning the protease responsible for PrP degradation. The impact of lichens on prions in the environment remains unknown. We speculate that lichens could have the potential to degrade prions when they are shed from infected animals onto lichens or into environments where lichens are abundant. In addition, lichens are frequently consumed by cervids and many other animals and the effect of dietary lichens on prion disease transmission should also be considered.
-
Fundamental resource-allocating model in colleges and universities based on Immune Clone Algorithms
NASA Astrophysics Data System (ADS)
Ye, Mengdie
2017-05-01
In this thesis we will seek the combination of antibodies and antigens converted from the optimal course arrangement and make an analogy with Immune Clone Algorithms. According to the character of the Algorithms, we apply clone, clone gene and clone selection to arrange courses. Clone operator can combine evolutionary search and random search, global search and local search. By cloning and clone mutating candidate solutions, we can find the global optimal solution quickly.
-
Pharmacological characterization of canine melancortin-4 receptor and its natural variant V213F.
Yan, J; Tao, Y-X
2011-08-01
Dogs have become one of the most important companion animals in modern society. However, it is estimated that 20% to 40% of owned dogs are obese, suggesting that obesity has become one of the most important canine health problem. In addition, obesity in dogs also leads to type II diabetes. Because the melanocortin-4 receptor (MC4R) has been shown to be essential in maintaining energy homeostasis in several different species, including rodents and humans, we initiated studies toward elucidating the roles of MC4R in obesity pathogenesis in dogs. Canine MC4R has been cloned, and a missense variant V213F was identified. We designed primers and successfully cloned canine MC4R and generated the variant V213F by site-directed mutagenesis. The objective of this study was to investigate the pharmacological properties of canine MC4R and its natural variant V213F. We measured ligand binding and signaling properties with the use of both natural and synthetic ligands. Human MC4R was also included in the experiments for comparison. Both wild-type canine MC4R and its natural variant V213F functioned normally in terms of binding and signaling. Of the ligands we used, [Nle(4), D-Phe(7)]-α-melanocyte-stimulating hormone is the most potent ligand. We conclude that the cloned canine MC4R is a functional receptor, and the natural variant V213F does not have any functional defect and therefore is not likely to cause obesity in dogs. Copyright © 2011 Elsevier Inc. All rights reserved.
-
Sakurai, Tetsuya; Plata, Germán; Rodríguez-Zapata, Fausto; Seki, Motoaki; Salcedo, Andrés; Toyoda, Atsushi; Ishiwata, Atsushi; Tohme, Joe; Sakaki, Yoshiyuki; Shinozaki, Kazuo; Ishitani, Manabu
2007-01-01
Background Cassava, an allotetraploid known for its remarkable tolerance to abiotic stresses is an important source of energy for humans and animals and a raw material for many industrial processes. A full-length cDNA library of cassava plants under normal, heat, drought, aluminum and post harvest physiological deterioration conditions was built; 19968 clones were sequence-characterized using expressed sequence tags (ESTs). Results The ESTs were assembled into 6355 contigs and 9026 singletons that were further grouped into 10577 scaffolds; we found 4621 new cassava sequences and 1521 sequences with no significant similarity to plant protein databases. Transcripts of 7796 distinct genes were captured and we were able to assign a functional classification to 78% of them while finding more than half of the enzymes annotated in metabolic pathways in Arabidopsis. The annotation of sequences that were not paired to transcripts of other species included many stress-related functional categories showing that our library is enriched with stress-induced genes. Finally, we detected 230 putative gene duplications that include key enzymes in reactive oxygen species signaling pathways and could play a role in cassava stress response features. Conclusion The cassava full-length cDNA library here presented contains transcripts of genes involved in stress response as well as genes important for different areas of cassava research. This library will be an important resource for gene discovery, characterization and cloning; in the near future it will aid the annotation of the cassava genome. PMID:18096061
-
Peng, Jinbiao; Han, Hongxiao; Hong, Yang; Wang, Yan; Guo, Fanji; Shi, Yaojun; Fu, Zhiqiang; Liu, Jinming; Cheng, Guofeng; Lin, Jiaojiao
2010-03-01
