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Sample records for adult somatic cells

  1. Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer.

    PubMed

    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.

  2. Chinmo is sufficient to induce male fate in somatic cells of the adult Drosophila ovary.

    PubMed

    Ma, Qing; de Cuevas, Margaret; Matunis, Erika L

    2016-03-01

    Sexual identity is continuously maintained in specific differentiated cell types long after sex determination occurs during development. In the adult Drosophila testis, the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo) acts with the canonical male sex determinant DoublesexM (Dsx(M)) to maintain the male identity of somatic cyst stem cells and their progeny. Here we find that ectopic expression of chinmo is sufficient to induce a male identity in adult ovarian somatic cells, but it acts through a Dsx(M)-independent mechanism. Conversely, the feminization of the testis somatic stem cell lineage caused by loss of chinmo is enhanced by expression of the canonical female sex determinant Dsx(F), indicating that chinmo acts in parallel with the canonical sex determination pathway to maintain the male identity of testis somatic cells. Consistent with this finding, ectopic expression of female sex determinants in the adult testis disrupts tissue morphology. The miRNA let-7 downregulates chinmo in many contexts, and ectopic expression of let-7 in the adult testis is sufficient to recapitulate the chinmo loss-of-function phenotype, but we find no apparent phenotypes upon removal of let-7 in the adult ovary or testis. Our finding that chinmo is necessary and sufficient to promote a male identity in adult gonadal somatic cells suggests that the sexual identity of somatic cells can be reprogrammed in the adult Drosophila ovary as well as in the testis. PMID:26811385

  3. Adult somatic stem cells in the human parasite, Schistosoma mansoni

    PubMed Central

    Collins, James J.; Wang, Bo; Lambrus, Bramwell G.; Tharp, Marla; Iyer, Harini; Newmark, Phillip A.

    2013-01-01

    Summary Schistosomiasis is among the most prevalent human parasitic diseases, affecting more than 200 million people worldwide1. The etiological agents of this disease are trematode flatworms (Schistosoma) that live and lay eggs within the vasculature of the host. These eggs lodge in host tissues, causing inflammatory responses that are the primary cause of morbidity. Because these parasites can live and reproduce within human hosts for decades2, elucidating the mechanisms that promote their longevity is of fundamental importance. Although adult pluripotent stem cells, called neoblasts, drive long-term homeostatic tissue maintenance in long-lived free-living flatworms3,4 (e.g., planarians), and neoblast-like cells have been described in some parasitic tapeworms5, little is known about whether similar cell types exist in any trematode species. Here, we describe a population of neoblast-like cells in the trematode Schistosoma mansoni. These cells resemble planarian neoblasts morphologically and share their ability to proliferate and differentiate into derivatives of multiple germ layers. Capitalizing on available genomic resources6,7 and RNAseq-based gene expression profiling, we find that these schistosome neoblast-like cells express a fibroblast growth factor receptor ortholog. Using RNA interference we demonstrate that this gene is required for the maintenance of these neoblast-like cells. Our observations suggest that adaptation of developmental strategies shared by free-living ancestors to modern-day schistosomes likely contributed to the success of these animals as long-lived obligate parasites. We expect that future studies deciphering the function of these neoblast-like cells will have important implications for understanding the biology of these devastating parasites. PMID:23426263

  4. Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells.

    PubMed

    Yamada, Mitsutoshi; Johannesson, Bjarki; Sagi, Ido; Burnett, Lisa Cole; Kort, Daniel H; Prosser, Robert W; Paull, Daniel; Nestor, Michael W; Freeby, Matthew; Greenberg, Ellen; Goland, Robin S; Leibel, Rudolph L; Solomon, Susan L; Benvenisty, Nissim; Sauer, Mark V; Egli, Dieter

    2014-06-26

    The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.

  5. Reprogramming of somatic cells.

    PubMed

    Rajasingh, Johnson

    2012-01-01

    Reprogramming of adult somatic cells into pluripotent stem cells may provide an attractive source of stem cells for regenerative medicine. It has emerged as an invaluable method for generating patient-specific stem cells of any cell lineage without the use of embryonic stem cells. A revolutionary study in 2006 showed that it is possible to convert adult somatic cells directly into pluripotent stem cells by using a limited number of pluripotent transcription factors and is called as iPS cells. Currently, both genomic integrating viral and nonintegrating nonviral methods are used to generate iPS cells. However, the viral-based technology poses increased risk of safety, and more studies are now focused on nonviral-based technology to obtain autologous stem cells for clinical therapy. In this review, the pros and cons of the present iPS cell technology and the future direction for the successful translation of this technology into the clinic are discussed.

  6. Cloning adult farm animals: a review of the possibilities and problems associated with somatic cell nuclear transfer.

    PubMed

    Edwards, J L; Schrick, F N; McCracken, M D; van Amstel, S R; Hopkins, F M; Welborn, M G; Davies, C J

    2003-08-01

    In 1997, Wilmut et al. announced the birth of Dolly, the first ever clone of an adult animal. To date, adult sheep, goats, cattle, mice, pigs, cats and rabbits have been cloned using somatic cell nuclear transfer. The ultimate challenge of cloning procedures is to reprogram the somatic cell nucleus for development of the early embryo. The cell type of choice for reprogramming the somatic nucleus is an enucleated oocyte. Given that somatic cells are easily obtained from adult animals, cultured in the laboratory and then genetically modified, cloning procedures are ideal for introducing specific genetic modifications in farm animals. Genetic modification of farm animals provides a means of studying genes involved in a variety of biological systems and disease processes. Moreover, genetically modified farm animals have created a new form of 'pharming' whereby farm animals serve as bioreactors for production of pharmaceuticals or organ donors. A major limitation of cloning procedures is the extreme inefficiency for producing live offspring. Dolly was the only live offspring produced after 277 attempts. Similar inefficiencies for cloning adult animals of other species have been described by others. Many factors related to cloning procedures and culture environment contribute to the death of clones, both in the embryonic and fetal periods as well as during neonatal life. Extreme inefficiencies of this magnitude, along with the fact that death of the surrogate may occur, continue to raise great concerns with cloning humans.

  7. Cloning adult farm animals: a review of the possibilities and problems associated with somatic cell nuclear transfer.

    PubMed

    Edwards, J L; Schrick, F N; McCracken, M D; van Amstel, S R; Hopkins, F M; Welborn, M G; Davies, C J

    2003-08-01

    In 1997, Wilmut et al. announced the birth of Dolly, the first ever clone of an adult animal. To date, adult sheep, goats, cattle, mice, pigs, cats and rabbits have been cloned using somatic cell nuclear transfer. The ultimate challenge of cloning procedures is to reprogram the somatic cell nucleus for development of the early embryo. The cell type of choice for reprogramming the somatic nucleus is an enucleated oocyte. Given that somatic cells are easily obtained from adult animals, cultured in the laboratory and then genetically modified, cloning procedures are ideal for introducing specific genetic modifications in farm animals. Genetic modification of farm animals provides a means of studying genes involved in a variety of biological systems and disease processes. Moreover, genetically modified farm animals have created a new form of 'pharming' whereby farm animals serve as bioreactors for production of pharmaceuticals or organ donors. A major limitation of cloning procedures is the extreme inefficiency for producing live offspring. Dolly was the only live offspring produced after 277 attempts. Similar inefficiencies for cloning adult animals of other species have been described by others. Many factors related to cloning procedures and culture environment contribute to the death of clones, both in the embryonic and fetal periods as well as during neonatal life. Extreme inefficiencies of this magnitude, along with the fact that death of the surrogate may occur, continue to raise great concerns with cloning humans. PMID:12846674

  8. Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras.

    PubMed

    Keighren, Margaret A; Flockhart, Jean H; West, John D

    2016-05-15

    The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1(-/-) null mouse embryos die but a previous study showed that some homozygous Gpi1(-/-) null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1(-/-)↔Gpi1(c/c) chimaera with functional Gpi1(-/-) null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1(-/-) null cells in adult Gpi1(-/-)↔Gpi1(c/c) chimaeras and determine if Gpi1(-/-) null germ cells are functional. Analysis of adult Gpi1(-/-)↔Gpi1(c/c) chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1(-/-) null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1(-/-) null oocytes in one female Gpi1(-/-)↔Gpi1(c/c) chimaera were functional and provided preliminary evidence that one male putative Gpi1(-/-)↔Gpi1(c/c) chimaera produced functional spermatozoa from homozygous Gpi1(-/-) null germ cells. Although the male chimaera was almost certainly Gpi1(-/-)↔Gpi1(c/c), this part of the study is considered preliminary because only blood was typed for GPI. Gpi1(-/-) null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1(-/-) null germ cells, it successfully identified functional Gpi1(-/-) null oocytes and revealed that some Gpi1(-/-) null cells could survive in many adult tissues.

  9. Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras

    PubMed Central

    Keighren, Margaret A.; Flockhart, Jean H.

    2016-01-01

    ABSTRACT The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1−/− null mouse embryos die but a previous study showed that some homozygous Gpi1−/− null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1−/−↔Gpi1c/c chimaera with functional Gpi1−/− null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1−/− null cells in adult Gpi1−/−↔Gpi1c/c chimaeras and determine if Gpi1−/− null germ cells are functional. Analysis of adult Gpi1−/−↔Gpi1c/c chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1−/− null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1−/− null oocytes in one female Gpi1−/−↔Gpi1c/c chimaera were functional and provided preliminary evidence that one male putative Gpi1−/−↔Gpi1c/c chimaera produced functional spermatozoa from homozygous Gpi1−/− null germ cells. Although the male chimaera was almost certainly Gpi1−/−↔Gpi1c/c, this part of the study is considered preliminary because only blood was typed for GPI. Gpi1−/− null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1−/− null germ cells, it successfully identified functional Gpi1−/− null oocytes and revealed that some Gpi1−/− null cells could survive in many adult tissues. PMID:27103217

  10. Somatic stem cells express Piwi and Vasa genes in an adult ctenophore: ancient association of "germline genes" with stemness.

    PubMed

    Alié, Alexandre; Leclère, Lucas; Jager, Muriel; Dayraud, Cyrielle; Chang, Patrick; Le Guyader, Hervé; Quéinnec, Eric; Manuel, Michaël

    2011-02-01

    Stem cells are essential for animal development and adult tissue homeostasis, and the quest for an ancestral gene fingerprint of stemness is a major challenge for evolutionary developmental biology. Recent studies have indicated that a series of genes, including the transposon silencer Piwi and the translational activator Vasa, specifically involved in germline determination and maintenance in classical bilaterian models (e.g., vertebrates, fly, nematode), are more generally expressed in adult multipotent stem cells in other animals like flatworms and hydras. Since the progeny of these multipotent stem cells includes both somatic and germinal derivatives, it remains unclear whether Vasa, Piwi, and associated genes like Bruno and PL10 were ancestrally linked to stemness, or to germinal potential. We have investigated the expression of Vasa, two Piwi paralogues, Bruno and PL10 in Pleurobrachia pileus, a member of the early-diverging phylum Ctenophora, the probable sister group of cnidarians. These genes were all expressed in the male and female germlines, and with the exception of one of the Piwi paralogues, they showed similar expression patterns within somatic territories (tentacle root, comb rows, aboral sensory complex). Cytological observations and EdU DNA-labelling and long-term retention experiments revealed concentrations of stem cells closely matching these gene expression areas. These stem cell pools are spatially restricted, and each specialised in the production of particular types of somatic cells. These data unveil important aspects of cell renewal within the ctenophore body and suggest that Piwi, Vasa, Bruno, and PL10 belong to a gene network ancestrally acting in two distinct contexts: (i) the germline and (ii) stem cells, whatever the nature of their progeny.

  11. Cloning of calves from various somatic cell types of male and female adult, newborn and fetal cows.

    PubMed

    Kato, Y; Tani, T; Tsunoda, Y

    2000-11-01

    Twenty-four calves were cloned from six somatic cell types of female and male adult, newborn and fetal cows. The clones were derived from female cumulus (n = 3), oviduct (n = 2) and uterine (n = 2) cells, female and male skin cells (n = 10), and male ear (n = 5) and liver (n = 2) cells. On the basis of the number of cloned embryos transferred (n = 172) to surrogate cows, the overall rate of success was 14%, but based on the number of surrogate mothers that became pregnant (n = 50), the success rate was 48%. Cell nuclei from uterus, ear and liver cells, which have not been tested previously, developed into newborn calves after nuclear transfer into enucleated oocytes. To date, seven female and six male calves have survived: six of the females were from adult cells (cumulus (n = 3), oviduct (n = 2) and skin (n = 1) cells) and one was from newborn skin cells, whereas the male calves were derived from adult ear cells (n = 3), newborn liver and skin cells (n = 2), and fetal cells (n = 1). Clones derived from adult cells frequently aborted in the later stages of pregnancy and calves developing to term showed a higher number of abnormalities than did those derived from newborn or fetal cells. The telomeric DNA lengths in the ear cells of three male calves cloned from the ear cells of a bull aged 10 years were similar to those of the original bull. However, the telomeric DNA lengths from the white blood cells of the clones, although similar to those in an age-matched control, were shorter than those of the original bull, which indicates that telomeric shortening varies among tissues.

  12. Direct reprogramming of adult somatic cells toward adventitious root formation in forest tree species: the effect of the juvenile–adult transition

    PubMed Central

    Díaz-Sala, Carmen

    2014-01-01

    Cellular plasticity refers, among others, to the capability of differentiated cells to switch the differentiation process and acquire new fates. One way by which plant cell plasticity is manifested is through de novo regeneration of organs from somatic differentiated cells in an ectopic location. However, switching the developmental program of adult cells prior to organ regeneration is difficult in many plant species, especially in forest tree species. In these species, a decline in the capacity to regenerate shoots, roots, or embryos from somatic differentiated cells is associated with tree age and maturation. The decline in the ability to form adventitious roots from stem cuttings is one of the most dramatic effects of maturation, and has been the subject of investigations on the basic nature of the process. Cell fate switches, both in plants and animals, are characterized by remarkable changes in the pattern of gene expression, as cells switch from the characteristic expression pattern of a somatic cell to a new one directing a new developmental pathway. Therefore, determining the way by which cells reset their gene expression pattern is crucial to understand cellular plasticity. The presence of specific cellular signaling pathways or tissue-specific factors underlying the establishment, maintenance, and redirection of gene expression patterns in the tissues involved in adventitious root formation could be crucial for cell fate switch and for the control of age-dependent cellular plasticity. PMID:25071793

  13. Definition of three somatic adult cell nuclear transplant methods in zebrafish (Danio rerio): before, during and after egg activation by sperm fertilization.

    PubMed

    Pérez-Camps, M; Cardona-Costa, J; Francisco-Simao, M; García-Ximénez, F

    2010-02-01

    Zebrafish somatic nuclear transplant has only been attempted using preactivated eggs. In this work, three methods to carry out the nuclear transplant using adult cells before, during and after the egg activation/fertilization were developed in zebrafish with the aim to be used in reprogramming studies. The donor nucleus from somatic adult cells was inserted: (method A) in the central region of the egg and subsequently fertilized; (method B) in the incipient animal pole at the same time that the egg was fertilized; and (method C) in the completely defined animal pole after fertilization. Larval and adult specimens were obtained using the three methods. Technical aspects related to temperature conditions, media required, egg activation/fertilization, post-ovulatory time of the transplant, egg aging, place of the donor nucleus injection in each methodology are presented. In conclusion, the technical approach developed in this work can be used in reprogramming studies.

  14. Bovine somatic cell nuclear transfer.

    PubMed

    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. PMID:20336522

  15. Somatic Cell Reprogramming into Cardiovascular Lineages

    PubMed Central

    Chen, Jenny X.; Plonowska, Karolina; Wu, Sean M.

    2015-01-01

    Ischemic cardiac disease is the leading cause of death in the developed world. The inability of the adult mammalian heart to adequately repair itself has motivated stem cell researchers to explore various strategies to regenerate cardiomyocytes after myocardial infarction. Over the past century, progressive gains in our knowledge about the cellular mechanisms governing fate determination have led to recent advances in cellular reprogramming. The identification of specific factors capable of inducing pluripotent phenotype in somatic cells as well as factors that can directly reprogram somatic cells into cardiomyocytes suggests the potential for these approaches to translate into clinical therapies in the future. While conceptually appealing, the field of cell lineage reprogramming is in its infancy and further research will be needed to improve the efficiency of the reprogramming process and the fidelity of the reprogrammed cells to their in vivo counterpart. PMID:24764131

  16. Human adult bone marrow-derived somatic cell therapy results in functional recovery and axonal plasticity following stroke in the rat.

    PubMed

    Andrews, E M; Tsai, S-Y; Johnson, S C; Farrer, J R; Wagner, J P; Kopen, G C; Kartje, G L

    2008-06-01

    Stroke is the leading cause of adult disability in the United States. To date there is no satisfactory treatment for stroke once neuronal damage has occurred. Human adult bone marrow-derived somatic cells (hABM-SC) represent a homogenous population of CD49c/CD90 co-positive, non-hematopoietic cells that have been shown to secrete therapeutically relevant trophic factors and to support axonal growth in a rodent model of spinal cord injury. Here we demonstrate that treatment with hABM-SC after ischemic stroke in adult rats results in recovery of forelimb function on a skilled motor test, and that this recovery is positively correlated with increased axonal outgrowth of the intact, uninjured corticorubral tract. While the complete mechanism of repair is still unclear, we conclude that enhancement of structural neuroplasticity from uninjured brain areas is one mechanism by which hABM-SC treatment after stroke leads to functional recovery. PMID:18440506

  17. Recent advancements in cloning by somatic cell nuclear transfer

    PubMed Central

    Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

    2013-01-01

    Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model. PMID:23166393

  18. Recent advancements in cloning by somatic cell nuclear transfer.

    PubMed

    Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

    2013-01-01

    Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.

  19. 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.

  20. More Frequent than Desired: Midgut Stem Cell Somatic Mutations.

    PubMed

    Li, Qi; Ip, Y Tony

    2015-12-01

    The accumulation of somatic mutations in adult stem cells contributes to the decline of tissue functions and cancer initiation. In this issue of Cell Stem Cell, Siudeja et al. (2015) investigate the rate and mechanism of naturally occurring mutations in Drosophila midgut intestinal stem cells during aging and find high-frequency mutations arising from multiple mechanisms. PMID:26637937

  1. Five classic articles in somatic cell reprogramming.

    PubMed

    Park, In-Hyun

    2010-09-01

    Research on somatic cell reprogramming has progressed significantly over the past few decades, from nuclear transfer into frogs' eggs in 1952 to the derivation of human-induced pluripotent stem (iPS) cells in the present day. In this article, I review five landmark papers that have laid the foundation for current efforts to apply somatic cell reprogramming in the clinic. PMID:20885901

  2. Cloning by somatic cell nuclear transfer.

    PubMed

    Fulka, J; First, N L; Loi, P; Moor, R M

    1998-10-01

    The birth of the first cloned mammals, produced by the introduction of somatic cell nuclei into enucleated oocytes, was an impressive and surprising development. Although the ethical debate has been intense, the important scientific questions raised by this work have been inadequately discussed and are still unresolved. In this essay we address three questions about nuclear transplantation in the eggs of mice and domestic animals. First, why were the recent experiments on somatic cell cloning successful, when so many others have failed? Second, were these exceptional cases, or is somatic cloning now open to all? Third, what are the future possibilities for increasing the efficiency and wider applicability of the cloning process?

  3. Human somatic cell nuclear transfer and cloning.

    PubMed

    2012-10-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer (cloning)," last published in Fertil Steril 2000;74:873-6.

  4. MicroRNA-mediated somatic cell reprogramming.

    PubMed

    Kuo, Chih-Hao; Ying, Shao-Yao

    2013-02-01

    Since the first report of induced pluripotent stem cells (iPSCs) using somatic cell nuclear transfer (SCNT), much focus has been placed on iPSCs due to their great therapeutic potential for diseases such as abnormal development, degenerative disorders, and even cancers. Subsequently, Takahashi and Yamanaka took a novel approach by using four defined transcription factors to generate iPSCs in mice and human fibroblast cells. Scientists have since been trying to refine or develop better approaches to reprogramming, either by using different combinations of transcription factors or delivery methods. However, recent reports showed that the microRNA expression pattern plays a crucial role in somatic cell reprogramming and ectopic introduction of embryonic stem cell-specific microRNAs revert cells back to an ESC-like state, although, the exact mechanism underlying this effect remains unclear. This review describes recent work that has focused on microRNA-mediated approaches to somatic cell reprogramming as well as some of the pros and cons to these approaches and a possible mechanism of action. Based on the pivotal role of microRNAs in embryogenesis and somatic cell reprogramming, studies in this area must continue in order to gain a better understanding of the role of microRNAs in stem cells regulation and activity. PMID:22961769

  5. Cloned mice derived from somatic cell nuclei.

    PubMed

    Hosaka, K; Ohi, S; Ando, A; Kobayashi, M; Sato, K

    2000-12-01

    In 1997, a cloned sheep "Dolly" was produced by nuclear transfer of somatic cell. The first birth of cloned mice derived from some somatic cells were succeeded in 1998. At present, it is shown that somatic cells, cumulus cells, fibroblasts and Sertoli cells can be used to the study of cloned animal as nuclear donor. In this study investigation was designed to compare with efficiency on the production of cloned embryos by using the microinjection and the electrofusion methods for nuclear transfer. Oocyte enucleation was performed with a micromanipulator. The oocyte was held by holding pipette, and was enucleated using a beveled pipette. Microinjection method: Cell's nucleus injection was carried out by piezo-micromanipulator. Cytochalasin B treated cumulus cell was aspirated into a injection pipette, and was broken its plasma membrane using the injection pipette. Then, the cumulus cell was injected into the enucleated ooplasm directly. Electrofusion method: The cell was aspirated into a beveled pipette, and then an aspirated cell was inserted into perivitelline space. Then, the pair of enucleated oocyte and cell was fused using electrical cell fusion apparatus. The reconstituted embryos were activated after nuclear transfer using St2+. Reconstituted embryos had been produced by the microinjection showed the embryonic development to over 8-cell stages. But, the rate of fragmentation of reconstituted embryos by the microinjection showed a little high rate in comparison with the electrofusion. When some reconstituted embryos by the microinjection were transplanted to pseudopregnant females' oviduct, 9 fetuses were observed at 14 days post coitum. PMID:11329940

  6. Somatic cell nuclear transfer in mammals: progress and applications.

    PubMed

    Colman, A

    Somatic nuclear transfer has been performed with frogs since the early 1960s, yet it has proved impossible to generate an adult frog using an adult cell as nuclear donor. After some initial skepticism, the birth of sheep, cows, goats, and mice using this technique with fetal or adult cell donors is now established fact. The success with adult mammalian cell donors extends the historic work in frogs by attesting to the totipotency of nuclei in at least some adult, differentiated cell types. Because the technique offers a developmental read out of the totality of genetic and molecular lifetime changes accumulated by the nucleus of a single somatic cell, basic research applications are seen in the fields of ageing, cancer, X chromosome inactivation, and imprinting. The prospect of a method for gene targeting in livestock holds particular promise for commercial applications; whilst for humans, the use of nuclear transfer to provide diverse populations of customized stem cells for therapeutic purposes presents a tantalizing future goal.