The present study was intend to clone and express the cDNA encoding Cyclophilin B (CyPB) of Schistosoma japonicum, its preliminary biological function and further immunoprotective effect against schistosome infection in mice. RT-PCR technique was applied to amplify a full-length cDNA encoding protein Cyclophilin B (Sj CyPB) from schistosomula cDNA. The expression profiles of Sj CyPB were determined by Real-time PCR using the template cDNAs isolated from 7, 13, 18, 23, 32 and 42 days parasites. The cDNA containing the Open Reading Frame of CyPB was then subcloned into a pGEX-6P-1 vector and transformed into competent Escherichia coli BL21 for expressing. The recombinant protein was renaturated, purified and its antigenicity were detected by Western blotting, and the immunoprotective effect induced by recombinant Sj CyPB was evaluated in Balb/C mice. The cDNA containing the ORF of Sj CyPB was cloned with the length of 672 base pairs, encoding 223 amino acids. Real-time PCR analysis revealed that the gene had the highest expression in 18-day schistosomula, suggesting that Sj CyPB was schistosomula differentially expressed gene. The recombinant protein showed a good antigenicity detected by Western blotting. Animal experiment indicated that the vaccination of recombinant CyPB protein in mice led to 31.5% worm and 41.01% liver egg burden reduction, respectively, compared with those of the control. A full-length cDNA differentially expressed in schistosomula was obtained. The recombinant Sj CyPB protein could induce partial protection against schistosome infection.
-
FUNGWITHAYA, Punpichaya; BRIKSHAVANA, Pasakorn; CHANCHAITHONG, Pattrarat; PRAPASARAKUL, Nuvee
2016-01-01
This study aimed to investigate the spread of methicillin-resistant coagulase-positive staphylococci (MRCoPS) among veterinary staff, hand-touch sites and surgical tissue during cystotomy operations on cats and dogs that were patients, and to analyze the genetic relatedness and antimicrobial resistance profiles of the isolates. Human and environmental samples were obtained from the nasal passageways of 12 surgeons and veterinary assistants and from 29 hand-touch sites of instruments in operative units and subjected to bacterial isolation and enumeration. Swab samples were collected in triplicate from 29 dogs and three cats at the site of incision, from the incision area, from the peritoneum during surgery and from the peritoneum before suture. MRCoPS were identified by mecA gene detection and characterized by their antibiogram profile, SCCmec type and pulsed-field gel electrophoresis. Twenty-four staphylococci were isolated, derived from one veterinary assistant, 12 operating room floor areas and hand-touch sites, three dogs and one cat. Methicillin-resistant S. pseudintermedius (MRSP) was found on an electric clipper and rebreathing circuits in the operating room. Three dogs were positive for MRSP during surgery, and one methicillin-resistant S. aureus (MRSA) was detected in a cat. All MRCoPS were resistant to doxycycline, erythromycin, clindamycin and enrofloxacin, but no patients developed surgical site infections. According to their genotypic patterns, the clones obtained from the environment and human sources differed from the animal clones. Despite intensive hygienic management, a variety of MRCoPS clones were present within the surgical unit and during surgery. PMID:27990009
-
Fungwithaya, Punpichaya; Brikshavana, Pasakorn; Chanchaithong, Pattrarat; Prapasarakul, Nuvee
2017-02-28
This study aimed to investigate the spread of methicillin-resistant coagulase-positive staphylococci (MRCoPS) among veterinary staff, hand-touch sites and surgical tissue during cystotomy operations on cats and dogs that were patients, and to analyze the genetic relatedness and antimicrobial resistance profiles of the isolates. Human and environmental samples were obtained from the nasal passageways of 12 surgeons and veterinary assistants and from 29 hand-touch sites of instruments in operative units and subjected to bacterial isolation and enumeration. Swab samples were collected in triplicate from 29 dogs and three cats at the site of incision, from the incision area, from the peritoneum during surgery and from the peritoneum before suture. MRCoPS were identified by mecA gene detection and characterized by their antibiogram profile, SCCmec type and pulsed-field gel electrophoresis. Twenty-four staphylococci were isolated, derived from one veterinary assistant, 12 operating room floor areas and hand-touch sites, three dogs and one cat. Methicillin-resistant S. pseudintermedius (MRSP) was found on an electric clipper and rebreathing circuits in the operating room. Three dogs were positive for MRSP during surgery, and one methicillin-resistant S. aureus (MRSA) was detected in a cat. All MRCoPS were resistant to doxycycline, erythromycin, clindamycin and enrofloxacin, but no patients developed surgical site infections. According to their genotypic patterns, the clones obtained from the environment and human sources differed from the animal clones. Despite intensive hygienic management, a variety of MRCoPS clones were present within the surgical unit and during surgery.