  7. Genetic improvement of mastitis through selection on somatic cell count.

    PubMed

    Shook, G E

    1993-11-01

    Heredity influences both clinical mastitis and somatic cell score. Intramammary infection is the major cause of elevated somatic cell score. A nationwide program of genetic evaluation of dairy cattle for somatic cell score is being developed. Proper selection of artificial insemination sires, considering their genetic merit for both milk production and somatic cell score, will reduce the genetic increase in mastitis susceptibility that accompanies selection for high production. PMID:8242460

  8. Metaplasia and somatic cell reprogramming.

    PubMed

    Slack, J M W

    2009-01-01

    The nature and occurrence of metaplasia is briefly reviewed. A theory of how metaplasia is initiated is presented, depending on the idea that it represents an alteration in the combination of developmental transcription factors that are expressed. Two examples of experimental metaplasia, provoked by over-expression of specific transcription factors, are presented: the transformation of B lymphocytes to macrophages, and of pancreatic exocrine cells to hepatocytes. The formation of induced pluripotential stem cells (iPS cells) is considered an example of the same process, in which the destination state is the embryonic stem cell. It is noted that iPS cell production is a stochastic process, depending on selection to obtain the desired cell type. It is proposed that analogous technology, using the appropriate transcription factors, could be used to bring about transformation to cell types other than embryonic stem cells. PMID:18855879

  9. Reprogramming of Somatic Cells Towards Pluripotency by Cell Fusion.

    PubMed

    Malinowski, Andrzej R; Fisher, Amanda G

    2016-01-01

    Pluripotent reprogramming can be dominantly induced in a somatic nucleus upon fusion with a pluripotent cell such as embryonic stem (ES) cell. Cell fusion between ES cells and somatic cells results in the formation of heterokaryons, in which the somatic nuclei begin to acquire features of the pluripotent partner. The generation of interspecies heterokaryons between mouse ES- and human somatic cells allows an experimenter to distinguish the nuclear events occurring specifically within the reprogrammed nucleus. Therefore, cell fusion provides a simple and rapid approach to look at the early nuclear events underlying pluripotent reprogramming. Here, we describe a polyethylene glycol (PEG)-mediated cell fusion protocol to generate interspecies heterokaryons and intraspecies hybrids between ES cells and B lymphocytes or fibroblasts. PMID:27659994

  10. Reprogramming of Somatic Cells Towards Pluripotency by Cell Fusion.

    PubMed

    Malinowski, Andrzej R; Fisher, Amanda G

    2016-01-01

    Pluripotent reprogramming can be dominantly induced in a somatic nucleus upon fusion with a pluripotent cell such as embryonic stem (ES) cell. Cell fusion between ES cells and somatic cells results in the formation of heterokaryons, in which the somatic nuclei begin to acquire features of the pluripotent partner. The generation of interspecies heterokaryons between mouse ES- and human somatic cells allows an experimenter to distinguish the nuclear events occurring specifically within the reprogrammed nucleus. Therefore, cell fusion provides a simple and rapid approach to look at the early nuclear events underlying pluripotent reprogramming. Here, we describe a polyethylene glycol (PEG)-mediated cell fusion protocol to generate interspecies heterokaryons and intraspecies hybrids between ES cells and B lymphocytes or fibroblasts.

  11. Mechanisms and models of somatic cell reprogramming

    PubMed Central

    Buganim, Yosef; Faddah, Dina A.; Jaenisch, Rudolf

    2014-01-01

    Conversion of somatic cells to pluripotency by defined factors is a long and complex process that yields embryonic stem cell-like cells that vary in their developmental potential. To improve the quality of resulting induced pluripotent stem cells (iPSCs), which is important for potential therapeutic applications, and to address fundamental questions about control of cell identity, molecular mechanisms of the reprogramming process must be understood. Here we discuss recent discoveries regarding the role of reprogramming factors in remodeling the genome, including new insights into the function of c-Myc, and describe the different phases, markers and emerging models of reprogramming. PMID:23681063

  12. Handmade somatic cell cloning in cattle.

    PubMed

    Vajta, Gàbor; Lewis, Ian M; Tecirlioglu, R Tayfur

    2006-01-01

    Apart from the biological and ethical problems, technical difficulties also hamper the improvement and widespread application of somatic cell nuclear transfer (NT). Recently introduced zona-free procedures may offer a solution for the latter problem. The most radical approach of these techniques is the so-called handmade cloning (HMC). It does not require micromanipulators because the manipulations required for both enucleation and nucleus transfer are performed by hand. The HMC technique includes manual bisection of zona-free oocytes, selection of cytoplasts by staining, and the simultaneous fusion of the somatic cell with two cytoplasts to produce a cloned embryo. HMC is a rapid and efficient technique that suits large-scale NT programs. It requires less expertise and time than traditional NT methods and the cost of equipment is significantly less. Production efficiency is high and embryo quality, in terms of pregnancy rates and live births, is not compromised. Although HMC has been developed particularly for bovine NT, the technique is applicable to other species. The method may become a useful tool for both experimental and commercial somatic cell cloning because it allows for standardization of procedures and provides the possibility of automation.

  13. Hemoglobins, programmed cell death and somatic embryogenesis.

    PubMed

    Hill, Robert D; Huang, Shuanglong; Stasolla, Claudio

    2013-10-01

    Programmed cell death (PCD) is a universal process in all multicellular organisms. It is a critical component in a diverse number of processes ranging from growth and differentiation to response to stress. Somatic embryogenesis is one such process where PCD is significantly involved. Nitric oxide is increasingly being recognized as playing a significant role in regulating PCD in both mammalian and plant systems. Plant hemoglobins scavenge NO, and evidence is accumulating that events that modify NO levels in plants also affect hemoglobin expression. Here, we review the process of PCD, describing the involvement of NO and plant hemoglobins in the process. NO is an effector of cell death in both plants and vertebrates, triggering the cascade of events leading to targeted cell death that is a part of an organism's response to stress or to tissue differentiation and development. Expression of specific hemoglobins can alter this response in plants by scavenging the NO, thus, interrupting the death process. Somatic embryogenesis is used as a model system to demonstrate how cell-specific expression of different classes of hemoglobins can alter the embryogenic process, affecting hormone synthesis, cell metabolite levels and genes associated with PCD and embryogenic competence. We propose that plant hemoglobins influence somatic embryogenesis and PCD through cell-specific expression of a distinct plant hemoglobin. It is based on the premise that both embryogenic competence and PCD are strongly influenced by cellular NO levels. Increases in cellular NO levels result in elevated Zn(2+) and reactive-oxygen species associated with PCD, but they also result in decreased expression of MYC2, a transcription factor that is a negative effector of indoleacetic acid synthesis, a hormone that positively influences embryogenic competence. Cell-specific hemoglobin expression reduces NO levels as a result of NO scavenging, resulting in cell survival.

  14. Blastocysts derivation from somatic cell fusion with premature oocytes (prematuration somatic cell fusion).

    PubMed

    Saadeldin, Islam M; Khoirinaya, Candrani; Kim, Su Jin; Moon, Joon Ho; Almadaly, Essam; Lee, Byeong Chun

    2016-02-01

    This study was undertaken to investigate the development of immature oocytes after their fusion with male somatic cells expressing red fluorescence protein (RFP). RFP-expressing cells were fused with immature oocytes, matured in vitro and then parthenogenetically activated. Somatic nuclei showed spindle formation, 1st polar body extrusion after in vitro maturation and protruded the 2nd polar body after parthenogenetic activation. RFP was expressed in the resultant embryos; two-cell stage and blastocysts. Chromosomal analysis showed aneuploidy in 81.82% of the resulting blastocysts while 18.18% of the resulting blastocysts were diploid. Among eight RFP-expressing blastocysts, Xist mRNAs was detected in six while Sry mRNA was detected in only one blastocyst. We propose "prematuration somatic cell fusion" as an approach to generate embryos using somatic cells instead of spermatozoa. The current approach, if improved, would assist production of embryos for couples where the male partner is sterile, however, genetic and chromosomal analysis of the resultant embryos are required before transfer to the mothers.

  15. Repression of somatic cell fate in the germline.

    PubMed

    Robert, Valérie J; Garvis, Steve; Palladino, Francesca

    2015-10-01

    Germ cells must transmit genetic information across generations, and produce gametes while also maintaining the potential to form all cell types after fertilization. Preventing the activation of somatic programs is, therefore, crucial to the maintenance of germ cell identity. Studies in Caenorhabditis elegans, Drosophila melanogaster, and mouse have revealed both similarities and differences in how somatic gene expression is repressed in germ cells, thereby preventing their conversion into somatic tissues. This review will focus on recent developments in our understanding of how global or gene-specific transcriptional repression, chromatin regulation, and translational repression operate in the germline to maintain germ cell identity and repress somatic differentiation programs. PMID:26043973

  16. Changes in somatic cell structure during senescence of Volvox carteri.

    PubMed

    Pommerville, J C; Kochert, G D

    1981-06-01

    Senescence of the terminally differentiated somatic cells of the green alga, Volvox carteri f. weismannia, was investigated by light, fluorescence, and electron microscopy. Viability of the somatic cell population, as determined by trypan blue or erythrosin B exclusion, showed a sharp reduction beginning 144 h after the somatic cells had lost the ability to divide. This increased mortality rate was correlated at the light microscopic level with a retraction of the somatic cell cytoplasm, a reduction in chloroplast autofluorescence (and total chlorophyll content), and a decline in the number of vacuoles which could be localized with 9-aminoacridine fluorescence microscopy. Nuclear fluorescence with acridine orange remained unaffected during this time. Lipid bodies increased in older cells, and total lipid analysis showed a sharp increase beginning 96 h after the somatic cells had stopped dividing. Electron microscopic comparison between young (48--72 h) and old (168 h) somatic cells showed a disorganization of chloroplast structure, a decline in the number of cytoplasmic ribosomes, and, substantiating the light microscopy, and accumulation of lipid bodies in the cytoplasm of the older cells. The results demonstrate progressive changes in somatic cell structure with age and are suggestive of cells under nutrient stress even though they are in nutrient medium. Therefore senescence and death of the V. carteri somatic cells may be caused, in part, by an inability to take up or utilize nutrients present in the culture medium. PMID:7285941

  17. Advances in Reprogramming Somatic Cells to Induced Pluripotent Stem Cells

    PubMed Central

    Patel, Minal; Yang, Shuying

    2010-01-01

    Traditionally, nuclear reprogramming of cells has been performed by transferring somatic cell nuclei into oocytes, by combining somatic and pluripotent cells together through cell fusion and through genetic integration of factors through somatic cell chromatin. All of these techniques changes gene expression which further leads to a change in cell fate. Here we discuss recent advances in generating induced pluripotent stem cells, different reprogramming methods and clinical applications of iPS cells. Viral vectors have been used to transfer transcription factors (Oct4, Sox2, c-myc, Klf4, and nanog) to induce reprogramming of mouse fibroblasts, neural stem cells, neural progenitor cells, keratinocytes, B lymphocytes and meningeal membrane cells towards pluripotency. Human fibroblasts, neural cells, blood and keratinocytes have also been reprogrammed towards pluripotency. In this review we have discussed the use of viral vectors for reprogramming both animal and human stem cells. Currently, many studies are also involved in finding alternatives to using viral vectors carrying transcription factors for reprogramming cells. These include using plasmid transfection, piggyback transposon system and piggyback transposon system combined with a non viral vector system. Applications of these techniques have been discussed in detail including its advantages and disadvantages. Finally, current clinical applications of induced pluripotent stem cells and its limitations have also been reviewed. Thus, this review is a summary of current research advances in reprogramming cells into induced pluripotent stem cells. PMID:20336395

  18. Stem cells and somatic cells: reprogramming and plasticity.

    PubMed

    Estrov, Zeev

    2009-01-01

    Recent seminal discoveries have significantly advanced the field of stem cell research and received worldwide attention. Improvements in somatic cell nuclear transfer (SCNT) technology, enabling the cloning of Dolly the sheep, and the derivation and differentiation of human embryonic stem cells raised hopes that normal cells could be generated to replace diseased or injured tissue. At the same time, in vitro and in vivo studies demonstrated that somatic cells of one tissue are capable of generating cells of another tissue. It was theorized that any cell might be reprogrammed, by exposure to a new environment, to become another cell type. This concept contradicts two established hypotheses: (1) that only specific tissues are generated from the endoderm, mesoderm, and ectoderm and (2) that tissue cells arise from a rare population of tissue-specific stem cells in a hierarchical fashion. SCNT, cell fusion experiments, and most recent gene transfer studies also contradict these hypotheses, as they demonstrate that mature somatic cells can be reprogrammed to regain pluripotent (or even totipotent) stem cell capacity. On the basis of the stem cell theory, hierarchical cancer stem cell differentiation models have been proposed. Cancer cell plasticity is an established phenomenon that supports the notion that cellular phenotype and function might be altered. Therefore, mechanisms of cellular plasticity should be exploited and the clinical significance of the cancer stem cell theory cautiously assessed. PMID:19778860

  19. A protocol for adult somatic cell nuclear transfer in medaka fish (Oryzias latipes) with a high rate of viable clone formation.

    PubMed

    Bubenshchikova, Ekaterina; Kaftanovskaya, Elena; Adachi, Tomoko; Hashimoto, Hisashi; Kinoshita, Masato; Wakamatsu, Yuko

    2013-12-01

    Previously, we successfully generated fully grown, cloned medaka (the Japanese rice fish, Oryzias latipes) using donor nuclei from primary culture cells of adult caudal fin tissue and nonenucleated recipient eggs that were heat shock-treated to induce diploidization of the nuclei. However, the mechanism of clone formation using this method is unknown, and the rate of adult clone formation is not high enough for studies in basic and applied sciences. To gain insight into the mechanism and increase the success rate of this method of clone formation, we tested two distinct nuclear transfer protocols. In one protocol, the timing of transfer of donor nuclei was changed, and in the other, the size of the donor cells was changed; each protocol was based on our original methodology. Ultimately, we obtained an unexpectedly high rate of adult clone formation using the protocol that differed with respect to the timing of donor nuclei transfer. Specifically, 17% of the transplants that developed to the blastula stage ultimately developed into adult clones. The success rate with this method was 13 times higher than that obtained using the original method. Analyses focusing on the reasons for this high success rate of clone formation will help to elucidate the mechanism of clone formation that occurs with this method.

  20. Progress in the reprogramming of somatic cells.

    PubMed

    Ma, Tianhua; Xie, Min; Laurent, Timothy; Ding, Sheng

    2013-02-01

    Pluripotent stem cells can differentiate into nearly all types of cells in the body. This unique potential provides significant promise for cell-based therapies to restore tissues or organs destroyed by injuries, degenerative diseases, aging, or cancer. The discovery of induced pluripotent stem cell (iPSC) technology offers a possible strategy to generate patient-specific pluripotent stem cells. However, because of concerns about the specificity, efficiency, kinetics, and safety of iPSC reprogramming, improvements or fundamental changes in this process are required before their effective clinical use. A chemical approach is regarded as a promising strategy to improve and change the iPSC process. Dozens of small molecules have been identified that can functionally replace reprogramming factors and significantly improve iPSC reprogramming. In addition to the prospect of deriving patient-specific tissues and organs from iPSCs, another attractive strategy for regenerative medicine is transdifferentiation-the direct conversion of one somatic cell type to another. Recent studies revealed a new paradigm of transdifferentiation: using transcription factors used in iPSC generation to induce transdifferentiation or called iPSC transcription factor-based transdifferentiation. This type of transdifferentiation not only reveals and uses the developmentally plastic intermediates generated during iPSC reprogramming but also produces a wide range of cells, including expandable tissue-specific precursor cells. Here, we review recent progress of small molecule approaches in the generation of iPSCs. In addition, we summarize the new concept of iPSC transcription factor-based transdifferentiation and discuss its application in generating various lineage-specific cells, especially cardiovascular cells.

  1. Chemically Induced Reprogramming of Somatic Cells to Pluripotent Stem Cells and Neural Cells

    PubMed Central

    Biswas, Dhruba; Jiang, Peng

    2016-01-01

    The ability to generate transplantable neural cells in a large quantity in the laboratory is a critical step in the field of developing stem cell regenerative medicine for neural repair. During the last few years, groundbreaking studies have shown that cell fate of adult somatic cells can be reprogrammed through lineage specific expression of transcription factors (TFs)-and defined culture conditions. This key concept has been used to identify a number of potent small molecules that could enhance the efficiency of reprogramming with TFs. Recently, a growing number of studies have shown that small molecules targeting specific epigenetic and signaling pathways can replace all of the reprogramming TFs. Here, we provide a detailed review of the studies reporting the generation of chemically induced pluripotent stem cells (ciPSCs), neural stem cells (ciNSCs), and neurons (ciN). We also discuss the main mechanisms of actions and the pathways that the small molecules regulate during chemical reprogramming. PMID:26861316

  2. Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning.

    PubMed

    Niemann, Heiner; Tian, X Cindy; King, W Allan; Lee, Rita S F

    2008-02-01

    The birth of 'Dolly', the first mammal cloned from an adult donor cell, has sparked a flurry of research activities to improve cloning technology and to understand the underlying mechanism of epigenetic reprogramming of the transferred somatic cell nucleus. Especially in ruminants, somatic cell nuclear transfer (SCNT) is frequently associated with pathological changes in the foetal and placental phenotype and has significant consequences for development both before and after birth. The most critical factor is epigenetic reprogramming of the transferred somatic cell nucleus from its differentiated status into the totipotent state of the early embryo. This involves an erasure of the gene expression program of the respective donor cell and the establishment of the well-orchestrated sequence of expression of an estimated number of 10 000-12 000 genes regulating embryonic and foetal development. The following article reviews the present knowledge on the epigenetic reprogramming of the transferred somatic cell nucleus, with emphasis on DNA methylation, imprinting, X-chromosome inactivation and telomere length restoration in bovine development. Additionally, we briefly discuss other approaches towards epigenetic nuclear reprogramming, including the fusion of somatic and embryonic stem cells and the overexpression of genes crucial in the formation and maintenance of the pluripotent status. Improvements in our understanding of this dramatic epigenetic reprogramming event will be instrumental in realising the great potential of SCNT for basic biological research and for various agricultural and biomedical applications.

  3. Depression, Health, and Somatic Complaints in Older Adults.

    ERIC Educational Resources Information Center

    Mahurin, Kathleen A.; Gatz, Margaret

    Although depression is considered to be common in the elderly, reliable rates of prevalence are lacking. Studies have shown that age differences on measures of depressive symptomatology can be attributed to higher levels of somatic complaints. In order to examine whether the association between somatic and depressive symptoms varies as a function…

  4. Mouse cloning and somatic cell reprogramming using electrofused blastomeres.

    PubMed

    Riaz, Amjad; Zhao, Xiaoyang; Dai, Xiangpeng; Li, Wei; Liu, Lei; Wan, Haifeng; Yu, Yang; Wang, Liu; Zhou, Qi

    2011-05-01

    Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

  5. Somatic embryogenesis - Stress-induced remodeling of plant cell fate.

    PubMed

    Fehér, Attila

    2015-04-01

    Plants as sessile organisms have remarkable developmental plasticity ensuring heir continuous adaptation to the environment. An extreme example is somatic embryogenesis, the initiation of autonomous embryo development in somatic cells in response to exogenous and/or endogenous signals. In this review I briefly overview the various pathways that can lead to embryo development in plants in addition to the fertilization of the egg cell and highlight the importance of the interaction of stress- and hormone-regulated pathways during the induction of somatic embryogenesis. Somatic embryogenesis can be initiated in planta or in vitro, directly or indirectly, and the requirement for dedifferentiation as well as the way to achieve developmental totipotency in the various systems is discussed in light of our present knowledge. The initiation of all forms of the stress/hormone-induced in vitro as well as the genetically provoked in planta somatic embryogenesis requires extensive and coordinated genetic reprogramming that has to take place at the chromatin level, as the embryogenic program is under strong epigenetic repression in vegetative plant cells. Our present knowledge on chromatin-based mechanisms potentially involved in the somatic-to-embryogenic developmental transition is summarized emphasizing the potential role of the chromatin to integrate stress, hormonal, and developmental pathways leading to the activation of the embryogenic program. The role of stress-related chromatin reorganization in the genetic instability of in vitro cultures is also discussed. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.

  6. Cloning animals by somatic cell nuclear transfer--biological factors.

    PubMed

    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.

  7. Somatic Embryogenesis of Lilium from Microbulb Transverse Thin Cell Layers.

    PubMed

    Marinangeli, Pablo

    2016-01-01

    A reliable somatic embryogenesis protocol is a prerequisite for application of other plant biotechniques. Several protocols were reported for genus Lilium, with variable success. Between them, transverse Thin Cell Layers (tTCL) were used efficiently to induce indirect somatic embryogenesis of Lilium. Somatic embryogenesis potential is dependent on the genotype, explant, and culture medium composition, especially as for plant growth regulators and environmental conditions. Usually, the process comprises three phases: embryogenic callus induction, embryogenic callus proliferation and somatic embryo germination. Somatic embryo germination can be achieved in light or dark. In the first case, complete plantlets are formed, with green leaves and pseudobulb in the base. In darkness, microbulbs are formed from single somatic embryos or clusters. A last phase of microbulb enlargement allows plantlets or microbulbs to increase their biomass. These enlarged microbulbs do not need special acclimatization conditions when transferred to soil and quickly produce sturdy plants. This chapter describes a protocol for somatic embryogenesis of Lilium using tTCL from microbulbs.

  8. Somatic Embryogenesis of Lilium from Microbulb Transverse Thin Cell Layers.

    PubMed

    Marinangeli, Pablo

    2016-01-01

    A reliable somatic embryogenesis protocol is a prerequisite for application of other plant biotechniques. Several protocols were reported for genus Lilium, with variable success. Between them, transverse Thin Cell Layers (tTCL) were used efficiently to induce indirect somatic embryogenesis of Lilium. Somatic embryogenesis potential is dependent on the genotype, explant, and culture medium composition, especially as for plant growth regulators and environmental conditions. Usually, the process comprises three phases: embryogenic callus induction, embryogenic callus proliferation and somatic embryo germination. Somatic embryo germination can be achieved in light or dark. In the first case, complete plantlets are formed, with green leaves and pseudobulb in the base. In darkness, microbulbs are formed from single somatic embryos or clusters. A last phase of microbulb enlargement allows plantlets or microbulbs to increase their biomass. These enlarged microbulbs do not need special acclimatization conditions when transferred to soil and quickly produce sturdy plants. This chapter describes a protocol for somatic embryogenesis of Lilium using tTCL from microbulbs. PMID:26619874

  9. Cell therapy using induced pluripotent stem cells or somatic stem cells: this is the question.

    PubMed

    Somoza, Rodrigo A; Rubio, Francisco J

    2012-05-01

    A lot of effort has been developed to bypass the use of embryonic stem cells (ES) in human therapies, because of several concerns and ethical issues. Some unsolved problems of using stem cells for human therapies, excluding the human embryonic origin, are: how to regulate cell plasticity and proliferation, immunological compatibility, potential adverse side-effects when stem cells are systemically administrated, and the in vivo signals to rule out a specific cell fate after transplantation. Currently, it is known that almost all tissues of an adult organism have somatic stem cells (SSC). Whereas ES are primary involved in the genesis of new tissues and organs, SSC are involved in regeneration processes, immuno-regulatory and homeostasis mechanisms. Although the differentiating potential of ES is higher than SSC, several studies suggest that some types of SSC, such as mesenchymal stem cells (MSC), can be induced epigenetically to differentiate into tissue-specific cells of different lineages. This unexpected pluripotency and the variety of sources that they come from, can make MSC-like cells suitable for the treatment of diverse pathologies and injuries. New hopes for cell therapy came from somatic/mature cells and the discovery that could be reprogrammed to a pluripotent stage similar to ES, thus generating induced pluripotent stem cells (iPS). For this, it is necessary to overexpress four main reprogramming factors, Sox2, Oct4, Klf4 and c-Myc. The aim of this review is to analyze the potential and requirements of cellular based tools in human therapy strategies, focusing on the advantage of using MSC over iPS.

  10. Somatic cells count in cow's bulk tank milk.

    PubMed

    Olechnowicz, Jan; Jaśkowski, Jedrzej M

    2012-06-01

    The objective of this study was therefore to present factors affecting somatic cell counts in bovine bulk milk as a result of intramammary infections as well as non-infectious factors. The paper presents also the impact of on-farm management practices on the level of bulk milk somatic cell counts and presents quality indicators in bulk tank milk. At the farm level bulk milk bacterial infection takes place through three main sources: bacterial contamination from the external surface of the udder and teats, from the surface of the milking equipment, and from mastitis microorganisms within the udder. The threshold of 200,000 cells/ml identifies bacteriological negative quarters of the udder. The counts of mammary pathogens in bulk tank milk are relatively low, on average not exceeding 1,000 cfu/ml. Environmental pathogens predominate in bulk tank milk samples with somatic cells count <300 × 10(3) ml. PMID:22230979

  11. [Product safety analysis of somatic cell cloned bovine].

    PubMed

    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.

  12. From Somatic Cells to Oocytes: A Novel Yolk Protein Produced by Ovarian Somatic Cells in a Stony Coral, Euphyllia ancora.

    PubMed

    Shikina, Shinya; Chiu, Yi-Ling; Lee, Yan-Horn; Chang, Ching-Fong

    2015-09-01

    To gain a better understanding of how corals form their eggs at both the molecular and cellular levels, we performed a differential screen (suppression subtractive hybridization) to identify genes related to oocyte development in a stony coral, Euphyllia ancora. Through the course of screening, a novel gene that contains three alternate repeats of fibronectin domain 2 and epidermal growth factor (EGF)-like domains, as well as an additional calcium-binding EGF-like domain (EGF-CA), was identified and tentatively named euphy after the scientific name of the coral, E. ancora. Quantitative RT-PCR revealed that expression levels of euphy increased in female colonies as the coral approached reproductive season. Tissue distribution analysis followed by mRNA in situ hybridization revealed that euphy is highly expressed in the ovarian (mesenterial) somatic cells in the body of E. ancora. Staining of tissue sections with an antibody against euphy protein (Euphy) revealed Euphy immunoreactivity in both ovarian somatic cells and oocytes. Subsequent Western blotting demonstrated the presence of abundant Euphy in unfertilized mature eggs. These results indicate that Euphy produced in the ovarian somatic cells is transported to and accumulates within oocytes as a yolk protein during oogenesis. We previously showed that two major yolk proteins, vitellogenin and egg protein, are similarly produced by ovarian somatic cells. Hence, the present study uncovered the third ovarian somatic-derived yolk protein in corals. Our data provide new information that contributes to a more comprehensive understanding of coral egg formation.

  13. Somatic cell reprogramming-free generation of genetically modified pigs.

    PubMed

    Tanihara, Fuminori; Takemoto, Tatsuya; Kitagawa, Eri; Rao, Shengbin; Do, Lanh Thi Kim; Onishi, Akira; Yamashita, Yukiko; Kosugi, Chisato; Suzuki, Hitomi; Sembon, Shoichiro; Suzuki, Shunichi; Nakai, Michiko; Hashimoto, Masakazu; Yasue, Akihiro; Matsuhisa, Munehide; Noji, Sumihare; Fujimura, Tatsuya; Fuchimoto, Dai-Ichiro; Otoi, Takeshige

    2016-09-01

    Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.

  14. Somatic cell reprogramming-free generation of genetically modified pigs.

    PubMed

    Tanihara, Fuminori; Takemoto, Tatsuya; Kitagawa, Eri; Rao, Shengbin; Do, Lanh Thi Kim; Onishi, Akira; Yamashita, Yukiko; Kosugi, Chisato; Suzuki, Hitomi; Sembon, Shoichiro; Suzuki, Shunichi; Nakai, Michiko; Hashimoto, Masakazu; Yasue, Akihiro; Matsuhisa, Munehide; Noji, Sumihare; Fujimura, Tatsuya; Fuchimoto, Dai-Ichiro; Otoi, Takeshige

    2016-09-01

    Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs. PMID:27652340

  15. Somatic cell reprogramming-free generation of genetically modified pigs

    PubMed Central

    Tanihara, Fuminori; Takemoto, Tatsuya; Kitagawa, Eri; Rao, Shengbin; Do, Lanh Thi Kim; Onishi, Akira; Yamashita, Yukiko; Kosugi, Chisato; Suzuki, Hitomi; Sembon, Shoichiro; Suzuki, Shunichi; Nakai, Michiko; Hashimoto, Masakazu; Yasue, Akihiro; Matsuhisa, Munehide; Noji, Sumihare; Fujimura, Tatsuya; Fuchimoto, Dai-ichiro; Otoi, Takeshige

    2016-01-01

    Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.

  16. Somatic cell reprogramming-free generation of genetically modified pigs

    PubMed Central

    Tanihara, Fuminori; Takemoto, Tatsuya; Kitagawa, Eri; Rao, Shengbin; Do, Lanh Thi Kim; Onishi, Akira; Yamashita, Yukiko; Kosugi, Chisato; Suzuki, Hitomi; Sembon, Shoichiro; Suzuki, Shunichi; Nakai, Michiko; Hashimoto, Masakazu; Yasue, Akihiro; Matsuhisa, Munehide; Noji, Sumihare; Fujimura, Tatsuya; Fuchimoto, Dai-ichiro; Otoi, Takeshige

    2016-01-01

    Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs. PMID:27652340

  17. Classification of neurons by dendritic branching pattern. A categorisation based on Golgi impregnation of spinal and cranial somatic and visceral afferent and efferent cells in the adult human.

    PubMed

    Abdel-Maguid, T E; Bowsher, D

    1984-06-01

    Neurons from adult human brainstem and spinal cord, fixed by immersion in formalin, were impregnated by a Golgi method and examined in sections 100 micron thick. Objective numerical criteria were used to classify completely impregnated neurons. Only the parameters mentioned below were found to be valid. Neurons in 100 micron sections were classified on the basis of (i) the primary dendrite number, indicated by a Roman numeral and called group; (ii) the dendritic branching pattern, comprising the highest branching order seen, indicated by an Arabic numeral and called category; the lowest dendritic branching order observed in complete neurons, indicated by an upper case letter and called class; and the number of branching orders seen between the two preceding, indicated by a lower case letter and called subclass. On the basis of the above characteristics, all neurons seen in the grey matter of the spinal cord and cranial nerve nuclei could be classified into thirteen 'families'. The results of other investigations (Abdel-Maguid & Bowsher, 1979, 1984) showed that this classification has functional value. PMID:6204961

  18. Clock-like mutational processes in human somatic cells

    SciTech Connect

    Alexandrov, Ludmil B.; Jones, Philip H.; Wedge, David C.; Sale, Julian E.; Campbell, Peter J.; Nik-Zainal, Serena; Stratton, Michael R.

    2015-11-09

    During the course of a lifetime, somatic cells acquire mutations. Different mutational processes may contribute to the mutations accumulated in a cell, with each imprinting a mutational signature on the cell's genome. Some processes generate mutations throughout life at a constant rate in all individuals, and the number of mutations in a cell attributable to these processes will be proportional to the chronological age of the person. Using mutations from 10,250 cancer genomes across 36 cancer types, we investigated clock-like mutational processes that have been operating in normal human cells. Two mutational signatures show clock-like properties. Both exhibit different mutation rates in different tissues. However, their mutation rates are not correlated, indicating that the underlying processes are subject to different biological influences. For one signature, the rate of cell division may influence its mutation rate. This paper provides the first survey of clock-like mutational processes operating in human somatic cells.

  19. The histone chaperone CAF-1 safeguards somatic cell identity

    PubMed Central

    Cheloufi, Sihem; Elling, Ulrich; Hopfgartner, Barbara; Jung, Youngsook L; Murn, Jernej; Ninova, Maria; Hubmann, Maria; Badeaux, Aimee I; Ang, Cheen Euong; Tenen, Danielle; Wesche, Daniel J; Abazova, Nadezhda; Hogue, Max; Tasdemir, Nilgun; Brumbaugh, Justin; Rathert, Philipp; Jude, Julian; Ferrari, Francesco; Blanco, Andres; Fellner, Michaela; Wenzel, Daniel; Zinner, Marietta; Vidal, Simon E; Bell, Oliver; Stadtfeld, Matthias; Chang, Howard Y.; Almouzni, Genevieve; Lowe, Scott W; Rinn, John; Wernig, Marius; Aravin, Alexei; Shi, Yang; Park, Peter; Penninger, Josef M; Zuber, Johannes; Hochedlinger, Konrad

    2016-01-01

    Cellular differentiation involves profound remodeling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNAi screens targeting chromatin factors during transcription factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSCs). Remarkably, subunits of the chromatin assembly factor-1 (CAF-1) complex emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPSC formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 as a novel regulator of somatic cell identity during transcription factor-induced cell fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting. PMID:26659182

  20. Reshaping the Transcriptional Frontier: Epigenetics and Somatic Cell Nuclear Transfer

    PubMed Central

    LONG, CHARLES R.; WESTHUSIN, MARK E.; GOLDING, MICHAEL C.

    2014-01-01

    SUMMARY Somatic-cell nuclear transfer (SCNT) experiments have paved the way to the field of cellular reprogramming. The demonstrated ability to clone over 20 different species to date has proven that the technology is robust but very inefficient, and is prone to developmental anomalies. Yet, the offspring from cloned animals exhibit none of the abnormalities of their parents, suggesting the low efficiency and high developmental mortality are epigenetic in origin. The epigenetic barriers to reprogramming somatic cells into a totipotent embryo capable of developing into a viable offspring are significant and varied. Despite their intimate relationship, chromatin structure and transcription are often not uniformly reprogramed after nuclear transfer, and many cloned embryos develop gene expression profiles that are hybrids between the donor cell and an embryonic blastomere. Recent advances in cellular reprogramming suggest that alteration of donor-cell chromatin structure towards that found in an normal embryo is actually the rate-limiting step in successful development of SCNT embryos. Here we review the literature relevant to the transformation of a somatic-cell nucleus into an embryo capable of full-term development. Interestingly, while resetting somatic transcription and associated epigenetic marks are absolutely required for development of SCNT embryos, life does not demand perfection. PMID:24167064

  1. Somatic cell nuclear transfer: Past, present and future perspectives.

    PubMed

    Campbell, K H S; Fisher, P; Chen, W C; Choi, I; Kelly, R D W; Lee, J-H; Xhu, J

    2007-09-01

    It is now over a decade since the birth, in 1996, of Dolly the first animal to be produced by nuclear transfer using an adult derived somatic cell as nuclear donor. Since this time similar techniques have been successfully applied to a range of species producing live offspring and allowing the development of transgenic technologies for agricultural, biotechnological and medical uses. However, though applicable to a range of species, overall, the efficiencies of development of healthy offspring remain low. The low frequency of successful development has been attributed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Many studies have demonstrated that such reprogramming occurs by epigenetic mechanisms not involving alterations in DNA sequence, however, at present the molecular mechanisms underlying reprogramming are poorly defined. Since the birth of Dolly many studies have attempted to improve the frequency of development, this review will discuss the process of animal production by nuclear transfer and in particular changes in the methodology which have increased development and survival, simplified or increased robustness of the technique. Although much of the discussion is applicable across species, for simplicity we will concentrate primarily on published data for cattle, sheep, pigs and mice. PMID:17610946

  2. Oocyte-somatic cells interactions, lessons from evolution

    PubMed Central

    2012-01-01

    Background Despite the known importance of somatic cells for oocyte developmental competence acquisition, the overall mechanisms underlying the acquisition of full developmental competence are far from being understood, especially in non-mammalian species. The present work aimed at identifying key molecular signals from somatic origin that would be shared by vertebrates. Results Using a parallel transcriptomic analysis in 4 vertebrate species - a teleost fish, an amphibian, and two mammals - at similar key steps of developmental competence acquisition, we identified a large number of species-specific differentially expressed genes and a surprisingly high number of orthologous genes exhibiting similar expression profiles in the 3 tetrapods and in the 4 vertebrates. Among the evolutionary conserved players participating in developmental competence acquisition are genes involved in key processes such as cellular energy metabolism, cell-to-cell communications, and meiosis control. In addition, we report many novel molecular actors from somatic origin that have never been studied in the vertebrate ovary. Interestingly, a significant number of these new players actively participate in Drosophila oogenesis. Conclusions Our study provides a comprehensive overview of evolutionary-conserved mechanisms from somatic origin participating in oocyte developmental competence acquisition in 4 vertebrates. Together our results indicate that despite major differences in ovarian follicular structure, some of the key players from somatic origin involved in oocyte developmental competence acquisition would be shared, not only by vertebrates, but also by metazoans. The conservation of these mechanisms during vertebrate evolution further emphasizes the important contribution of the somatic compartment to oocyte quality and paves the way for future investigations aiming at better understanding what makes a good egg. PMID:23083410

  3. Clock-like mutational processes in human somatic cells

    PubMed Central

    Alexandrov, Ludmil B.; Jones, Philip H.; Wedge, David C.; Sale, Julian E.; Campbell, Peter J.; Nik-Zainal, Serena; Stratton, Michael R.

    2016-01-01

    During the course of a lifetime somatic cells acquire mutations. Different mutational processes may contribute to the mutations accumulated in a cell, with each imprinting a mutational signature on the cell’s genome. Some processes generate mutations throughout life at a constant rate in all individuals and the number of mutations in a cell attributable to these processes will be proportional to the chronological age of the person. Using mutations from 10,250 cancer genomes across 36 cancer types, we investigated clock-like mutational processes that have been operating in normal human cells. Two mutational signatures show clock-like properties. Both exhibit different mutation rates in different tissues. However, their mutation rates are not correlated indicating that the underlying processes are subject to different biological influences. For one signature, the rate of cell division may influence its mutation rate. This study provides the first survey of clock-like mutational processes operative in human somatic cells. PMID:26551669

  4. Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

    PubMed

    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.

  5. Somatic cell nuclear transfer cloning: practical applications and current legislation.

    PubMed

    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.

  6. Bovine ooplasm partially remodels primate somatic nuclei following somatic cell nuclear transfer.

    PubMed

    Wang, Kai; Beyhan, Zeki; Rodriguez, Ramon M; Ross, Pablo J; Iager, Amy E; Kaiser, German G; Chen, Ying; Cibelli, Jose B

    2009-03-01

    Interspecies somatic cell nuclear transfer (iSCNT) has the potential to become a useful tool to address basic questions about the nucleus-cytoplasm interactions between species. It has also been proposed as an alternative for the preservation of endangered species and to derive autologous embryonic stem cells. Using chimpanzee/ bovine iSCNT as our experimental model we studied the early epigenetic events that take place soon after cell fusion until embryonic genome activation (EGA). Our analysis suggested partial EGA in iSCNT embryos at the eight-cell stage, as indicated by Br-UTP incorporation and expression of chimpanzee embryonic genes. Oct4, Stella, Crabp1, CCNE2, CXCL6, PTGER4, H2AFZ, c-MYC, KLF4, and GAPDH transcripts were expressed, while Nanog, Glut1, DSC2, USF2, Adrbk1, and Lin28 failed to be activated. Although development of iSCNT embryos did not progress beyond the 8- to 16-cell stage, chromatin remodeling events, monitored by H3K27 methylation, H4K5 acetylation, and global DNA methylation, were similar in both intra- and interspecies SCNT embryos. However, bisulfite sequencing indicated incomplete demethylation of Oct4 and Nanog promoters in eight-cell iSCNT embryos. ATP production levels were significantly higher in bovine SCNT embryos than in iSCNT embryos, TUNEL assays did not reveal any difference in the apoptotic status of the nuclei from both types of embryos. Collectively, our results suggest that bovine ooplasm can partially remodel chimpanzee somatic nuclei, and provides insight into some of the current barriers iSCNT must overcome if further embryonic development is to be expected. PMID:19196039

  7. A New, Dynamic Era for Somatic Cell Nuclear Transfer?

    PubMed

    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). PMID:27118511

  8. Somatic cell nuclear transfer: pros and cons.

    PubMed

    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. PMID:20232594

  9. Somatic cell nuclear transfer: pros and cons.

    PubMed

    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.