-
Surpassing the no-cloning limit with a heralded hybrid linear amplifier for coherent states
Haw, Jing Yan; Zhao, Jie; Dias, Josephine; Assad, Syed M.; Bradshaw, Mark; Blandino, Rémi; Symul, Thomas; Ralph, Timothy C.; Lam, Ping Koy
2016-01-01
The no-cloning theorem states that an unknown quantum state cannot be cloned exactly and deterministically due to the linearity of quantum mechanics. Associated with this theorem is the quantitative no-cloning limit that sets an upper bound to the quality of the generated clones. However, this limit can be circumvented by abandoning determinism and using probabilistic methods. Here, we report an experimental demonstration of probabilistic cloning of arbitrary coherent states that clearly surpasses the no-cloning limit. Our scheme is based on a hybrid linear amplifier that combines an ideal deterministic linear amplifier with a heralded measurement-based noiseless amplifier. We demonstrate the production of up to five clones with the fidelity of each clone clearly exceeding the corresponding no-cloning limit. Moreover, since successful cloning events are heralded, our scheme has the potential to be adopted in quantum repeater, teleportation and computing applications. PMID:27782135
-
Human cloning: can it be made safe?
Rhind, Susan M; Taylor, Jane E; De Sousa, Paul A; King, Tim J; McGarry, Michelle; Wilmut, Ian
2003-11-01
There are continued claims of attempts to clone humans using nuclear transfer, despite the serious problems that have been encountered in cloning other mammals. It is known that epigenetic and genetic mechanisms are involved in clone failure, but we still do not know exactly how. Human reproductive cloning is unethical, but the production of cells from cloned embryos could offer many potential benefits. So, can human cloning be made safe?