  10. Reprogramming of two somatic nuclei in the same ooplasm leads to pluripotent embryonic stem cells.

    PubMed

    Pfeiffer, Martin J; Esteves, Telma C; Balbach, Sebastian T; Araúzo-Bravo, Marcos J; Stehling, Martin; Jauch, Anna; Houghton, Franchesca D; Schwarzer, Caroline; Boiani, Michele

    2013-11-01

    The conversion of the nuclear program of a somatic cell from a differentiated to an undifferentiated state can be accomplished by transplanting its nucleus to an enucleated oocyte (somatic cell nuclear transfer [SCNT]) in a process termed "reprogramming." This process achieves pluripotency and occasionally also totipotency. Exploiting the obstacle of tetraploidy to full development in mammals, we show that mouse ooplasts transplanted with two somatic nuclei simultaneously (double SCNT) support preimplantation development and derivation of novel tetraploid SCNT embryonic stem cells (tNT-ESCs). Although the double SCNT embryos do not recapitulate the expression pattern of the pluripotency-associated gene Oct4 in fertilized embryos, derivative tNT-ESCs have characteristics of genuine pluripotency: in vitro they differentiate into neurons, cardiomyocytes, and endodermal cells; in vivo, tNT-ESCs form teratomas, albeit at reduced rates compared to diploid counterparts. Global transcriptome analysis revealed only few specific alterations, for example, in the quantitative expression of gastrulation-associated genes. In conclusion, we have shown that the oocyte's reprogramming capacity is in excess of a single nucleus and that double nucleus-transplanted embryos and derivative ESCs are very similar to their diploid counterparts. These results have key implications for reprogramming studies based on pluripotency: while reprogramming in the tetraploid state was known from fusion-mediated reprogramming and from fetal and adult hepatocyte-derived induced pluripotent stem cells, we have now accomplished it with enucleated oocytes.

  11. Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells

    PubMed Central

    Ruiz, Sergio; Lopez-Contreras, Andres J.; Gabut, Mathieu; Marion, Rosa M.; Gutierrez-Martinez, Paula; Bua, Sabela; Ramirez, Oscar; Olalde, Iñigo; Rodrigo-Perez, Sara; Li, Han; Marques-Bonet, Tomas; Serrano, Manuel; Blasco, Maria A.; Batada, Nizar N.; Fernandez-Capetillo, Oscar

    2015-01-01

    The generation of induced pluripotent stem cells (iPSC) from adult somatic cells is one of the most remarkable discoveries in recent decades. However, several works have reported evidence of genomic instability in iPSC, raising concerns on their biomedical use. The reasons behind the genomic instability observed in iPSC remain mostly unknown. Here we show that, similar to the phenomenon of oncogene-induced replication stress, the expression of reprogramming factors induces replication stress. Increasing the levels of the checkpoint kinase 1 (CHK1) reduces reprogramming-induced replication stress and increases the efficiency of iPSC generation. Similarly, nucleoside supplementation during reprogramming reduces the load of DNA damage and genomic rearrangements on iPSC. Our data reveal that lowering replication stress during reprogramming, genetically or chemically, provides a simple strategy to reduce genomic instability on mouse and human iPSC. PMID:26292731

  12. Generation of embryonic stem cells from mouse adipose-tissue derived cells via somatic cell nuclear transfer.

    PubMed

    Qin, Yiren; Qin, Jilong; Zhou, Chikai; Li, Jinsong; Gao, Wei-Qiang

    2015-01-01

    Somatic cells can be reprogrammed into embryonic stem cells (ESCs) by nuclear transfer (NT-ESCs), or into induced pluripotent stem cells (iPSCs) by the "Yamanaka method." However, recent studies have indicated that mouse and human iPSCs are prone to epigenetic and transcriptional aberrations, and that NT-ESCs correspond more closely to ESCs derived from in vitro fertilized embryos than iPSCs. In addition, the procedure of NT-ESCs does not involve gene modification. Demonstration of generation of NT-ESCs using an easily-accessible source of adult cell types would be very important. Adipose tissue is a source of readily accessible donor cells and can be isolated from both males and females at different ages. Here we report that NT-ESCs can be generated from adipose tissue-derived cells (ADCs). At morphological, mRNA and protein levels, these NT-ESCs show classic ESC colonies, exhibit alkaline phosphatase (AP) activity, and display normal diploid karyotypes. Importantly, these cells express pluripotent markers including Oct4, Sox2, Nanog and SSEA-1. Furthermore, they can differentiate in vivo into various types of cells from 3 germinal layers by teratoma formation assays. This study demonstrates for the first time that ESCs can be generated from the adipose tissue by somatic cell nuclear transfer (SCNT) and suggests that ADCs can be a new donor-cell type for potential therapeutic cloning.

  13. Generation of embryonic stem cells from mouse adipose-tissue derived cells via somatic cell nuclear transfer

    PubMed Central

    Qin, Yiren; Qin, Jilong; Zhou, Chikai; Li, Jinsong; Gao, Wei-Qiang

    2015-01-01

    Somatic cells can be reprogrammed into embryonic stem cells (ESCs) by nuclear transfer (NT-ESCs), or into induced pluripotent stem cells (iPSCs) by the “Yamanaka method.” However, recent studies have indicated that mouse and human iPSCs are prone to epigenetic and transcriptional aberrations, and that NT-ESCs correspond more closely to ESCs derived from in vitro fertilized embryos than iPSCs. In addition, the procedure of NT-ESCs does not involve gene modification. Demonstration of generation of NT-ESCs using an easily-accessible source of adult cell types would be very important. Adipose tissue is a source of readily accessible donor cells and can be isolated from both males and females at different ages. Here we report that NT-ESCs can be generated from adipose tissue-derived cells (ADCs). At morphological, mRNA and protein levels, these NT-ESCs show classic ESC colonies, exhibit alkaline phosphatase (AP) activity, and display normal diploid karyotypes. Importantly, these cells express pluripotent markers including Oct4, Sox2, Nanog and SSEA-1. Furthermore, they can differentiate in vivo into various types of cells from 3 germinal layers by teratoma formation assays. This study demonstrates for the first time that ESCs can be generated from the adipose tissue by somatic cell nuclear transfer (SCNT) and suggests that ADCs can be a new donor-cell type for potential therapeutic cloning. PMID:25692793

  14. Clock-like mutational processes in human somatic cells

    DOE PAGES

    Alexandrov, Ludmil B.; Jones, Philip H.; Wedge, David C.; Sale, Julian E.; Campbell, Peter J.; Nik-Zainal, Serena; Stratton, Michael R.

    2015-11-09

    During the course of a lifetime, somatic cells acquire mutations. Different mutational processes may contribute to the mutations accumulated in a cell, with each imprinting a mutational signature on the cell's genome. Some processes generate mutations throughout life at a constant rate in all individuals, and the number of mutations in a cell attributable to these processes will be proportional to the chronological age of the person. Using mutations from 10,250 cancer genomes across 36 cancer types, we investigated clock-like mutational processes that have been operating in normal human cells. Two mutational signatures show clock-like properties. Both exhibit different mutationmore » rates in different tissues. However, their mutation rates are not correlated, indicating that the underlying processes are subject to different biological influences. For one signature, the rate of cell division may influence its mutation rate. This paper provides the first survey of clock-like mutational processes operating in human somatic cells.« less

  15. Somatic cell nuclear transfer in horses.

    PubMed

    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. PMID:18638143

  16. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming

    PubMed Central

    Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

    2016-01-01

    Summary Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated domain (TAD) repositioning and alterations of tissue-restricted genomic neighborhoods and chromatin loops, effectively erasing the somatic-cell-specific genome structures while establishing an embryonic stem-cell-like 3D genome. Yet, early passage iPSCs carry topological hallmarks that enable recognition of their cell of origin. These hallmarks are not remnants of somatic chromosome topologies. Instead, the distinguishing topological features are acquired during reprogramming, as we also find for cell-of-origin-dependent gene expression patterns. PMID:26971819

  17. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming.

    PubMed

    Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

    2016-05-01

    Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated domain (TAD) repositioning and alterations of tissue-restricted genomic neighborhoods and chromatin loops, effectively erasing the somatic-cell-specific genome structures while establishing an embryonic stem-cell-like 3D genome. Yet, early passage iPSCs carry topological hallmarks that enable recognition of their cell of origin. These hallmarks are not remnants of somatic chromosome topologies. Instead, the distinguishing topological features are acquired during reprogramming, as we also find for cell-of-origin-dependent gene expression patterns.

  18. Mutation of mitochondria genome: trigger of somatic cell transforming to cancer cell

    PubMed Central

    2010-01-01

    Nearly 80 years ago, scientist Otto Warburg originated a hypothesis that the cause of cancer is primarily a defect in energy metabolism. Following studies showed that mitochondria impact carcinogenesis to remodel somatic cells to cancer cells through modifying the genome, through maintenance the tumorigenic phenotype, and through apoptosis. And the Endosymbiotic Theory explains the origin of mitochondria and eukaryotes, on the other hands, the mitochondria also can fall back. Compared to chromosome genomes, the mitochondria genomes were not restricted by introns so they were mutated(fall back) easy. The result is that mitochondria lose function and internal environment of somatic cell become acid and evoked chromosome genomes to mutate, in the end somatic cells become cancer cells. It is the trigger of somatic cell transforming to cancer cell that mitochondria genome happen mutation and lose function. PMID:20181100

  19. Cloned ferrets produced by somatic cell nuclear transfer

    PubMed Central

    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

  20. Application of Somatic Embryogenesis in Woody Plants.

    PubMed Central

    Guan, Yuan; Li, Shui-Gen; Fan, Xiao-Fen; Su, Zhen-Hong

    2016-01-01

    Somatic embryogenesis is a developmental process where a plant somatic cell can dedifferentiate to a totipotent embryonic stem cell that has the ability to give rise to an embryo under appropriate conditions. This new embryo can further develop into a whole plant. In woody plants, somatic embryogenesis plays a critical role in clonal propagation and is a powerful tool for synthetic seed production, germplasm conservation, and cryopreservation. A key step in somatic embryogenesis is the transition of cell fate from a somatic cell to embryo cell. Although somatic embryogenesis has already been widely used in a number of woody species, propagating adult woody plants remains difficult. In this review, we focus on molecular mechanisms of somatic embryogenesis and its practical applications in economic woody plants. Furthermore, we propose a strategy to improve the process of somatic embryogenesis using molecular means. PMID:27446166

  1. Cloning Endangered Felids by Interspecies Somatic Cell Nuclear Transfer.

    PubMed

    Gómez, Martha C; Pope, C Earle

    2015-01-01

    In 2003, the first wild felid was produced by interspecies somatic cell nuclear transfer. Since then other wild felid clone offspring have been produced by using the same technique with minor modifications. This chapter describes detailed protocols used in our laboratory for (1) the isolation, culture, and preparation of fibroblast cells as donor nucleus, and (2) embryo reconstruction with domestic cat enucleated oocytes to produce cloned embryos that develop to the blastocyst stage in vitro and, after transfer into synchronized recipients, establish successful pregnancies.

  2. Interspecies Somatic Cell Nuclear Transfer: Advancements and Problems

    PubMed Central

    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

  3. Somatic cell genotoxicity at the glycophorin A locus in humans

    SciTech Connect

    Jensen, R.H.; Grant, S.G.; Langlois, R.G.; Bigbee, W.L.

    1990-12-28

    We have developed an assay for detecting variant erythrocytes that occur as a result of in vivo allele loss at the glycophorin A (GPA) locus on chromosome 4 in humans. This gene codes for an erythroid- specific cell surface glycoprotein, and with our assay we are able to detect rare variant erythrocytes that have lost expression of one of the two GPA alleles. Two distinctly different variant cell types are detected with this assay. One variant cell type (called N{O}) is hemizygous. Our assay also detects homozygous variant erythrocytes that have lost expression of the GPA(M) allele and express the GPA(N) allele at twice the heterozygous level. The results of this assay are an enumeration of the frequency of N{O} and NN variant cell types for each individual analyzed. These variant cell frequencies provide a measure of the amount of somatic cell genotoxicity that has occurred at the GPA locus. Such genotoxicity could be the result of (1) reactions of toxic chemicals to which the individual has been exposed, or (2) high energy radiation effects on erythroid precursor cells, or (3) errors in DNA replication or repair in these cells of the bone marrow. Thus, the GPA-based variant cell frequency can serve as a biodosimeter that indicates the amount of genotoxic exposure each individual has received. Because two very different kinds of variant cells are enumerated, different kinds of genotoxicity should be distinguishable. Results of the GPA somatic genotoxicity assay may also provide valuable information for cancer-risk estimation on each individual. 16 refs.

  4. Preparation and stability of milk somatic cell reference materials.

    PubMed

    Di Marzo, Larissa; Wojciechowski, Karen L; Barbano, David M

    2016-09-01

    Our objectives were to develop a method to produce milk somatic cell count (SCC) reference materials for calibration of electronic somatic cell count (ESCC) using gravity separation and to determine the effect of refrigerated storage (4°C) and freeze-thaw stability of the skim and whole milk SCC reference materials. Whole raw milk was high-temperature short-time pasteurized and split into 2 portions. One portion was gravity separated at 4°C for 22 h and the second portion was centrifugally separated to produce skim milk that was also gravity separated with somatic cells rising to the surface. After 22 h, stock solutions (low SCC skim milk, high SCC skim milk, high SCC whole milk) were prepared and preserved (bronopol). Two experiments were conducted, one to compare the shelf-life of skim and whole milk SCC standards at 4°C and one to determine the effect of freezing and thawing on SCC standards. Both experiments were replicated 3 times. Gravity separation was an effective approach to isolate and concentrate somatic cells from bovine milk and redistribute them in a skim or whole milk matrix to create a set of reference materials with a wider and more uniformly distributed range of SCC than current calibration sets. The liquid SCC reference materials stored using the common industry practice at 4°C were stable (i.e., fit for purpose, no large decrease in SCC) for a 2-wk period, whereas frozen and thawed reference materials may have a much longer useful life. A gradual decrease occurred in residual difference in ESCC (SCC × 1,000/mL) versus original assigned reference SCC over duration of refrigerated storage for both skim and whole milk SCC samples, indicating that milk ESCC of the preserved milks was gradually decreasing during 28 d of storage at 4°C by about 15,000 SCC/mL. No difference in the ESCC for skim milk was detected between refrigerated and frozen storage, whereas for whole milk the ESCC for frozen was lower than refrigerated samples. Future work is

  5. Preparation and stability of milk somatic cell reference materials.

    PubMed

    Di Marzo, Larissa; Wojciechowski, Karen L; Barbano, David M

    2016-09-01

    Our objectives were to develop a method to produce milk somatic cell count (SCC) reference materials for calibration of electronic somatic cell count (ESCC) using gravity separation and to determine the effect of refrigerated storage (4°C) and freeze-thaw stability of the skim and whole milk SCC reference materials. Whole raw milk was high-temperature short-time pasteurized and split into 2 portions. One portion was gravity separated at 4°C for 22 h and the second portion was centrifugally separated to produce skim milk that was also gravity separated with somatic cells rising to the surface. After 22 h, stock solutions (low SCC skim milk, high SCC skim milk, high SCC whole milk) were prepared and preserved (bronopol). Two experiments were conducted, one to compare the shelf-life of skim and whole milk SCC standards at 4°C and one to determine the effect of freezing and thawing on SCC standards. Both experiments were replicated 3 times. Gravity separation was an effective approach to isolate and concentrate somatic cells from bovine milk and redistribute them in a skim or whole milk matrix to create a set of reference materials with a wider and more uniformly distributed range of SCC than current calibration sets. The liquid SCC reference materials stored using the common industry practice at 4°C were stable (i.e., fit for purpose, no large decrease in SCC) for a 2-wk period, whereas frozen and thawed reference materials may have a much longer useful life. A gradual decrease occurred in residual difference in ESCC (SCC × 1,000/mL) versus original assigned reference SCC over duration of refrigerated storage for both skim and whole milk SCC samples, indicating that milk ESCC of the preserved milks was gradually decreasing during 28 d of storage at 4°C by about 15,000 SCC/mL. No difference in the ESCC for skim milk was detected between refrigerated and frozen storage, whereas for whole milk the ESCC for frozen was lower than refrigerated samples. Future work is

  6. Germ cell formation from embryonic stem cells and the use of somatic cell nuclei in oocytes.

    PubMed

    Pelosi, Emanuele; Forabosco, Antonino; Schlessinger, David

    2011-03-01

    Embryonic stem cells (ESCs) have remarkable properties of pluripotency and self-renewal, along with the retention of chromosomal integrity. Germ cells function as a kind of "transgenerational stem cells," transmitting genetic information from one generation to the next. The formation of putative primordial germ cells (PGCs) and germ cells from mouse and human ESCs (hESCs) has, in fact, been shown, and the apparent derivation of functional mouse male gametes has also been described. Additionally, investigators have successfully reprogrammed somatic nuclei into a pluripotent state by inserting them into ESCs or oocytes. This would enable the generation of ESCs genetically identical to the somatic cell donor and their use in cell therapy. However, these methodologies are still inefficient and their mechanisms poorly understood. Until full comprehension of these processes is obtained, clinical applications remain remote. Nevertheless, they represent promising tools in the future, enhancing methods of therapeutic cloning and infertility treatment.

  7. Mutational History of a Human Cell Lineage from Somatic to Induced Pluripotent Stem Cells

    PubMed Central

    Rouhani, Foad J.; Nik-Zainal, Serena; Wuster, Arthur; Li, Yilong; Conte, Nathalie; Koike-Yusa, Hiroko; Kumasaka, Natsuhiko; Vallier, Ludovic; Yusa, Kosuke; Bradley, Allan

    2016-01-01

    The accuracy of replicating the genetic code is fundamental. DNA repair mechanisms protect the fidelity of the genome ensuring a low error rate between generations. This sustains the similarity of individuals whilst providing a repertoire of variants for evolution. The mutation rate in the human genome has recently been measured to be 50–70 de novo single nucleotide variants (SNVs) between generations. During development mutations accumulate in somatic cells so that an organism is a mosaic. However, variation within a tissue and between tissues has not been analysed. By reprogramming somatic cells into induced pluripotent stem cells (iPSCs), their genomes and the associated mutational history are captured. By sequencing the genomes of polyclonal and monoclonal somatic cells and derived iPSCs we have determined the mutation rates and show how the patterns change from a somatic lineage in vivo through to iPSCs. Somatic cells have a mutation rate of 14 SNVs per cell per generation while iPSCs exhibited a ten-fold lower rate. Analyses of mutational signatures suggested that deamination of methylated cytosine may be the major mutagenic source in vivo, whilst oxidative DNA damage becomes dominant in vitro. Our results provide insights for better understanding of mutational processes and lineage relationships between human somatic cells. Furthermore it provides a foundation for interpretation of elevated mutation rates and patterns in cancer. PMID:27054363

  8. DNA replication licensing in somatic and germ cells.

    PubMed

    Eward, Kathryn Leigh; Obermann, Ellen C; Shreeram, S; Loddo, Marco; Fanshawe, Thomas; Williams, Craig; Jung, Hyo-Il; Prevost, A Toby; Blow, J Julian; Stoeber, Kai; Williams, Gareth H

    2004-11-15

    The DNA replication (or origin) licensing system ensures precise duplication of the genome in each cell cycle and is a powerful regulator of cell proliferation in metazoa. Studies in yeast, Drosophila melanogaster and Xenopus laevis have characterised the molecular machinery that constitutes the licensing system, but it remains to be determined how this important evolutionary conserved pathway is regulated in Homo sapiens. We have investigated regulation of the origin licensing factors Cdc6, Cdt1, Mcm2 and Geminin in human somatic and germ cells. Cdc6 and Cdt1 play an essential role in DNA replication initiation by loading the Mcm2-7 complex, which is required for unwinding the DNA helix, onto chromosomal origins. Geminin is a repressor of origin licensing that blocks Mcm2-7 loading onto origins. Our studies demonstrate that Cdc6, Cdt1 and Mcm2 play a central role in coordinating growth during the proliferation-differentiation switch in somatic self-renewing systems and that Cdc6 expression is rate-limiting for acquisition of replication competence in primary oocytes. In striking contrast, we show that proliferation control during male gametogenesis is not linked to Cdc6 or Mcm2, but appears to be coordinated by the negative regulator Geminin with Cdt1 becoming rate-limiting in late prophase. Our data demonstrate a striking sexual dimorphism in the mechanisms repressing origin licensing and preventing untimely DNA synthesis during meiosis I, implicating a pivotal role for Geminin in maintaining integrity of the male germline genome.

  9. Vitamin C modulates TET1 function during somatic cell reprogramming.

    PubMed

    Chen, Jiekai; Guo, Lin; Zhang, Lei; Wu, Haoyu; Yang, Jiaqi; Liu, He; Wang, Xiaoshan; Hu, Xiao; Gu, Tianpeng; Zhou, Zhiwei; Liu, Jing; Liu, Jiadong; Wu, Hongling; Mao, Shi-Qing; Mo, Kunlun; Li, Yingying; Lai, Keyu; Qi, Jing; Yao, Hongjie; Pan, Guangjin; Xu, Guo-Liang; Pei, Duanqing

    2013-12-01

    Vitamin C, a micronutrient known for its anti-scurvy activity in humans, promotes the generation of induced pluripotent stem cells (iPSCs) through the activity of histone demethylating dioxygenases. TET hydroxylases are also dioxygenases implicated in active DNA demethylation. Here we report that TET1 either positively or negatively regulates somatic cell reprogramming depending on the absence or presence of vitamin C. TET1 deficiency enhances reprogramming, and its overexpression impairs reprogramming in the context of vitamin C by modulating the obligatory mesenchymal-to-epithelial transition (MET). In the absence of vitamin C, TET1 promotes somatic cell reprogramming independent of MET. Consistently, TET1 regulates 5-hydroxymethylcytosine (5hmC) formation at loci critical for MET in a vitamin C-dependent fashion. Our findings suggest that vitamin C has a vital role in determining the biological outcome of TET1 function at the cellular level. Given its benefit to human health, vitamin C should be investigated further for its role in epigenetic regulation. PMID:24162740

  10. Cyclooxygenase and prostaglandins in somatic cell populations of the testis.

    PubMed

    Frungieri, Mónica B; Calandra, Ricardo S; Mayerhofer, Artur; Matzkin, María E

    2015-04-01

    Prostaglandins (PGs) are synthesized through the action of the rate-limiting enzyme cyclooxygenase (COX) and further specific enzymes. The development of Cox-deficient mice in the 1990s gave insights into the reproductive roles of PGs. Female Cox-knockout mice were subfertile or infertile. Interestingly, fertility was not affected in male mice deficient in Cox, suggesting that PGs may not be critical for the functioning of the testis. However, this conclusion has recently been challenged by observations of important roles for PGs in both physiological and pathological processes in the testis. The two key somatic cell types in the testis, Leydig and Sertoli cells, express the inducible isoenzyme COX2 and produce PGs. Testicular COX2 expression in these somatic cells is regulated by hormonal input (FSH, prolactin (PRL), and testosterone) as well as by IL1β. PGs modulate steroidogenesis in Leydig cells and glucose uptake in Sertoli cells. Hence, the COX2/PG system in Leydig and Sertoli cells acts as a local modulator of testicular activity, and consequently may regulate spermatogenic efficiency. In addition to its expression in Leydig and Sertoli cells, COX2 has been detected in the seminiferous tubule wall, and in testicular macrophages and mast cells of infertile patients. These observations highlight the possible relevance of PGs in testicular inflammation associated with idiopathic infertility. Collectively, these data indicate that the COX2/PG system plays crucial roles not only in testicular physiology (i.e., development, steroidogenesis, and spermatogenesis), but more importantly in the pathogenesis or maintenance of infertility status in the male gonad. Further studies of these actions could lead to new therapeutic approaches to idiopathic male infertility.

  11. Conversion of quiescent niche cells to somatic stem cells causes ectopic niche formation in the Drosophila testis

    PubMed Central

    Hétié, Phylis; de Cuevas, Margaret; Matunis, Erika

    2014-01-01

    Summary Adult stem cells reside in specialized regulatory microenvironments, or niches, where local signals ensure stem cell maintenance. The Drosophila testis contains a well-characterized niche wherein signals from post-mitotic hub cells promote maintenance of adjacent germline stem cells and somatic cyst stem cells (CySCs). Hub cells were considered to be terminally differentiated; here we show that they can give rise to CySCs. Genetic ablation of CySCs triggers hub cells to transiently exit quiescence, delaminate from the hub, and convert into functional CySCs. Ectopic Cyclin D-Cdk4 expression in hub cells is also sufficient to trigger their conversion into CySCs. In both cases, this conversion causes the formation of multiple ectopic niches over time. Therefore, our work provides a model for understanding how oncogenic mutations in quiescent niche cells could promote loss of quiescence, changes in cell fate, and aberrant niche expansion more generally. PMID:24746819

  12. Radiation-induced bystander signaling from somatic cells to germ cells in Caenorhabditis elegans.

    PubMed

    Guo, Xiaoying; Sun, Jie; Bian, Po; Chen, Lianyun; Zhan, Furu; Wang, Jun; Xu, An; Wang, Yugang; Hei, Tom K; Wu, Lijun

    2013-09-01

    Recently, radiation-induced bystander effects (RIBE) have been studied in mouse models in vivo, which clearly demonstrated bystander effects among somatic cells. However, there is currently no evidence for RIBE between somatic cells and germ cells in animal models in vivo. In the current study, the model animal Caenorhabditis elegans was used to investigate the bystander signaling from somatic cells to germ cells, as well as underlying mechanisms. C. elegans body size allows for precise microbeam irradiation and the abundant mutant strains for genetic dissection relative to currently adopted mouse models make it ideal for such analysis. Our results showed that irradiation of posterior pharynx bulbs and tails of C. elegans enhanced the level of germ cell apoptosis in bystander gonads. The irradiation of posterior pharynx bulbs also increased the level of DNA damage in bystander germ cells and genomic instability in the F1 progeny of irradiated worms, suggesting a potential carcinogenic risk in progeny even only somatic cells of parents are exposed to ionizing radiation (IR). It was also shown that DNA damage-induced germ cell death machinery and MAPK signaling pathways were both involved in the induction of germ cell apoptosis by microbeam induced bystander signaling, indicating a complex cooperation among multiple signaling pathways for bystander effects from somatic cells to germ cells.

  13. Somatically expressed germ-granule components, PGL-1 and PGL-3, repress programmed cell death in C. elegans.

    PubMed

    Al-Amin, Mohammad; Min, Hyemin; Shim, Yhong-Hee; Kawasaki, Ichiro

    2016-01-01

    We previously reported that germline apoptosis in C. elegans increased by loss of PGL-1 and PGL-3, members of a family of constitutive germ-granule components, from germ cells in adult hermaphrodite gonads. In this study, we found that somatic apoptosis was reduced in synthetic multivulva class B (synMuv B) mutants due to ectopic expression of PGL-1 and PGL-3 in the soma. In synMuv B-mutant somatic cells, CED-4 expression level was reduced due to ectopic expression of PGL-1. Furthermore, in contrast to wild type, somatic apoptosis in synMuv B mutants increased following DNA damage in a SIR-2.1-dependent manner. Intriguingly, somatic apoptosis was repressed not only in synMuv B mutants but also by ectopically expressing pgl-1 and/or pgl-3 transgenes in wild-type somatic cells. Our study demonstrates that germ-granule components, PGL-1 and PGL-3, can serve as negative regulators of apoptosis not only in the germline but also in the soma in C. elegans. PMID:27650246

  14. Somatically expressed germ-granule components, PGL-1 and PGL-3, repress programmed cell death in C. elegans

    PubMed Central

    Al-Amin, Mohammad; Min, Hyemin; Shim, Yhong-Hee; Kawasaki, Ichiro

    2016-01-01

    We previously reported that germline apoptosis in C. elegans increased by loss of PGL-1 and PGL-3, members of a family of constitutive germ-granule components, from germ cells in adult hermaphrodite gonads. In this study, we found that somatic apoptosis was reduced in synthetic multivulva class B (synMuv B) mutants due to ectopic expression of PGL-1 and PGL-3 in the soma. In synMuv B-mutant somatic cells, CED-4 expression level was reduced due to ectopic expression of PGL-1. Furthermore, in contrast to wild type, somatic apoptosis in synMuv B mutants increased following DNA damage in a SIR-2.1-dependent manner. Intriguingly, somatic apoptosis was repressed not only in synMuv B mutants but also by ectopically expressing pgl-1 and/or pgl-3 transgenes in wild-type somatic cells. Our study demonstrates that germ-granule components, PGL-1 and PGL-3, can serve as negative regulators of apoptosis not only in the germline but also in the soma in C. elegans. PMID:27650246

  15. Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency.

    PubMed

    Kallingappa, Prasanna K; Turner, Pavla M; Eichenlaub, Michael P; Green, Andria L; Oback, Fleur C; Chibnall, Alice M; Wells, David N; Oback, Björn

    2016-07-01

    Reprogramming by nuclear transfer (NT) cloning forces cells to lose their lineage-specific epigenetic marks and reacquire totipotency. This process often produces molecular anomalies that compromise clone development. We hypothesized that quiescence alters the epigenetic status of somatic NT donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) fibroblasts versus nonstarved mitotically selected (G1) controls. We show that G0 chromatin contains reduced levels of Polycomb group proteins EED, SUZ12, PHC1, and RING2, as well as histone variant H2A.Z. Using quantitative confocal immunofluorescence microscopy and fluorometric enzyme-linked immunosorbent assay, we further show that G0 induced DNA and histone hypomethylation, specifically at H3K4me3, H3K9me2/3 and H3K27me3, but not H3K9me1. Collectively, these changes resulted in a more relaxed G0 chromatin state. Following NT, G0 donors developed into blastocysts that retained H3K9me3 hypomethylation, both in the inner cell mass and trophectoderm. G0 blastocysts from different cell types and cell lines developed significantly better into adult offspring. In conclusion, serum starvation induced epigenetic changes, specifically hypotrimethylation, that provide a mechanistic correlate for increased somatic cell reprogrammability. PMID:27281704

  16. Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells.

    PubMed

    Mitalipov, Shoukhrat M; Yeoman, Richard R; Nusser, Kevin D; Wolf, Don P

    2002-05-01

    Production of genetically identical nonhuman primates would reduce the number of animals required for biomedical research and dramatically impact studies pertaining to immune system function, such as development of the human-immunodeficiency-virus vaccine. Our long-term goal is to develop robust somatic cell cloning and/or twinning protocols in the rhesus macaque. The objective of this study was to determine the developmental competence of nuclear transfer (NT) embryos derived from embryonic blastomeres (embryonic cell NT) or fetal fibroblasts (somatic cell NT) as a first step in the production of rhesus monkeys by somatic cell cloning. Development of cleaved embryos up to the 8-cell stage was similar among embryonic and somatic cell NT embryos and comparable to controls created by intracytoplasmic sperm injection (ICSI; mean +/- SEM, 81 +/- 5%, 88 +/- 7%, and 87 +/- 4%, respectively). However, significantly lower rates of development to the blastocyst stage were observed with somatic cell NT embryos (1%) in contrast to embryonic cell NT (34 +/- 15%) or ICSI control embryos (46 +/- 6%). Development of somatic cell NT embryos was not markedly affected by donor cell treatment, timing of activation, or chemical activation protocol. Transfer of embryonic, but not of somatic cell NT embryos, into recipients resulted in term pregnancy. Future efforts will focus on optimizing the production of somatic cell NT embryos that develop in high efficiency to the blastocyst stage in vitro.

  17. Method for somatic cell nuclear transfer in zebrafish.

    PubMed

    Siripattarapravat, K; Prukudom, S; Cibelli, J

    2016-01-01

    This chapter presents a detailed methodology for somatic cell nuclear transfer-cloning of zebrafish. We aim to place the reader in a virtual lab experience to assist acquisition of the technical skills required for reproducing the published protocol. All materials, including catalog numbers for reagents and techniques for their preparation, are provided. Our protocols describe laser inactivation of egg chromosomes, the transfer of a cell through the oocyte micropyle, and spontaneous activation of the reconstructed embryo. High-quality eggs are the key to cloning success, and Chinook salmon ovarian fluid is indispensable for keeping eggs arrested at the metaphase of meiosis II. This protocol continues to be refined by our laboratory. However, naive investigators should be able to apply it in its present form to generate cloned zebrafish. PMID:27443929

  18. Evolving proteins in mammalian cells using somatic hypermutation.

    PubMed

    Wang, Lei; Tsien, Roger Y

    2006-01-01

    We describe a new method to mutate target genes through somatic hypermutation (SHM) and to evolve proteins directly in living mammalian cells. Target genes are expressed under the control of an inducible promoter in a B-cell line that hypermutates its immunoglobulin (Ig) V genes constitutively. Mutations can be introduced into the target gene through SHM upon transcription. Mutant genes are then expressed and selected or screened for desired properties in cells. Identified cells are subjected to another round of mutation and selection or screening. This process can be iterated easily for numerous rounds, and multiple reinforcing mutations can be accumulated to produce desirable phenotypes. This approach bypasses labor-intensive in vitro mutagenesis and samples a large protein sequence space. In this protocol a monomeric red fluorescent protein (mRFP1.2) was evolved in Ramos cells to afford a mutant (mPlum) with far-red emission. This method can be adapted to evolve other eukaryotic proteins and to be used in other cells able to perform SHM. For each round of evolution, it takes approximately 1 d to mutate the target gene, approximately 0.5-1 d to select or screen, and 2-4 d to propagate the cells for the next round depending on how many cells are collected. PMID:17406421

  19. Essential elements for translation: the germline factor Vasa functions broadly in somatic cells

    PubMed Central

    Yajima, Mamiko; Wessel, Gary M.

    2015-01-01

    ABSTRACT Vasa is a conserved RNA-helicase found in the germ lines of all metazoans tested. Whereas Vasa presence is often indicated as a metric for germline determination in animals, it is also expressed in stem cells of diverse origin. Recent research suggests, however, that Vasa has a much broader function, including a significant role in cell cycle regulation. Results herein indicate that Vasa is utilized widely, and often induced transiently, during development in diverse somatic cells and adult precursor tissues. We identified that Vasa in the sea urchin is essential for: (1) general mRNA translation during embryogenesis, (2) developmental re-programming upon manipulations to the embryo and (3) larval wound healing. We also learned that Vasa interacted with mRNAs in the perinuclear area and at the spindle in an Importin-dependent manner during cell cycle progression. These results suggest that, when present, Vasa functions are essential to contributing to developmental regulation. PMID:25977366

  20. Somatic cell nuclear transfer: Infinite reproduction of a unique diploid genome

    SciTech Connect

    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.

  1. Somatic cell nuclear transfer: infinite reproduction of a unique diploid genome.

    PubMed

    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. PMID:18346729

  2. Comparison of semiautomated method with official optical somatic cell counting method III for determining somatic cells in milk.

    PubMed

    Mochrie, R D; Dickey, D A

    1984-01-01

    The new method specifying the Fossomatic-90 differs from the official method, 46.105-46.109, in that the modified instrument includes a halogen lamp; a semiconductor photoelectric detector; a less expensive, bench-top cabinet; manual injection of a larger sample, and a reduced capacity. The new instrument was compared with 2 optical somatic cell counters in routine use. On each of 3 days, 12 subsamples were prepared for each of 5 cell count levels from AM milk with half kept fresh and half preserved with 0.05% potassium dichromate. Subsamples were refrigerated and read 30+ h post-collection. Duplicate sets were read in random order on each machine daily (CV 0.77%). Two sets of slides read by 2 technicians each (strip reticle on 2 smears/slide) gave geometric mean direct microscopic somatic cell count (DMSCC) levels of 296, 526, 772, 930, and 1438 th/mL. Within-technician CV values (from day-level means) ranged from 1.68 to 2.28%. Geometric mean cells in th/mL on the new machine were significantly higher than those on the other two (674 vs 621) and were closer to the DMSCC (694). On the new machine, cell counts were 8.5% greater than on the original machines, were only 2.9% lower than the DMSCC, and showed no significant evidence of bias. Preserved samples averaged slightly greater than fresh (5.3%) but only on the original machines. Carryover by covariance analysis was insignificant. Except for cell levels, high machine precision (error CV value of 1.18%) gave differences with statistical but not practical significance, even for regulatory laboratories. PMID:6378874

  3. Gnotobiotic Miniature Pig Interbreed Somatic Cell Nuclear Transfer for Xenotransplantation.

    PubMed

    Hwang, Jeong Ho; Kim, Sang Eun; Gupta, Mukesh Kumar; Lee, HoonTaek

    2016-08-01

    Transgenic animal producing technology has improved consistently over the last couple of decades. Among the available methods, somatic cell nuclear transfer (SCNT) technology was officially the most popular. However, SCNT has low efficiency and requires a highly skilled individual. Additionally, the allo-SCNT nuclear reprogramming mechanism is poorly understood in the gnotobiotic miniature pig, which is a candidate for xenotransplantation, making sampling in oocytes very difficult compared to commercial hybrid pigs. Therefore, interbreed SCNT (ibSCNT), which is a combination of miniature pig and commercial pig (Landrace based), was analyzed and was found to be similar to SCNT in terms of the rate of blastocyst formation (12.6% ± 2.9% vs. 15.5% ± 2.2%; p > 0.05). However, a significantly lower fusion rate was observed in the ibSCNT compared to normal SCNT with Landrace pig somatic cells (29.6% ± 0.8% vs. 65.0% ± 4.9%). Thus, the optimization of fusion parameters was necessary for efficient SCNT. Our results further revealed that ibSCNT by the whole-cell intracytoplasmic injection (WCICI) method had a significantly higher blastocyst forming efficiency than the electrofusion method (31.1 ± 8.5 vs. 15.5% ± 2.2%). The nuclear remodeling and the pattern of changes in acetylation at H3K9 residue were similar in both SCNT and ibSCNT embryos. PMID:27459580

  4. Histone Demethylase Expression Enhances Human Somatic Cell Nuclear Transfer Efficiency and Promotes Derivation of Pluripotent Stem Cells.

    PubMed

    Chung, Young Gie; Matoba, Shogo; Liu, Yuting; Eum, Jin Hee; Lu, Falong; Jiang, Wei; Lee, Jeoung Eun; Sepilian, Vicken; Cha, Kwang Yul; Lee, Dong Ryul; Zhang, Yi

    2015-12-01

    The extremely low efficiency of human embryonic stem cell (hESC) derivation using somatic cell nuclear transfer (SCNT) limits its potential application. Blastocyst formation from human SCNT embryos occurs at a low rate and with only some oocyte donors. We previously showed in mice that reduction of histone H3 lysine 9 trimethylation (H3K9me3) through ectopic expression of the H3K9me3 demethylase Kdm4d greatly improves SCNT embryo development. Here we show that overexpression of a related H3K9me3 demethylase KDM4A improves human SCNT, and that, as in mice, H3K9me3 in the human somatic cell genome is an SCNT reprogramming barrier. Overexpression of KDM4A significantly improves the blastocyst formation rate in human SCNT embryos by facilitating transcriptional reprogramming, allowing efficient derivation of SCNT-derived ESCs using adult Age-related Macular Degeneration (AMD) patient somatic nuclei donors. This conserved mechanistic insight has potential applications for improving SCNT in a variety of contexts, including regenerative medicine. PMID:26526725

  5. Regenerative therapy for neuronal diseases with transplantation of somatic stem cells.

    PubMed

    Kanno, Hiroshi

    2013-10-26

    Pluripotent stem cells, which are capable of differentiating in various species of cells, are hoped to be donor cells in transplantation in regenerative medicine. Embryonic stem (ES) cells and induced pluripotent stem cells have the potential to differentiate in approximately all species of cells. However, the proliferating ability of these cells is high and the cancer formation ability is also recognized. In addition, ethical problems exist in using ES cells. Somatic stem cells with the ability to differentiate in various species of cells have been used as donor cells for neuronal diseases, such as amyotrophic lateral sclerosis, spinal cord injury, Alzheimer disease, cerebral infarction and congenital neuronal diseases. Human mesenchymal stem cells derived from bone marrow, adipose tissue, dermal tissue, umbilical cord blood and placenta are usually used for intractable neuronal diseases as somatic stem cells, while neural progenitor/stem cells and retinal progenitor/stem cells are used for a few congenital neuronal diseases and retinal degenerative disease, respectively. However, non-treated somatic stem cells seldom differentiate to neural cells in recipient neural tissue. Therefore, the contribution to neuronal regeneration using non-treated somatic stem cells has been poor and various differential trials, such as the addition of neurotrophic factors, gene transfer, peptide transfer for neuronal differentiation of somatic stem cells, have been performed. Here, the recent progress of regenerative therapies using various somatic stem cells is described.

  6. The somatic genomic landscape of chromophobe renal cell carcinoma.

    PubMed

    Davis, Caleb F; Ricketts, Christopher J; Wang, Min; Yang, Lixing; Cherniack, Andrew D; Shen, Hui; Buhay, Christian; Kang, Hyojin; Kim, Sang Cheol; Fahey, Catherine C; Hacker, Kathryn E; Bhanot, Gyan; Gordenin, Dmitry A; Chu, Andy; Gunaratne, Preethi H; Biehl, Michael; Seth, Sahil; Kaipparettu, Benny A; Bristow, Christopher A; Donehower, Lawrence A; Wallen, Eric M; Smith, Angela B; Tickoo, Satish K; Tamboli, Pheroze; Reuter, Victor; Schmidt, Laura S; Hsieh, James J; Choueiri, Toni K; Hakimi, A Ari; Chin, Lynda; Meyerson, Matthew; Kucherlapati, Raju; Park, Woong-Yang; Robertson, A Gordon; Laird, Peter W; Henske, Elizabeth P; Kwiatkowski, David J; Park, Peter J; Morgan, Margaret; Shuch, Brian; Muzny, Donna; Wheeler, David A; Linehan, W Marston; Gibbs, Richard A; Rathmell, W Kimryn; Creighton, Chad J

    2014-09-01

    We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) on the basis of multidimensional and comprehensive characterization, including mtDNA and whole-genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared with other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT upregulation in cancer distinct from previously observed amplifications and point mutations.

  7. The somatic genomic landscape of chromophobe renal cell carcinoma

    PubMed Central

    Davis, Caleb F.; Ricketts, Christopher; Wang, Min; Yang, Lixing; Cherniack, Andrew D.; Shen, Hui; Buhay, Christian; Kang, Hyojin; Kim, Sang Cheol; Fahey, Catherine C.; Hacker, Kathryn E.; Bhanot, Gyan; Gordenin, Dmitry A.; Chu, Andy; Gunaratne, Preethi H.; Biehl, Michael; Seth, Sahil; Kaipparettu, Benny A.; Bristow, Christopher A.; Donehower, Lawrence A.; Wallen, Eric M.; Smith, Angela B.; Tickoo, Satish K.; Tamboli, Pheroze; Reuter, Victor; Schmidt, Laura S.; Hsieh, James J.; Choueiri, Toni K.; Hakimi, A. Ari; Chin, Lynda; Meyerson, Matthew; Kucherlapati, Raju; Park, Woong-Yang; Robertson, A. Gordon; Laird, Peter W.; Henske, Elizabeth P.; Kwiatkowski, David J.; Park, Peter J.; Morgan, Margaret; Shuch, Brian; Muzny, Donna; Wheeler, David A.; Linehan, W. Marston; Gibbs, Richard A.; Rathmell, W. Kimryn; Creighton, Chad J.