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Bartkiewicz, Karol; Miranowicz, Adam
We find an optimal quantum cloning machine, which clones qubits of arbitrary symmetrical distribution around the Bloch vector with the highest fidelity. The process is referred to as phase-independent cloning in contrast to the standard phase-covariant cloning for which an input qubit state is a priori better known. We assume that the information about the input state is encoded in an arbitrary axisymmetric distribution (phase function) on the Bloch sphere of the cloned qubits. We find analytical expressions describing the optimal cloning transformation and fidelity of the clones. As an illustration, we analyze cloning of qubit state described by themore » von Mises-Fisher and Brosseau distributions. Moreover, we show that the optimal phase-independent cloning machine can be implemented by modifying the mirror phase-covariant cloning machine for which quantum circuits are known.« less
-
Pedersen, Rebecca; Andersen, Anders Daniel; Hermann-Bank, Marie Louise; Stagsted, Jan; Boye, Mette
2013-01-01
The aim of this study was to investigate the effect of high-far-high-energy diet on cloned and non-cloned domestic pigs of both lean and obese phenotype and to evaluate if the lean cloned pigs had a lower inter-individual variation as compared with non-cloned pigs. The microbiota of colon and terminal ileum was investigated in cloned and non-cloned pigs that received a high-far-high-energy diet with either restricted or ad libitum access to feed, resulting in lean and obese phenotypes, respectively. The fecal microbiota of lean pigs was investigated by terminal restriction fragment length polymorphism (T-RFLP). The intestinal microbiota of lean and obese cloned and non-cloned pigs was analyzed by quantitative real time PCR and a novel high-throughput qPCR platform (Fluidigm). Principal component analysis (PCA) of the T-RFLP profiles revealed that lean cloned and non-cloned pigs had a different overall composition of their gut microbiota. The colon of lean cloned pigs contained relatively more bacteria belonging to the phylum Firmicutes and less from the phylum Bacteroidetes than obese cloned pigs as estimated by qPCR. Fluidigm qPCR results revealed differences in specific bacterial groups in the gut microbiota of both lean and obese pigs. Our results suggest that high-far-high-energy diet is associated with changes in the gut microbiota even in the absence of obesity. Overall, the cloned pigs had a different gut microbiota from that of non-cloned pigs. To our knowledge this is the first study to investigate the gut microbiota of cloned domestic pigs of lean and obese phenotype. PMID:23974297
-
Pedersen, Rebecca; Andersen, Anders Daniel; Hermann-Bank, Marie Louise; Stagsted, Jan; Boye, Mette
2013-01-01
The aim of this study was to investigate the effect of high-far-high-energy diet on cloned and non-cloned domestic pigs of both lean and obese phenotype and to evaluate if the lean cloned pigs had a lower inter-individual variation as compared with non-cloned pigs. The microbiota of colon and terminal ileum was investigated in cloned and non-cloned pigs that received a high-far-high-energy diet with either restricted or ad libitum access to feed, resulting in lean and obese phenotypes, respectively. The fecal microbiota of lean pigs was investigated by terminal restriction fragment length polymorphism (T-RFLP). The intestinal microbiota of lean and obese cloned and non-cloned pigs was analyzed by quantitative real time PCR and a novel high-throughput qPCR platform (Fluidigm). Principal component analysis (PCA) of the T-RFLP profiles revealed that lean cloned and non-cloned pigs had a different overall composition of their gut microbiota. The colon of lean cloned pigs contained relatively more bacteria belonging to the phylum Firmicutes and less from the phylum Bacteroidetes than obese cloned pigs as estimated by qPCR. Fluidigm qPCR results revealed differences in specific bacterial groups in the gut microbiota of both lean and obese pigs. Our results suggest that high-far-high-energy diet is associated with changes in the gut microbiota even in the absence of obesity. Overall, the cloned pigs had a different gut microbiota from that of non-cloned pigs. To our knowledge this is the first study to investigate the gut microbiota of cloned domestic pigs of lean and obese phenotype.