    2014-01-01

    Summary We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) based on multidimensional and comprehensive characterization, including mitochondrial DNA (mtDNA) and whole genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared to other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT up-regulation in cancer distinct from previously-observed amplifications and point mutations. PMID:25155756

  8. Somatic Stem Cells and Their Dysfunction in Endometriosis

    PubMed Central

    Djokovic, Dusan; Calhaz-Jorge, Carlos

    2015-01-01

    Emerging evidence indicates that somatic stem cells (SSCs) of different types prominently contribute to endometrium-associated disorders such as endometriosis. We reviewed the pertinent studies available on PubMed, published in English language until December 2014 and focused on the involvement of SSCs in the pathogenesis of this common gynecological disease. A concise summary of the data obtained from in vitro experiments, animal models, and human tissue analyses provides insights into the SSC dysregulation in endometriotic lesions. In addition, a set of research results is presented supporting that SSC-targeting, in combination with hormonal therapy, may result in improved control of the disease, while a more in-depth characterization of endometriosis SSCs may contribute to the development of early-disease diagnostic tests with increased sensitivity and specificity. Key message: Seemingly essential for the establishment and progression of endometriotic lesions, dysregulated SSCs, and associated molecular alterations hold a promise as potential endometriosis markers and therapeutic targets. PMID:25593975

  9. The somatic genomic landscape of chromophobe renal cell carcinoma.

    PubMed

    Davis, Caleb F; Ricketts, Christopher J; Wang, Min; Yang, Lixing; Cherniack, Andrew D; Shen, Hui; Buhay, Christian; Kang, Hyojin; Kim, Sang Cheol; Fahey, Catherine C; Hacker, Kathryn E; Bhanot, Gyan; Gordenin, Dmitry A; Chu, Andy; Gunaratne, Preethi H; Biehl, Michael; Seth, Sahil; Kaipparettu, Benny A; Bristow, Christopher A; Donehower, Lawrence A; Wallen, Eric M; Smith, Angela B; Tickoo, Satish K; Tamboli, Pheroze; Reuter, Victor; Schmidt, Laura S; Hsieh, James J; Choueiri, Toni K; Hakimi, A Ari; Chin, Lynda; Meyerson, Matthew; Kucherlapati, Raju; Park, Woong-Yang; Robertson, A Gordon; Laird, Peter W; Henske, Elizabeth P; Kwiatkowski, David J; Park, Peter J; Morgan, Margaret; Shuch, Brian; Muzny, Donna; Wheeler, David A; Linehan, W Marston; Gibbs, Richard A; Rathmell, W Kimryn; Creighton, Chad J

    2014-09-01

    We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) on the basis of multidimensional and comprehensive characterization, including mtDNA and whole-genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared with other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT upregulation in cancer distinct from previously observed amplifications and point mutations. PMID:25155756

  10. Finnish Sixth Graders as Victims of Adult, Peer, and Co-Occurring Adult and Peer Violence: Depression, Somatization, and Violent Ideation in Relation to Victimization

    ERIC Educational Resources Information Center

    Uusitalo-Malmivaara, Lotta

    2013-01-01

    This study examined the experiences of peer and adult victimization of 737 12-year-old Finnish students. Of the respondents, 28.4% had experienced peer or adult, or both kinds of violence. Peer violence was the most common type of violence, while adult violence was rare. The associations between victimization and depression, somatization and…

  11. Factors affecting the development of somatic cell nuclear transfer embryos in Cattle.

    PubMed

    Akagi, Satoshi; Matsukawa, Kazutsugu; Takahashi, Seiya

    2014-01-01

    Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle.

  12. Deterministic direct reprogramming of somatic cells to pluripotency.

    PubMed

    Rais, Yoach; Zviran, Asaf; Geula, Shay; Gafni, Ohad; Chomsky, Elad; Viukov, Sergey; Mansour, Abed AlFatah; Caspi, Inbal; Krupalnik, Vladislav; Zerbib, Mirie; Maza, Itay; Mor, Nofar; Baran, Dror; Weinberger, Leehee; Jaitin, Diego A; Lara-Astiaso, David; Blecher-Gonen, Ronnie; Shipony, Zohar; Mukamel, Zohar; Hagai, Tzachi; Gilad, Shlomit; Amann-Zalcenstein, Daniela; Tanay, Amos; Amit, Ido; Novershtern, Noa; Hanna, Jacob H

    2013-10-01

    Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.

  13. Using somatic-cell nuclear transfer to study aging.

    PubMed

    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.

  14. Using somatic-cell nuclear transfer to study aging.

    PubMed

    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. PMID:23929101

  15. Production of the first cloned camel by somatic cell nuclear transfer.

    PubMed

    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.

  16. Netrin-1 regulates somatic cell reprogramming and pluripotency maintenance

    PubMed Central

    Ozmadenci, Duygu; Féraud, Olivier; Markossian, Suzy; Kress, Elsa; Ducarouge, Benjamin; Gibert, Benjamin; Ge, Jian; Durand, Isabelle; Gadot, Nicolas; Plateroti, Michela; Bennaceur-Griscelli, Annelise; Scoazec, Jean-Yves; Gil, Jesus; Deng, Hongkui; Bernet, Agnes; Mehlen, Patrick; Lavial, Fabrice

    2015-01-01

    The generation of induced pluripotent stem (iPS) cells holds great promise in regenerative medicine. The use of the transcription factors Oct4, Sox2, Klf4 and c-Myc for reprogramming is extensively documented, but comparatively little is known about soluble molecules promoting reprogramming. Here we identify the secreted cue Netrin-1 and its receptor DCC, described for their respective survival/death functions in normal and oncogenic contexts, as reprogramming modulators. In various somatic cells, we found that reprogramming is accompanied by a transient transcriptional repression of Netrin-1 mediated by an Mbd3/Mta1/Chd4-containing NuRD complex. Mechanistically, Netrin-1 imbalance induces apoptosis mediated by the receptor DCC in a p53-independent manner. Correction of the Netrin-1/DCC equilibrium constrains apoptosis and improves reprogramming efficiency. Our work also sheds light on Netrin-1's function in protecting embryonic stem cells from apoptosis mediated by its receptor UNC5b, and shows that the treatment with recombinant Netrin-1 improves the generation of mouse and human iPS cells. PMID:26154507

  17. Current methods for inducing pluripotency in somatic cells.

    PubMed

    Tavernier, Geertrui; Mlody, Barbara; Demeester, Jo; Adjaye, James; De Smedt, Stefaan C

    2013-05-28

    The groundbreaking discovery of reprogramming fibroblasts towards pluripotency merely by introducing four transcription factors (OCT4, SOX2, KLF4 and c-MYC) by means of retroviral transduction has created a promising revolution in the field of regenerative medicine. These so-called induced pluripotent stem cells (iPSCs) can provide a cell source for disease-modelling, drug-screening platforms, and transplantation strategies to treat incurable degenerative diseases, while circumventing the ethical issues and immune rejections associated with the use of non-autologous embryonic stem cells. The risk of insertional mutagenesis, caused both by the viral and transgene nature of the technique has proven to be the major limitation for iPSCs to be used in a clinical setting. In view of this, a variety of alternative techniques have been developed to induce pluripotency in somatic cells. This review provides an overview on current reprogramming protocols, discusses their pros and cons and future challenges to provide safe and transgene-free iPSCs.

  18. Replication-defective vectors of reticuloendotheliosis virus transduce exogenous genes into somatic stem cells of the unincubated chicken embryo

    SciTech Connect

    Bosselman, R.A.; Hsu, R.Y.; Boggs, T.; Hu, S.; Bruszewski, J.; Ou, S.; Souza, L.; Kozar, L.; Martin, F.; Nicolson, M.

    1989-06-01

    Replication-defective vectors derived from reticuloendotheliosis virus were used to transduce exogenous genes into early somatic stem cells of the chicken embryo. One of these vectors transduced and expressed the chicken growth hormone coding sequence. The helper cell line, C3, was used to generate stocks of vector containing about 10/sup 4/ transducing units per ml. Injection of 5- to 20-..mu..l volumes of vector directly beneath the blastoderm of unincubated chicken embryos led to infection of somatic stem cells. Infected embryos and adults contained unrearranged integrated proviral DNAs. Embryos expressed the transduced chicken growth hormone gene and contained high levels of serum growth hormone. Blood, brain, muscle, testis, and semen contained from individuals injected as embryos contained vector DNA. Replication-defective vectors of the reticuloendotheliosis virus transduced exogenous genes into chicken embryonic stem cells in vivo.

  19. A microdroplet cell culture based high frequency somatic embryogenesis system for pigeonpea, Cajanus cajan (L.) Millsp.

    PubMed

    Kumar, Nagan Udhaya; Gnanaraj, Muniraj; Sindhujaa, Vajravel; Viji, Maluventhen; Manoharan, Kumariah

    2015-09-01

    A protocol for high frequency production of somatic embryos was worked out in pigeonpea, Cajanus cajan (L.) Millsp. The protocol involved sequential employment of embryogenic callus cultures, low density cell suspension cultures and a novel microdroplet cell culture system. The microdroplet cell cultures involved culture of a single cell in 10 μI of Murashige and Skoog's medium supplemented with phytohormones, growth factors and phospholipid precursors. By employing the microdroplet cell cultures, single cells in isolation were grown into cell clones which developed somatic embryos. Further, 2,4-dichlorophenoxyacetic acid, kinetin, polyethylene glycol, putrescine, spermine, spermidine, choline chloride, ethanolamine and LiCl were supplemented to the low density cell suspension cultures and microdroplet cell cultures to screen for their cell division and somatic embryogenesis activity. Incubation of callus or the inoculum employed for low density cell suspension cultures and microdroplet cell cultures with polyethylene glycol was found critical for induction of somatic embryogenesis. Somatic embryogenesis at a frequency of 1.19, 3.16 and 6.51 per 10(6) cells was achieved in the callus, low density cell suspension cultures and microdroplet cell cultures, respectively. Advantages of employing microdroplet cell cultures for high frequency production of somatic embryos and its application in genetic transformation protocols are discussed. PMID:26548080

  20. A microdroplet cell culture based high frequency somatic embryogenesis system for pigeonpea, Cajanus cajan (L.) Millsp.

    PubMed

    Kumar, Nagan Udhaya; Gnanaraj, Muniraj; Sindhujaa, Vajravel; Viji, Maluventhen; Manoharan, Kumariah

    2015-09-01

    A protocol for high frequency production of somatic embryos was worked out in pigeonpea, Cajanus cajan (L.) Millsp. The protocol involved sequential employment of embryogenic callus cultures, low density cell suspension cultures and a novel microdroplet cell culture system. The microdroplet cell cultures involved culture of a single cell in 10 μI of Murashige and Skoog's medium supplemented with phytohormones, growth factors and phospholipid precursors. By employing the microdroplet cell cultures, single cells in isolation were grown into cell clones which developed somatic embryos. Further, 2,4-dichlorophenoxyacetic acid, kinetin, polyethylene glycol, putrescine, spermine, spermidine, choline chloride, ethanolamine and LiCl were supplemented to the low density cell suspension cultures and microdroplet cell cultures to screen for their cell division and somatic embryogenesis activity. Incubation of callus or the inoculum employed for low density cell suspension cultures and microdroplet cell cultures with polyethylene glycol was found critical for induction of somatic embryogenesis. Somatic embryogenesis at a frequency of 1.19, 3.16 and 6.51 per 10(6) cells was achieved in the callus, low density cell suspension cultures and microdroplet cell cultures, respectively. Advantages of employing microdroplet cell cultures for high frequency production of somatic embryos and its application in genetic transformation protocols are discussed.

  1. The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells.

    PubMed

    Keam, Simon P; Young, Paul E; McCorkindale, Alexandra L; Dang, Thurston H Y; Clancy, Jennifer L; Humphreys, David T; Preiss, Thomas; Hutvagner, Gyorgy; Martin, David I K; Cropley, Jennifer E; Suter, Catherine M

    2014-08-01

    The Piwi-piRNA pathway is active in animal germ cells where its functions are required for germ cell maintenance and gamete differentiation. Piwi proteins and piRNAs have been detected outside germline tissue in multiple phyla, but activity of the pathway in mammalian somatic cells has been little explored. In particular, Piwi expression has been observed in cancer cells, but nothing is known about the piRNA partners or the function of the system in these cells. We have surveyed the expression of the three human Piwi genes, Hiwi, Hili and Hiwi2, in multiple normal tissues and cancer cell lines. We find that Hiwi2 is ubiquitously expressed; in cancer cells the protein is largely restricted to the cytoplasm and is associated with translating ribosomes. Immunoprecipitation of Hiwi2 from MDAMB231 cancer cells enriches for piRNAs that are predominantly derived from processed tRNAs and expressed genes, species which can also be found in adult human testis. Our studies indicate that a Piwi-piRNA pathway is present in human somatic cells, with an uncharacterised function linked to translation. Taking this evidence together with evidence from primitive organisms, we propose that this somatic function of the pathway predates the germline functions of the pathway in modern animals.

  2. Psychic and somatic symptoms of depression among young adults, institutionalized aged and noninstitutionalized aged.

    PubMed

    Zemore, R; Eames, N

    1979-09-01

    Beck Depression Inventory scores were obtained from 48 elderly who had been residing in homes for the aged for more than one year, 31 elderly residing in the community and waiting to enter an old-age home, and 424 young adults enrolled in a fist-year psychology course. The residents of old-age homes reported no more symptoms of depression than the waiting-list controls, a finding that provides no support for the hypothesis that the institional nature of old-age homes increases depression in the elderly. Both the institutionalized and noninstitutionalized aged reported more somatic symptoms of depression than the young adults, but no greater cognitive or affective symptoms of depression. These results were interpreted as providing no support for the widely belief that the aged are more depressed than any other age group. Finally, it was argued that somatic complaints can be valid indicators of depression in the elderly if normative differences between young and old are taken into account.

  3. Established epigenetic modifications determine the expression of developmentally regulated globin genes in somatic cell hybrids.

    PubMed Central

    Stanworth, S J; Roberts, N A; Sharpe, J A; Sloane-Stanley, J A; Wood, W G

    1995-01-01

    Somatic cell hybrids generated from transgenic mouse cells have been used to examine the developmental regulation of human gamma-to-beta-globin gene switching. In hybrids between mouse erythroleukemia (MEL) cells and transgenic erythroblasts taken at various stages of development, there was regulated expression of the human fetal gamma and adult beta genes, reproducing the in vivo pattern prior to fusion. Hybrids formed from embryonic blood cells produced predominantly gamma mRNA, whereas beta gene expression was observed in adult hybrids and a complete range of intermediate patterns was found in fetal liver hybrids. The adult environment of the MEL cells, therefore, did not appear to influence selective transcription from this gene complex. Irradiation of the embryonic erythroid cells prior to fusion resulted in hybrids containing only small fragments of donor chromosomes, but the pattern of gene expression did not differ from that of unirradiated hybrids. This finding suggests that continued expression of trans-acting factors from the donor erythroblasts is not necessary for continued expression of the human gamma gene in MEL cells. These results contrast with the lack of developmental regulation of the cluster after transfection of naked DNA into MEL cells and suggest that epigenetic processes established during normal development result in the gene cluster adopting a developmental stage-specific, stable conformation which is maintained through multiple rounds of replication and transcription in the MEL cell hybrids. On prolonged culture, hybrids that initially expressed only the gamma transgene switched to beta gene expression. The time period of switching, from approximately 10 to > 40 weeks, was similar to that seen previously in human fetal erythroblast x MEL cell hybrids but in this case bore no relationship to the time of in vivo switching. It seems unlikely, therefore, that switching in these hybrids is regulated by a developmental clock. PMID:7623793

  4. Effective donor cell fusion conditions for production of cloned dogs by somatic cell nuclear transfer.

    PubMed

    Park, JungEun; Oh, HyunJu; Hong, SoGun; Kim, MinJung; Kim, GeonA; Koo, OkJae; Kang, SungKeun; Jang, Goo; Lee, ByeongChun

    2011-03-01

    As shown by the birth of the first cloned dog 'Snuppy', a protocol to produce viable cloned dogs has been reported. In order to evaluate optimum fusion conditions for improving dog cloning efficiency, in vivo matured oocytes were reconstructed with adult somatic cells from a female Pekingese using different fusion conditions. Fusion with needle vs chamber methods, and with low vs high pulse strength was compared by evaluating fusion rate and in vivo development of canine cloned embryos. The fusion rates in the high voltage groups were significantly higher than in the low voltage groups regardless of fusion method (83.5 vs 66.1% for the needle fusion method, 67.4 vs 37.9% for the fusion chamber method). After embryo transfer, one each pregnancy was detected after using the needle fusion method with high and low voltage and in the chamber fusion method with high voltage, whereas no pregnancy was detected using the chamber method with low voltage. However, only the pregnancy from the needle fusion method with high voltage was maintained to term and one healthy puppy was delivered. The results of the present study demonstrated that two DC pulses of 3.8 to 4.0 kV/cm for 15 μsec using the needle fusion method were the most effective method for the production of cloned dogs under the conditions of this experiment.

  5. Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion

    SciTech Connect

    Robinson, Claire; Kolb, Andreas F.

    2009-02-01

    The transcriptional regulation of a gene is best analysed in the context of its normal chromatin surroundings. However, most somatic cells, in contrast to embryonic stem cells, are refractory to accurate modification by homologous recombination. We show here that it is possible to introduce precise genomic modifications in ES cells and to analyse the phenotypic consequences in differentiated cells by using a combination of gene targeting, site-specific recombination and somatic cell fusion. To provide a proof of principle, we have analysed the regulation of the casein gene locus in mammary gland cells derived from modified murine ES cells by somatic cell fusion. A {beta}-galactosidase reporter gene was inserted in place of the {beta}-casein gene and the modified ES cells, which do not express the reporter gene, were fused with the mouse mammary gland cell line HC11. The resulting cell clones expressed the {beta}-galactosidase gene to a similar extent and with similar hormone responsiveness as the endogenous gene. However, a reporter gene under the control of a minimal {beta}-casein promoter (encompassing the two consensus STAT5 binding sites which mediate the hormone response of the casein genes) was unable to replicate expression levels or hormone responsiveness of the endogenous gene when inserted into the same site of the casein locus. As expected, these results implicate sequences other than the STAT5 sites in the regulation of the {beta}-casein gene.

  6. Myc and SAGA rewire an alternative splicing network during early somatic cell reprogramming

    PubMed Central

    Hirsch, Calley L.; Coban Akdemir, Zeynep; Wang, Li; Jayakumaran, Gowtham; Trcka, Dan; Weiss, Alexander; Hernandez, J. Javier; Pan, Qun; Han, Hong; Xu, Xueping; Xia, Zheng; Salinger, Andrew P.; Wilson, Marenda; Vizeacoumar, Frederick; Datti, Alessandro; Li, Wei; Cooney, Austin J.; Barton, Michelle C.; Blencowe, Benjamin J.

    2015-01-01

    Embryonic stem cells are maintained in a self-renewing and pluripotent state by multiple regulatory pathways. Pluripotent-specific transcriptional networks are sequentially reactivated as somatic cells reprogram to achieve pluripotency. How epigenetic regulators modulate this process and contribute to somatic cell reprogramming is not clear. Here we performed a functional RNAi screen to identify the earliest epigenetic regulators required for reprogramming. We identified components of the SAGA histone acetyltransferase complex, in particular Gcn5, as critical regulators of reprogramming initiation. Furthermore, we showed in mouse pluripotent stem cells that Gcn5 strongly associates with Myc and that, upon initiation of somatic reprogramming, Gcn5 and Myc form a positive feed-forward loop that activates a distinct alternative splicing network and the early acquisition of pluripotency-associated splicing events. These studies expose a Myc–SAGA pathway that drives expression of an essential alternative splicing regulatory network during somatic cell reprogramming. PMID:25877919

  7. Amphibian and mammal somatic-cell cloning: different species, common results?

    PubMed

    Loi, Pasqualino; Fulka, Josef; Ptak, Grazyna

    2003-11-01

    Since the production of Dolly the sheep cloning methods for somatic cells have been thoroughly described and are becoming routine. However, the rate at which live clones are produced remains low in all mammalian species tested so far. Remarkably, irrespective of the cloning protocol or the donor-cell type, all clones display common abnormalities, particularly in the placenta. The process is also complicated by early mortality of somatic-cell clones and the founder mammalian clone, Dolly the sheep, died in February 2003 aged six years. Based on published data and on our own experience, our view is that mammalian somatic-cell cloning and the pioneer nuclear-transfer data from amphibians have much in common. We suggest that the only way to improve nuclear reprogramming is to modify the chromatin structure of somatic cells before nuclear transfer, to provide the oocyte with a chromosomal structure that is more compatible with the natural reprogramming machinery of the oocyte.