-
Distribution of quantum Fisher information in asymmetric cloning machines
Xiao, Xing; Yao, Yao; Zhou, Lei-Ming; Wang, Xiaoguang
2014-01-01
An unknown quantum state cannot be copied and broadcast freely due to the no-cloning theorem. Approximate cloning schemes have been proposed to achieve the optimal cloning characterized by the maximal fidelity between the original and its copies. Here, from the perspective of quantum Fisher information (QFI), we investigate the distribution of QFI in asymmetric cloning machines which produce two nonidentical copies. As one might expect, improving the QFI of one copy results in decreasing the QFI of the other copy. It is perhaps also unsurprising that asymmetric phase-covariant cloning outperforms universal cloning in distributing QFI since a priori information of the input state has been utilized. However, interesting results appear when we compare the distributabilities of fidelity (which quantifies the full information of quantum states), and QFI (which only captures the information of relevant parameters) in asymmetric cloning machines. Unlike the results of fidelity, where the distributability of symmetric cloning is always optimal for any d-dimensional cloning, we find that any asymmetric cloning outperforms symmetric cloning on the distribution of QFI for d ≤ 18, whereas some but not all asymmetric cloning strategies could be worse than symmetric ones when d > 18. PMID:25484234
-
Gao, Fan-Xiang; Wang, Yang; Zhang, Qi-Ya; Mou, Cheng-Yan; Li, Zhi; Deng, Yuan-Sheng; Zhou, Li; Gui, Jian-Fang
2017-07-24
Gibel carp is an important aquaculture species in China, and a herpesvirus, called as Carassius auratus herpesvirus (CaHV), has hampered the aquaculture development. Diverse gynogenetic clones of gibel carp have been identified or created, and some of them have been used as aquaculture varieties, but their resistances to herpesvirus and the underlying mechanism remain unknown. To reveal their susceptibility differences, we firstly performed herpesvirus challenge experiments in three gynogenetic clones of gibel carp, including the leading variety clone A + , candidate variety clone F and wild clone H. Three clones showed distinct resistances to CaHV. Moreover, 8772, 8679 and 10,982 differentially expressed unigenes (DEUs) were identified from comparative transcriptomes between diseased individuals and control individuals of clone A + , F and H, respectively. Comprehensive analysis of the shared DEUs in all three clones displayed common defense pathways to the herpesvirus infection, activating IFN system and suppressing complements. KEGG pathway analysis of specifically changed DEUs in respective clones revealed distinct immune responses to the herpesvirus infection. The DEU numbers identified from clone H in KEGG immune-related pathways, such as "chemokine signaling pathway", "Toll-like receptor signaling pathway" and others, were remarkably much more than those from clone A + and F. Several IFN-related genes, including Mx1, viperin, PKR and others, showed higher increases in the resistant clone H than that in the others. IFNphi3, IFI44-like and Gig2 displayed the highest expression in clone F and IRF1 uniquely increased in susceptible clone A + . In contrast to strong immune defense in resistant clone H, susceptible clone A + showed remarkable up-regulation of genes related to apoptosis or death, indicating that clone A + failed to resist virus offensive and evidently induced apoptosis or death. Our study is the first attempt to screen distinct resistances and immune responses of three gynogenetic gibel carp clones to herpesvirus infection by comprehensive transcriptomes. These differential DEUs, immune-related pathways and IFN system genes identified from susceptible and resistant clones will be beneficial to marker-assisted selection (MAS) breeding or molecular module-based resistance breeding in gibel carp.
-
Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes
Throop, Andrea L.; LaBaer, Joshua
2015-01-01
The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular recombinational cloning technologies, including the commercially available Gateway® (Life Technologies) and In-Fusion® (Clontech) cloning technologies. PMID:25827088
-
Molecular cloning and characterization of SoxB2 gene from Zhikong scallop Chlamys farreri
NASA Astrophysics Data System (ADS)
He, Yan; Bao, Zhenmin; Guo, Huihui; Zhang, Yueyue; Zhang, Lingling; Wang, Shi; Hu, Jingjie; Hu, Xiaoli
2013-11-01
The Sox proteins play critical roles during the development of animals, including sex determination and central nervous system development. In this study, the SoxB2 gene was cloned from a mollusk, the Zhikong scallop ( Chlamys farreri), and characterized with respect to phylogeny and tissue distribution. The full-length cDNA and genomic DNA sequences of C. farreri SoxB2 ( Cf SoxB2) were obtained by rapid amplification of cDNA ends and genome walking, respectively, using a partial cDNA fragment from the highly conserved DNA-binding domain, i.e., the High Mobility Group (HMG) box. The full-length cDNA sequence of Cf SoxB2 was 2 048 bp and encoded 268 amino acids protein. The genomic sequence was 5 551 bp in length with only one exon. Several conserved elements, such as the TATA-box, GC-box, CAAT-box, GATA-box, and Sox/sry-sex/testis-determining and related HMG box factors, were found in the promoter region. Furthermore, real-time quantitative reverse transcription PCR assays were carried out to assess the mRNA expression of Cf SoxB 2 in different tissues. SoxB2 was highly expressed in the mantle, moderately in the digestive gland and gill, and weakly expressed in the gonad, kidney and adductor muscle. In male and female gonads at different developmental stages of reproduction, the expression levels of Cf SoxB2 were similar. Considering the specific expression and roles of SoxB 2 in other animals, in particular vertebrates, and the fact that there are many pallial nerves in the mantle, cerebral ganglia in the digestive gland and gill nerves in gill, we propose a possible essential role in nervous tissue function for Sox B 2 in C. farreri.
-
Godoy, Daniel; Randle, Gaynor; Simpson, Andrew J; Aanensen, David M; Pitt, Tyrone L; Kinoshita, Reimi; Spratt, Brian G
2003-05-01
A collection of 147 isolates of Burkholderia pseudomallei, B. mallei, and B. thailandensis was characterized by multilocus sequence typing (MLST). The 128 isolates of B. pseudomallei, the causative agent of melioidosis, were obtained from diverse geographic locations, from humans and animals with disease, and from the environment and were resolved into 71 sequence types. The utility of the MLST scheme for epidemiological investigations was established by analyzing isolates from captive marine mammals and birds and from humans in Hong Kong with melioidosis. MLST gave a level of resolution similar to that given by pulsed-field gel electrophoresis and identified the same three clones causing disease in animals, each of which was also associated with disease in humans. The average divergence between the alleles of B. thailandensis and B. pseudomallei was 3.2%, and there was no sharing of alleles between these species. Trees constructed from differences in the allelic profiles of the isolates and from the concatenated sequences of the seven loci showed that the B. pseudomallei isolates formed a cluster of closely related lineages that were fully resolved from the cluster of B. thailandensis isolates, confirming their separate species status. However, isolates of B. mallei, the causative agent of glanders, recovered from three continents over a 30-year period had identical allelic profiles, and the B. mallei isolates clustered within the B. pseudomallei group of isolates. Alleles at six of the seven loci in B. mallei were also present within B. pseudomallei isolates, and B. mallei is a clone of B. pseudomallei that, on population genetics grounds, should not be given separate species status.
-
Li, Hong Lian; Gu, Xiao Hui; Li, Bi Jun; Chen, Xiao; Lin, Hao Ran; Xia, Jun Hong
2017-01-01
Hypoxia is a major cause of fish morbidity and mortality in the aquatic environment. Hypoxia-inducible factors are very important modulators in the transcriptional response to hypoxic stress. In this study, we characterized and conducted functional analysis of hypoxia-inducible factor HIF1α and its inhibitor HIF1αn in Nile tilapia (Oreochromis niloticus). By cloning and Sanger sequencing, we obtained the full length cDNA sequences for HIF1α (2686bp) and HIF1αn (1308bp), respectively. The CDS of HIF1α includes 15 exons encoding 768 amino acid residues and the CDS of HIF1αn contains 8 exons encoding 354 amino acid residues. The complete CDS sequences of HIF1α and HIF1αn cloned from tilapia shared very high homology with known genes from other fishes. HIF1α show differentiated expression in different tissues (brain, heart, gill, spleen, liver) and at different hypoxia exposure times (6h, 12h, 24h). HIF1αn expression level under hypoxia is generally increased (6h, 12h, 24h) and shows extremely highly upregulation in brain tissue under hypoxia. A functional determination site analysis in the protein sequences between fish and land animals identified 21 amino acid sites in HIF1α and 2 sites in HIF1αn as significantly associated sites (α = 0.05). Phylogenetic tree-based positive selection analysis suggested 22 sites in HIF1α as positively selected sites with a p-value of at least 95% for fish lineages compared to the land animals. Our study could be important for clarifying the mechanism of fish adaptation to aquatic hypoxia environment.