  8. An unregulated regulator: Vasa expression in the development of somatic cells and in tumorigenesis.

    PubMed

    Poon, Jessica; Wessel, Gary M; Yajima, Mamiko

    2016-07-01

    Growing evidence in diverse organisms shows that genes originally thought to function uniquely in the germ line may also function in somatic cells, and in some cases even contribute to tumorigenesis. Here we review the somatic functions of Vasa, one of the most conserved "germ line" factors among metazoans. Vasa expression in somatic cells is tightly regulated and often transient during normal development, and appears to play essential roles in regulation of embryonic cells and regenerative tissues. Its dysregulation, however, is believed to be an important element of tumorigenic cell regulation. In this perspectives paper, we propose how some conserved functions of Vasa may be selected for somatic cell regulation, including its potential impact on efficient and localized translational activities and in some cases on cellular malfunctioning and tumorigenesis. PMID:27179696

  9. Amphibian and mammal somatic-cell cloning: different species, common results?

    PubMed

    Loi, Pasqualino; Fulka, Josef; Ptak, Grazyna

    2003-11-01

    Since the production of Dolly the sheep cloning methods for somatic cells have been thoroughly described and are becoming routine. However, the rate at which live clones are produced remains low in all mammalian species tested so far. Remarkably, irrespective of the cloning protocol or the donor-cell type, all clones display common abnormalities, particularly in the placenta. The process is also complicated by early mortality of somatic-cell clones and the founder mammalian clone, Dolly the sheep, died in February 2003 aged six years. Based on published data and on our own experience, our view is that mammalian somatic-cell cloning and the pioneer nuclear-transfer data from amphibians have much in common. We suggest that the only way to improve nuclear reprogramming is to modify the chromatin structure of somatic cells before nuclear transfer, to provide the oocyte with a chromosomal structure that is more compatible with the natural reprogramming machinery of the oocyte. PMID:14573358

  10. Somatic stem cells and the kinetics of mutagenesis and carcinogenesis

    PubMed Central

    Cairns, John

    2002-01-01

    There is now strong experimental evidence that epithelial stem cells arrange their sister chromatids at mitosis such that the same template DNA strands stay together through successive divisions; DNA labeled with tritiated thymidine in infancy is still present in the stem cells of adult mice even though these cells are incorporating (and later losing) bromodeoxyuridine [Potten, C. S., Owen, G., Booth, D. & Booth, C. (2002) J. Cell Sci.115, 2381–2388]. But a cell that preserves “immortal strands” will avoid the accumulation of replication errors only if it inhibits those pathways for DNA repair that involve potentially error-prone resynthesis of damaged strands, and this appears to be a property of intestinal stem cells because they are extremely sensitive to the lethal effects of agents that damage DNA. It seems that the combination, in the stem cell, of immortal strands and the choice of death rather than error-prone repair makes epithelial stem cell systems resistant to short exposures to DNA-damaging agents, because the stem cell accumulates few if any errors, and any errors made by the daughters are destined to be discarded. This paper discusses these issues and shows that they lead to a model that explains the strange kinetics of mutagenesis and carcinogenesis in adult mammalian tissues. Coincidentally, the model also can explain why cancers arise even though the spontaneous mutation rate of differentiated mammalian cells is not high enough to generate the multiple mutations needed to form a cancer and why loss of nucleotide-excision repair does not significantly increase the frequency of the common internal cancers. PMID:12149477

  11. Murine somatic cell nuclear transfer using reprogrammed donor cells expressing male germ cell-specific genes.

    PubMed

    Kang, Hoin; Park, Jong Im; Roh, Sangho

    2016-01-01

    In vivo-matured mouse oocytes were enucleated, and a single murine embryonic fibroblast (control or reprogrammed by introducing extracts from murine testis tissue, which showed expression of male germ cell-specific genes) was injected into the cytoplasm of the oocytes. The rate of blastocyst development and expression levels of Oct-4, Eomes and Cdx-2 were not significantly different in both experimental groups. However, the expression levels of Nanog, Sox9 and Glut-1 were significantly increased when reprogrammed cells were used as donor nuclei. Increased expression of Nanog can be supportive of complete reprogramming of somatic cell nuclear transfer murine embryos. The present study suggested that donor cells expressing male germ cell-specific genes can be reconstructed and can develop into embryos with normal high expression of developmentally essential genes. PMID:26369430

  12. Production of somatic chimera chicks by injection of bone marrow cells into recipient blastoderms.

    PubMed

    Heo, Young Tae; Lee, Sung Ho; Kim, Teoan; Kim, Nam Hyung; Lee, Hoon Taek

    2012-01-01

    Several types of cells, including blastoderm cells, primordial germ cells, and embryonic germ cells were injected into early-stage recipient embryos to produce chimera avians and to gain insights into cell development. However, a limited number of studies of avian adult stem cells have also been conducted. This study is, to the best of our knowledge, the first to evaluate chicken bone marrow cells' (chBMC) ability to differentiate into multiple cell lineages and capability to generate chimera chicks. We induced random differentiation of chBMCs in vitro and injected immunologically selected pluripotent cells in chBMCs into the blastoderms of recipient eggs. The multipotency of BMCs from the barred Plymouth rock (BPR) was confirmed via AP staining, RT-PCR, immunocytochemistry, and FACS using specific markers, such as Oct-4 and SSEA-1, 3 and 4. Isolated chBMCs were found to be able to induce in vitro differentiation to multiple cell lineages. Approximately 5,000 chBMCs were injected into the blastoderms of white leghorn (WL) recipients and proved able to contribute to the generation of somatic chimera chicks with a frequency of 2.7% (2 of 73). Confirmation of chimerism in hatched chicks was achieved via PCR analysis using D-loop-specific primers of BPR and WL. Our study demonstrated the successful production of chimera chicks using chBMC. Therefore, we propose that the use of adult chBMCs may constitute a new possible approach to the production of chimera poultry, and may provide helpful studies in avian developmental biology.

  13. Human Endometrial Side Population Cells Exhibit Genotypic, Phenotypic and Functional Features of Somatic Stem Cells

    PubMed Central

    Cervelló, Irene; Gil-Sanchis, Claudia; Mas, Aymara; Delgado-Rosas, Francisco; Martínez-Conejero, José Antonio; Galán, Amparo; Martínez-Romero, Alicia; Martínez, Sebastian; Navarro, Ismael; Ferro, Jaime; Horcajadas, José Antonio; Esteban, Francisco José; O'Connor, José Enrique; Pellicer, Antonio; Simón, Carlos

    2010-01-01

    During reproductive life, the human endometrium undergoes around 480 cycles of growth, breakdown and regeneration should pregnancy not be achieved. This outstanding regenerative capacity is the basis for women's cycling and its dysfunction may be involved in the etiology of pathological disorders. Therefore, the human endometrial tissue must rely on a remarkable endometrial somatic stem cells (SSC) population. Here we explore the hypothesis that human endometrial side population (SP) cells correspond to somatic stem cells. We isolated, identified and characterized the SP corresponding to the stromal and epithelial compartments using endometrial SP genes signature, immunophenotyping and characteristic telomerase pattern. We analyzed the clonogenic activity of SP cells under hypoxic conditions and the differentiation capacity in vitro to adipogenic and osteogenic lineages. Finally, we demonstrated the functional capability of endometrial SP to develop human endometrium after subcutaneous injection in NOD-SCID mice. Briefly, SP cells of human endometrium from epithelial and stromal compartments display genotypic, phenotypic and functional features of SSC. PMID:20585575

  14. Chromosome microduplication in somatic cells decreases the genetic stability of human reprogrammed somatic cells and results in pluripotent stem cells.

    PubMed

    Yu, Yang; Chang, Liang; Zhao, Hongcui; Li, Rong; Fan, Yong; Qiao, Jie

    2015-01-01

    Human pluripotent stem cells, including cloned embryonic and induced pluripotent stem cells, offer a limitless cellular source for regenerative medicine. However, their derivation efficiency is limited, and a large proportion of cells are arrested during reprogramming. In the current study, we explored chromosome microdeletion/duplication in arrested and established reprogrammed cells. Our results show that aneuploidy induced by somatic cell nuclear transfer technology is a key factor in the developmental failure of cloned human embryos and primary colonies from implanted cloned blastocysts and that expression patterns of apoptosis-related genes are dynamically altered. Overall, ~20%-53% of arrested primary colonies in induced plurpotent stem cells displayed aneuploidy, and upregulation of P53 and Bax occurred in all arrested primary colonies. Interestingly, when somatic cells with pre-existing chromosomal mutations were used as donor cells, no cloned blastocysts were obtained, and additional chromosomal mutations were detected in the resulting iPS cells following long-term culture, which was not observed in the two iPS cell lines with normal karyotypes. In conclusion, aneuploidy induced by the reprogramming process restricts the derivation of pluripotent stem cells, and, more importantly, pre-existing chromosomal mutations enhance the risk of genome instability, which limits the clinical utility of these cells. PMID:25965553

  15. Human somatic cell nuclear transfer and reproductive cloning: an Ethics Committee opinion.

    PubMed

    2016-04-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer and cloning," last published in Fertil Steril 2012;98:804-7. PMID:26746137

  16. Human somatic cell nuclear transfer and reproductive cloning: an Ethics Committee opinion.

    PubMed

    2016-04-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer and cloning," last published in Fertil Steril 2012;98:804-7.

  17. Chromatin diminution in the copepod Mesocyclops edax: diminution of tandemly repeated DNA families from somatic cells.

    PubMed

    Drouin, Guy

    2006-06-01

    Chromatin diminution, i.e., the loss of selected chromosomal regions during the differentiation of early embryonic cells into somatic cells, has been described in taxa as varied as ciliates, copepods, insects, nematodes, and hagfish. The nature of the eliminated DNA has been extensively studied in ciliate, nematode, and hagfish species. However, the small size of copepods, which makes it difficult to obtain enough DNA from early embryonic cells for cloning and sequencing, has limited such studies. Here, to identify the sequences eliminated from the somatic cells of a copepod species that undergoes chromatin diminution, we randomly amplified DNA fragments from germ line and somatic line cells of Mesocyclops edax, a freshwater cyclopoid copepod. Of 47 randomly amplified germ line clones, 45 (96%) contained short, tandemly repeated sequences composed of either 2 bp CA-repeats, 8 bp CAAATAGA-repeats, or 9 bp CAAATTAAA-repeats. In contrast, of 83 randomly amplified somatic line clones, only 47 (57%) contained such short, tandemly repeated sequences. As previously observed in some nematode species, our results therefore show that there is partial elimination of chromosomal regions containing (CAAATAGA and CAAATTAAA) repeated sequences during the chromatin diminution observed in the somatic cells of M. edax. We speculate that chromatin diminution might have evolved repeatedly by recruitment of RNAi-related mechanisms to eliminate nonfunctional tandemly repeated DNA sequences from the somatic genome of some species.

  18. Somatic cell nuclear transfer-derived embryonic stem cell lines in humans: pros and cons.

    PubMed

    Langerova, Alena; Fulka, Helena; Fulka, Josef

    2013-12-01

    The recent paper, published by Mitalipov's group in Cell (Tachibana et al., 2013 ), reporting the production of human somatic cell nuclear transfer (SCNT) embryonic stem cells (ESCs), opens again the debate if, in the era of induced pluripotent stem cells (iPSCs), the production of these cells is indeed necessary and, if so, whether they are different from ESCs produced from spare embryos and iPSCs. It is our opinion that these questions are very difficult to answer because it is still unclear whether and how normal ESCs differ from iPSCs. PMID:24180743

  19. Somatic cell nuclear transfer-derived embryonic stem cell lines in humans: pros and cons.

    PubMed

    Langerova, Alena; Fulka, Helena; Fulka, Josef

    2013-12-01

    The recent paper, published by Mitalipov's group in Cell (Tachibana et al., 2013 ), reporting the production of human somatic cell nuclear transfer (SCNT) embryonic stem cells (ESCs), opens again the debate if, in the era of induced pluripotent stem cells (iPSCs), the production of these cells is indeed necessary and, if so, whether they are different from ESCs produced from spare embryos and iPSCs. It is our opinion that these questions are very difficult to answer because it is still unclear whether and how normal ESCs differ from iPSCs.

  20. Self-renewal and pluripotency acquired through somatic reprogramming to human cancer stem cells.

    PubMed

    Nagata, Shogo; Hirano, Kunio; Kanemori, Michele; Sun, Liang-Tso; Tada, Takashi

    2012-01-01

    Human induced pluripotent stem cells (iPSCs) are reprogrammed by transient expression of transcription factors in somatic cells. Approximately 1% of somatic cells can be reprogrammed into iPSCs, while the remaining somatic cells are differentially reprogrammed. Here, we established induced pluripotent cancer stem-like cells (iCSCs) as self-renewing pluripotent cell clones. Stable iCSC lines were established from unstable induced epithelial stem cell (iESC) lines through re-plating followed by embryoid body formation and serial transplantation. iCSCs shared the expression of pluripotent marker genes with iPSCs, except for REX1 and LIN28, while exhibited the expression of somatic marker genes EMP1 and PPARγ. iESCs and iCSCs could generate teratomas with high efficiency by implantation into immunodeficient mice. The second iCSCs isolated from dissociated cells of teratoma from the first iCSCs were stably maintained, showing a gene expression profile similar to the first iCSCs. In the first and second iCSCs, transgene-derived Oct4, Sox2, Klf4, and c-Myc were expressed. Comparative global gene expression analyses demonstrated that the first iCSCs were similar to iESCs, and clearly different from human iPSCs and somatic cells. In iCSCs, gene expression kinetics of the core pluripotency factor and the Myc-related factor were pluripotent type, whereas the polycomb complex factor was somatic type. These findings indicate that pluripotent tumorigenicity can be conferred on somatic cells through up-regulation of the core pluripotency and Myc-related factors, prior to establishment of the iPSC molecular network by full reprogramming through down-regulation of the polycomb complex factor.

  1. Somatic tetraploidy in specific chick retinal ganglion cells induced by nerve growth factor

    PubMed Central

    Morillo, Sandra M.; Escoll, Pedro; de la Hera, Antonio; Frade, José M.

    2009-01-01

    A subset of neurons in the normal vertebrate nervous system contains double the normal amount of DNA in their nuclei. These neurons are all thought to derive from aberrant mitoses in neuronal precursor cells. Here we show that endogenous NGF induces DNA replication in a subpopulation of differentiating chick retinal ganglion cells that express both the neurotrophin receptor p75 and the E2F1 transcription factor, but that lack the retinoblastoma protein. Many of these neurons avoid G2/M transition and remain alive in the retina as tetraploid cells with large cell somas and extensive dendritic trees, and most of them express β2 nicotinic acetylcholine receptor subunits, a specific marker of retinal ganglion cells innervating lamina F in the stratum-griseum-et-fibrosum-superficiale of the tectal cortex. Tetraploid neurons were also observed in the adult mouse retina. Thus, a developmental program leading to somatic tetraploidy in specific retinal neurons exists in vertebrates. This program might occur in other vertebrate neurons during normal or pathological situations. PMID:20018664

  2. Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning.

    PubMed

    Skrzyszowska, M; Smorag, Z; Słomski, R; Katska-Ksiazkiewicz, L; Kalak, R; Michalak, E; Wielgus, K; Lehmann, J; Lipiński, D; Szalata, M; Pławski, A; Samiec, M; Jura, J; Gajda, B; Ryńska, B; Pieńkowski, M

    2006-06-01

    A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.

  3. Adult Stem Cells and Diseases of Aging

    PubMed Central

    Boyette, Lisa B.; Tuan, Rocky S.

    2014-01-01

    Preservation of adult stem cells pools is critical for maintaining tissue homeostasis into old age. Exhaustion of adult stem cell pools as a result of deranged metabolic signaling, premature senescence as a response to oncogenic insults to the somatic genome, and other causes contribute to tissue degeneration with age. Both progeria, an extreme example of early-onset aging, and heritable longevity have provided avenues to study regulation of the aging program and its impact on adult stem cell compartments. In this review, we discuss recent findings concerning the effects of aging on stem cells, contributions of stem cells to age-related pathologies, examples of signaling pathways at work in these processes, and lessons about cellular aging gleaned from the development and refinement of cellular reprogramming technologies. We highlight emerging therapeutic approaches to manipulation of key signaling pathways corrupting or exhausting adult stem cells, as well as other approaches targeted at maintaining robust stem cell pools to extend not only lifespan but healthspan. PMID:24757526

  4. BINNING SOMATIC MUTATIONS BASED ON BIOLOGICAL KNOWLEDGE FOR PREDICTING SURVIVAL: AN APPLICATION IN RENAL CELL CARCINOMA

    PubMed Central

    Kim, Dokyoon; Li, Ruowang; Dudek, Scott M.; Wallace, John R.; Ritchie, Marylyn D.

    2014-01-01

    Enormous efforts of whole exome and genome sequencing from hundreds to thousands of patients have provided the landscape of somatic genomic alterations in many cancer types to distinguish between driver mutations and passenger mutations. Driver mutations show strong associations with cancer clinical outcomes such as survival. However, due to the heterogeneity of tumors, somatic mutation profiles are exceptionally sparse whereas other types of genomic data such as miRNA or gene expression contain much more complete data for all genomic features with quantitative values measured in each patient. To overcome the extreme sparseness of somatic mutation profiles and allow for the discovery of combinations of somatic mutations that may predict cancer clinical outcomes, here we propose a new approach for binning somatic mutations based on existing biological knowledge. Through the analysis using renal cell carcinoma dataset from The Cancer Genome Atlas (TCGA), we identified combinations of somatic mutation burden based on pathways, protein families, evolutionary conversed regions, and regulatory regions associated with survival. Due to the nature of heterogeneity in cancer, using a binning strategy for somatic mutation profiles based on biological knowledge will be valuable for improved prognostic biomarkers and potentially for tailoring therapeutic strategies by identifying combinations of driver mutations. PMID:25592572

  5. Effects of Neuroendocrine CB1 Activity on Adult Leydig Cells

    PubMed Central

    Cobellis, Gilda; Meccariello, Rosaria; Chianese, Rosanna; Chioccarelli, Teresa; Fasano, Silvia; Pierantoni, Riccardo

    2016-01-01

    Endocannabinoids control male reproduction acting at central and local level via cannabinoid receptors. The cannabinoid receptor CB1 has been characterized in the testis, in somatic and germ cells of mammalian and non-mammalian animal models, and its activity related to Leydig cell differentiation, steroidogenesis, spermiogenesis, sperm quality, and maturation. In this short review, we provide a summary of the insights concerning neuroendocrine CB1 activity in male reproduction focusing on adult Leydig cell ontogenesis and steroid biosynthesis. PMID:27375550

  6. Hexavalent chromium induces apoptosis in male somatic and spermatogonial stem cells via redox imbalance

    PubMed Central

    Das, Joydeep; Kang, Min-Hee; Kim, Eunsu; Kwon, Deug-Nam; Choi, Yun-Jung; Kim, Jin-Hoi

    2015-01-01

    Hexavalent chromium [Cr(VI)], an environmental toxicant, causes severe male reproductive abnormalities. However, the actual mechanisms of toxicity are not clearly understood and have not been studied in detail. The present in vitro study aimed to investigate the mechanism of reproductive toxicity of Cr(VI) in male somatic cells (mouse TM3 Leydig cells and TM4 Sertoli cells) and spermatogonial stem cells (SSCs) because damage to or dysfunction of these cells can directly affect spermatogenesis, resulting in male infertility. Cr(VI) by inducing oxidative stress was cytotoxic to both male somatic cells and SSCs in a dose-dependent manner, and induced mitochondria-dependent apoptosis. Although the mechanism of Cr(VI)-induced cytotoxicity was similar in both somatic cells, the differences in sensitivity of TM3 and TM4 cells to Cr(VI) could be attributed, at least in part, to cell-specific regulation of P-AKT1, P-ERK1/2, and P-P53 proteins. Cr(VI) affected the differentiation and self-renewal mechanisms of SSCs, disrupted steroidogenesis in TM3 cells, while in TM4 cells, the expression of tight junction signaling and cell receptor molecules was affected as well as the secretory functions were impaired. In conclusion, our results show that Cr(VI) is cytotoxic and impairs the physiological functions of male somatic cells and SSCs. PMID:26355036

  7. [Isolation and characteristics of somatic cell hybrids of the Chinese hamster and American mink].

    PubMed

    Rubtsov, N B; Radzhabli, S I; Gradov, A A; Serov, O L

    1981-01-01

    The paper deals with obtaining somatic cell hybrids of Chinese hamster and mink by means of inactivated Sendy virus. 39 hybrid clones segregating mink chromosomes were formed by fusing Chinese hamster cells deficient in hypoxanthine phosphoribosyliransferase with normal cells of mink. Enzyme analyses of these hybrid clones revealed that in mink genes coding lactate dehydrogenase-A, lactate dehydrogenase-B, malate dehydrogenase-NAD (soluble), 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase are not syntenic. A possibility of successful utilization of these somatic cell hybrids for mapping mink genes is shown. PMID:6942558

  8. Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells.

    PubMed

    Ju, Young Seok; Tubio, Jose M C; Mifsud, William; Fu, Beiyuan; Davies, Helen R; Ramakrishna, Manasa; Li, Yilong; Yates, Lucy; Gundem, Gunes; Tarpey, Patrick S; Behjati, Sam; Papaemmanuil, Elli; Martin, Sancha; Fullam, Anthony; Gerstung, Moritz; Nangalia, Jyoti; Green, Anthony R; Caldas, Carlos; Borg, Åke; Tutt, Andrew; Lee, Ming Ta Michael; van't Veer, Laura J; Tan, Benita K T; Aparicio, Samuel; Span, Paul N; Martens, John W M; Knappskog, Stian; Vincent-Salomon, Anne; Børresen-Dale, Anne-Lise; Eyfjörd, Jórunn Erla; Flanagan, Adrienne M; Foster, Christopher; Neal, David E; Cooper, Colin; Eeles, Rosalind; Lakhani, Sunil R; Desmedt, Christine; Thomas, Gilles; Richardson, Andrea L; Purdie, Colin A; Thompson, Alastair M; McDermott, Ultan; Yang, Fengtang; Nik-Zainal, Serena; Campbell, Peter J; Stratton, Michael R

    2015-06-01

    Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements, and the features of the fusion fragments indicate that nonhomologous end joining and/or replication-dependent DNA double-strand break repair are the dominant mechanisms involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells. PMID:25963125

  9. Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells

    PubMed Central

    Ju, Young Seok; Tubio, Jose M.C.; Mifsud, William; Fu, Beiyuan; Davies, Helen R.; Ramakrishna, Manasa; Li, Yilong; Yates, Lucy; Gundem, Gunes; Tarpey, Patrick S.; Behjati, Sam; Papaemmanuil, Elli; Martin, Sancha; Fullam, Anthony; Gerstung, Moritz; Nangalia, Jyoti; Green, Anthony R.; Caldas, Carlos; Borg, Åke; Tutt, Andrew; Lee, Ming Ta Michael; van't Veer, Laura J.; Tan, Benita K.T.; Aparicio, Samuel; Span, Paul N.; Martens, John W.M.; Knappskog, Stian; Vincent-Salomon, Anne; Børresen-Dale, Anne-Lise; Eyfjörd, Jórunn Erla; Flanagan, Adrienne M.; Foster, Christopher; Neal, David E.; Cooper, Colin; Eeles, Rosalind; Lakhani, Sunil R.; Desmedt, Christine; Thomas, Gilles; Richardson, Andrea L.; Purdie, Colin A.; Thompson, Alastair M.; McDermott, Ultan; Yang, Fengtang; Nik-Zainal, Serena; Campbell, Peter J.; Stratton, Michael R.

    2015-01-01

    Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements, and the features of the fusion fragments indicate that nonhomologous end joining and/or replication-dependent DNA double-strand break repair are the dominant mechanisms involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells. PMID:25963125

  10. Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells.

    PubMed

    Ju, Young Seok; Tubio, Jose M C; Mifsud, William; Fu, Beiyuan; Davies, Helen R; Ramakrishna, Manasa; Li, Yilong; Yates, Lucy; Gundem, Gunes; Tarpey, Patrick S; Behjati, Sam; Papaemmanuil, Elli; Martin, Sancha; Fullam, Anthony; Gerstung, Moritz; Nangalia, Jyoti; Green, Anthony R; Caldas, Carlos; Borg, Åke; Tutt, Andrew; Lee, Ming Ta Michael; van't Veer, Laura J; Tan, Benita K T; Aparicio, Samuel; Span, Paul N; Martens, John W M; Knappskog, Stian; Vincent-Salomon, Anne; Børresen-Dale, Anne-Lise; Eyfjörd, Jórunn Erla; Flanagan, Adrienne M; Foster, Christopher; Neal, David E; Cooper, Colin; Eeles, Rosalind; Lakhani, Sunil R; Desmedt, Christine; Thomas, Gilles; Richardson, Andrea L; Purdie, Colin A; Thompson, Alastair M; McDermott, Ultan; Yang, Fengtang; Nik-Zainal, Serena; Campbell, Peter J; Stratton, Michael R

    2015-06-01

    Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements, and the features of the fusion fragments indicate that nonhomologous end joining and/or replication-dependent DNA double-strand break repair are the dominant mechanisms involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells.

  11. 40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following... cells in culture. 798.5300 Section 798.5300 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell...

  12. 40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following... cells in culture. 798.5300 Section 798.5300 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell...

  13. 40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following... cells in culture. 798.5300 Section 798.5300 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell...

  14. 40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following... cells in culture. 798.5300 Section 798.5300 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell...

  15. Somatic cell nuclear transfer (cloning): implications for the medical practitioner.

    PubMed

    Tong, W F; Ng, Y F; Ng, S C

    2002-07-01

    The current century will bring tremendous changes to the science and the practice of medicine. This century will be acknowledged as the century of Biology as the fusion of molecular genetics and experimental embryology pushes the barriers of science beyond perimeters that we have thought existed, as much as the past century was the century of Physics, with all the exact scientific calculations and predictions, resulting in electricity, nuclear power and quantum physics. The first major breakthrough has been the pioneering work of Wilmut and Campbell, first with the birth of Megan and Moran in 1995 (1), followed by the birth of Dolly the sheep, the first reported mammalian clone from a fully differentiated adult cell, reported in July 1996 (2). However, current cloning techniques are an extension of over 40 years of research using nuclei derived from non-human embryonic and fetal cells. However, following the birth of Dolly, the prospects of cloning technology have extended to ethically hazier areas of human cloning and embryonic stem cell research. This review hopes to bring the reader closer to the science and the ethics of this new technology, and what the implications are for the medical practitioner.

  16. Somatic cell nuclear transfer (cloning): implications for the medical practitioner.

    PubMed

    Tong, W F; Ng, Y F; Ng, S C

    2002-07-01

    The current century will bring tremendous changes to the science and the practice of medicine. This century will be acknowledged as the century of Biology as the fusion of molecular genetics and experimental embryology pushes the barriers of science beyond perimeters that we have thought existed, as much as the past century was the century of Physics, with all the exact scientific calculations and predictions, resulting in electricity, nuclear power and quantum physics. The first major breakthrough has been the pioneering work of Wilmut and Campbell, first with the birth of Megan and Moran in 1995 (1), followed by the birth of Dolly the sheep, the first reported mammalian clone from a fully differentiated adult cell, reported in July 1996 (2). However, current cloning techniques are an extension of over 40 years of research using nuclei derived from non-human embryonic and fetal cells. However, following the birth of Dolly, the prospects of cloning technology have extended to ethically hazier areas of human cloning and embryonic stem cell research. This review hopes to bring the reader closer to the science and the ethics of this new technology, and what the implications are for the medical practitioner. PMID:12437047

  17. Successful cloning of coyotes through interspecies somatic cell nuclear transfer using domestic dog oocytes.

    PubMed

    Hwang, Insung; Jeong, Yeon Woo; Kim, Joung Joo; Lee, Hyo Jeong; Kang, Mina; Park, Kang Bae; Park, Jung Hwan; Kim, Yeun Wook; Kim, Woo Tae; Shin, Taeyoung; Hyun, Sang Hwan; Jeung, Eui-Bae; Hwang, Woo Suk

    2013-01-01

    Interspecies somatic cell nuclear transfer (iSCNT) is an emerging assisted reproductive technology (ART) for preserving Nature's diversity. The scarcity of oocytes from some species makes utilisation of readily available oocytes inevitable. In the present study, we describe the successful cloning of coyotes (Canis latrans) through iSCNT using oocytes from domestic dogs (Canis lupus familiaris or dingo). Transfer of 320 interspecies-reconstructed embryos into 22 domestic dog recipients resulted in six pregnancies, from which eight viable offspring were delivered. Fusion rate and cloning efficiency during iSCNT cloning of coyotes were not significantly different from those observed during intraspecies cloning of domestic dogs. Using neonatal fibroblasts as donor cells significantly improved the cloning efficiency compared with cloning using adult fibroblast donor cells (P<0.05). The use of domestic dog oocytes in the cloning of coyotes in the present study holds promise for cloning other endangered species in the Canidae family using similar techniques. However, there are still limitations of the iSCNT technology, as demonstrated by births of morphologically abnormal coyotes and the clones' inheritance of maternal domestic dog mitochondrial DNA.

  18. Successful cloning of coyotes through interspecies somatic cell nuclear transfer using domestic dog oocytes.

    PubMed

    Hwang, Insung; Jeong, Yeon Woo; Kim, Joung Joo; Lee, Hyo Jeong; Kang, Mina; Park, Kang Bae; Park, Jung Hwan; Kim, Yeun Wook; Kim, Woo Tae; Shin, Taeyoung; Hyun, Sang Hwan; Jeung, Eui-Bae; Hwang, Woo Suk

    2013-01-01

    Interspecies somatic cell nuclear transfer (iSCNT) is an emerging assisted reproductive technology (ART) for preserving Nature's diversity. The scarcity of oocytes from some species makes utilisation of readily available oocytes inevitable. In the present study, we describe the successful cloning of coyotes (Canis latrans) through iSCNT using oocytes from domestic dogs (Canis lupus familiaris or dingo). Transfer of 320 interspecies-reconstructed embryos into 22 domestic dog recipients resulted in six pregnancies, from which eight viable offspring were delivered. Fusion rate and cloning efficiency during iSCNT cloning of coyotes were not significantly different from those observed during intraspecies cloning of domestic dogs. Using neonatal fibroblasts as donor cells significantly improved the cloning efficiency compared with cloning using adult fibroblast donor cells (P<0.05). The use of domestic dog oocytes in the cloning of coyotes in the present study holds promise for cloning other endangered species in the Canidae family using similar techniques. However, there are still limitations of the iSCNT technology, as demonstrated by births of morphologically abnormal coyotes and the clones' inheritance of maternal domestic dog mitochondrial DNA. PMID:23217630

  19. The use of marsupial x eutherian somatic cell hybrids to study marsupial cell surface antigens.

    PubMed

    Sykes, P J; Hope, R M

    1978-12-01

    Buck and Bodmer (1976) have developed a technique for identifying an antigen on the surface of human x mouse somatic cell hybrids, specified by a gene on a particular human chromosome. We have successfully adapted this technique to a study of marsupial cell surface antigens. Somatic cell hybrids between Macropus rufus (Marsupialia) lymphocytes and the mouse cell lines PG19 and 1R were injected intraperitoneally into mice of the same inbred strain from which the above cell lines were derived (C57B16J and C3H, respectively). The only identified M. rufus chromosome present in the hybrid cells was the X chromosome. The antisera, after adsorption with PG19 or 1R, were tested using indirect immunofluorescence, against the hybrid cells, and also against sub-clones (derived from hybrids) which had apparently lost the M. rufus X chromosome, or at least its long arm. The results of these tests showed that the absorbed antisera contained reactivity against an M. rufus cell surface antigen (or antigens). The reactions of one of the antisera were most simply interpreted by supposing that it was detecting an M. rufus X-lined antigen(s).

  20. Embryo production and possible species preservation by nuclear transfer of somatic cells isolated from bovine semen.

    PubMed

    Liu, Jie; Westhusin, Mark; Long, Charles; Johnson, Gregory; Burghardt, Robert; Kraemer, Duane

    2010-12-01

    Somatic cells in semen are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important when an animal has died and the only viable genetic material available is frozen semen. Usefulness of somatic cells obtained from fresh (cultured) and frozen (isolated, not cultured) bovine semen for nuclear transfer was evaluated. Twelve ejaculates were collected from nine bulls representing three breeds: Charolais, Brahman, and crossbred Rodeo bull. All samples were processed immediately and cell growth was obtained from seven of the twelve ejaculates (58.3%). Cells from three bulls (with the best growth rates) were evaluated by optical microscopy and used in cloning experiments. In culture, these cells exhibited classic epithelial morphology and expressed cytokeratin and vimentin, indicating they were of epithelial origin. When cells from the three bulls were used as donor cells, 15.9% (18/113), 34.5% (29/84), and 14.4% (13/90) of the fused embryos developed into blastocysts, respectively. Of the blastocyst stage embryos, 38.9% (7/18), 72.4% (21/29), and 61.5% (8/13) hatched, respectively. Somatic cells isolated (not cultured) from frozen bovine semen were also used in the cloning experiments. Although cleavage occurred, no compact morulae or blastocysts were obtained. In conclusion, epithelial cell growth was obtained from fresh bovine ejaculates with relatively high efficiency. Somatic cells from semen can be used as nucleus donors to produce cloned blastocyst-stage embryos.

  1. Development of buffalo (Bubalus bubalis) embryonic stem cell lines from somatic cell nuclear transferred blastocysts.

    PubMed

    Shah, Syed Mohmad; Saini, Neha; Ashraf, Syma; Singh, Manoj K; Manik, Radheysham; Singla, Suresh K; Palta, Prabhat; Chauhan, Manmohan Singh

    2015-11-01

    We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES) cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions. PMID:26987926

  2. In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.

    PubMed

    Gerger, R P C; Ribeiro, E S; Forell, F; Bertolini, L R; Rodrigues, J L; Ambrósio, C E; Miglino, M A; Mezzalira, A; Bertolini, M

    2010-02-18

    The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

  3. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells.

    PubMed

    Abyzov, Alexej; Mariani, Jessica; Palejev, Dean; Zhang, Ying; Haney, Michael Seamus; Tomasini, Livia; Ferrandino, Anthony F; Rosenberg Belmaker, Lior A; Szekely, Anna; Wilson, Michael; Kocabas, Arif; Calixto, Nathaniel E; Grigorenko, Elena L; Huttner, Anita; Chawarska, Katarzyna; Weissman, Sherman; Urban, Alexander Eckehart; Gerstein, Mark; Vaccarino, Flora M

    2012-12-20

    Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has been suspected of causing de novo copy number variation. To explore this issue, here we perform a whole-genome and transcriptome analysis of 20 human iPSC lines derived from the primary skin fibroblasts of seven individuals using next-generation sequencing. We find that, on average, an iPSC line manifests two copy number variants (CNVs) not apparent in the fibroblasts from which the iPSC was derived. Using PCR and digital droplet PCR, we show that at least 50% of those CNVs are present as low-frequency somatic genomic variants in parental fibroblasts (that is, the fibroblasts from which each corresponding human iPSC line is derived), and are manifested in iPSC lines owing to their clonal origin. Hence, reprogramming does not necessarily lead to de novo CNVs in iPSCs, because most of the line-manifested CNVs reflect somatic mosaicism in the human skin. Moreover, our findings demonstrate that clonal expansion, and iPSC lines in particular, can be used as a discovery tool to reliably detect low-frequency CNVs in the tissue of origin. Overall, we estimate that approximately 30% of the fibroblast cells have somatic CNVs in their genomes, suggesting widespread somatic mosaicism in the human body. Our study paves the way to understanding the fundamental question of the extent to which cells of the human body normally acquire structural alterations in their DNA post-zygotically.

  4. Reprogramming resistant genes: in-depth comparison of gene expressions among iPS, ES, and somatic cells

    PubMed Central

    Polouliakh, Natalia

    2012-01-01

    Transcription factor-based reprogramming reverts adult cells to an embryonic state, yielding potential for generating different tissue types. However, recent reports indicated the substantial differences in pattern of gene expression between induced pluripotent stem (iPS) cells and embryonic stem cells (ESC). In this study, we compare gene expression signatures of different iPS and ES cell lines and relate expression profiles of differently expressed genes to their expression status in somatic cells. As a result, we discovered that genes resistant to reprogramming comprise two major clusters, which are reprogramming dependent “Induced Genes” and somatic origin “Inherited Genes,” both exhibiting preferences in methylation marks. Closer look into the Induced Genes by means of the transcription regulation analysis predicted several groups of genes with various roles in reprogramming and transcription factor DNA binding model. We believe that our results are a helpful source for biologists for further improvement of iPS cell technology. PMID:23386832

  5. Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes.

    PubMed Central

    Klein, U.; Klein, G.; Ehlin-Henriksson, B.; Rajewsky, K.; Küppers, R.

    1995-01-01

    BACKGROUND: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells and their descendents. MATERIALS AND METHODS: Rearranged V kappa region genes from 10 kappa-expressing sporadic and endemic BL-derived cell lines (9 IgM and 1 IgG positive) and three kappa-expressing endemic BL biopsy specimens were amplified by polymerase chain reaction and sequenced. In addition, VH region gene sequences from these cell lines were determined. RESULTS: All BL cell lines and the three biopsy specimens carried somatically mutated V region genes. The average mutation frequency of rearranged V kappa genes from eight BL cell lines established from sporadic BL was 1.8%. A higher frequency (6%) was found in five endemic cases (three biopsy specimens and two BL cell lines). CONCLUSIONS: The detection of somatic mutations in the rearranged V region genes suggests that both sporadic and endemic BL represent a B-cell malignancy originating from germinal center B cells or their descendants. Interestingly, the mutation frequency detected in sporadic BL is in a range similar to that characteristic for IgM-expressing B cells in the human peripheral blood and for mu chain-expressing germinal center B cells, whereas the mutation frequency found in endemic BL is significantly higher. PMID:8529116

  6. Usp16 contributes to somatic stem-cell defects in Down's syndrome.

    PubMed

    Adorno, Maddalena; Sikandar, Shaheen; Mitra, Siddhartha S; Kuo, Angera; Nicolis Di Robilant, Benedetta; Haro-Acosta, Veronica; Ouadah, Youcef; Quarta, Marco; Rodriguez, Jacqueline; Qian, Dalong; Reddy, Vadiyala M; Cheshier, Samuel; Garner, Craig C; Clarke, Michael F

    2013-09-19

    Down's syndrome results from full or partial trisomy of chromosome 21. However, the consequences of the underlying gene-dosage imbalance on adult tissues remain poorly understood. Here we show that in Ts65Dn mice, which are trisomic for 132 genes homologous to genes on human chromosome 21, triplication of Usp16 reduces the self-renewal of haematopoietic stem cells and the expansion of mammary epithelial cells, neural progenitors and fibroblasts. In addition, Usp16 is associated with decreased ubiquitination of Cdkn2a and accelerated senescence in Ts65Dn fibroblasts. Usp16 can remove ubiquitin from histone H2A on lysine 119, a critical mark for the maintenance of multiple somatic tissues. Downregulation of Usp16, either by mutation of a single normal Usp16 allele or by short interfering RNAs, largely rescues all of these defects. Furthermore, in human tissues overexpression of USP16 reduces the expansion of normal fibroblasts and postnatal neural progenitors, whereas downregulation of USP16 partially rescues the proliferation defects of Down's syndrome fibroblasts. Taken together, these results suggest that USP16 has an important role in antagonizing the self-renewal and/or senescence pathways in Down's syndrome and could serve as an attractive target to ameliorate some of the associated pathologies.

  7. Usp16 contributes to somatic stem cell defects in Down syndrome

    PubMed Central

    Adorno, Maddalena; Sikandar, Shaheen; Mitra, Siddhartha S.; Kuo, Angera; Di Robilant, Benedetta Nicolis; Haro-Acosta, Veronica; Ouadah, Youcef; Quarta, Marco; Rodriguez, Jacqueline; Qian, Dalong; Reddy, Vadiyala M.; Cheshier, Samuel; Garner, Craig C.; Clarke, Michael F.

    2013-01-01

    SUMMARY Down syndrome (DS) results from full or partial trisomy of chromosome 21. However, the consequences of the underlying gene-dosage imbalance on adult tissues remain poorly understood. Here we show that in Ts65Dn mice, trisomic for 132 genes homologous to HSA21, triplication of Usp16 reduces self-renewal of hematopoietic stem cells and expansion of mammary epithelial cells, neural progenitors, and fibroblasts. Moreover, Usp16 is associated with decreased ubiquitination of Cdkn2a and accelerated senescence in Ts65Dn fibroblasts. Usp16 can remove ubiquitin from H2AK119, a critical mark for the maintenance of multiple somatic tissues. Downregulation of Usp16, either by mutation of a single normal USP16 allele or by shRNAs, largely rescues all these defects. Furthermore, in human tissues overexpression of USP16 reduces the expansion of normal fibroblasts and post-natal neural progenitors while downregulation of USP16 partially rescues the proliferation defects of DS fibroblasts. Taken together, these results suggest that USP16 plays an important role in antagonizing the self-renewal and/or senescence pathways in Down syndrome and could serve as an attractive target to ameliorate some of the associated pathologies. PMID:24025767

  8. No differences in sheep somatic cell nuclear transfer outcomes using serum-starved or actively growing donor granulosa cells.

    PubMed

    Peura, T T; Hartwich, K M; Hamilton, H M; Walker, S K

    2003-01-01

    The aim of this study was to compare serum-starved and non-starved donor cells in sheep nuclear transfer with a special emphasis on cloning outcomes. Sheep oocytes, derived either in vivo or in vitro, were fused with cultured serum-starved or actively growing adult granulosa cells. Resulting blastocysts were transferred to recipients fresh or after vitrification, and subsequent pregnancies followed to term. Donor cell treatment did not significantly affect preimplantation development, pregnancy rates, fetal loss or neonate survival rates. Of 22 lambs born, ten survived the immediate perinatal period but all succumbed at various timepoints within the first few weeks of life. The results of the study suggest that the use of serum-starved cells offers no advantages or disadvantages to cloning outcomes. Neither were significant differences in outcomes observed when using either in vivo- or in vitro-derived oocytes or embryos transferred fresh or after vitrification. Yet, these results continue to highlight problems associated with somatic cell cloning as indicated by offspring mortality. It remains unclear whether the high offspring mortality in the current study was related to species, associated with the cell lines used or the result of other causes. PMID:12921702

  9. Flow cytometric cell cycle analysis of somatic cells primary cultures established for bovine cloning.

    PubMed

    Katska, L; Bochenek, M; Kania, G; Ryñska, B; Smorag, Z

    2002-12-01

    An important factor governing developmental rates of somatic cloned embryos is the phase of the cell cycle of donor nuclei. The aim of this experiment was to investigate the distribution of cell cycle phases in bovine cumulus and fibroblast cells cultured using routine treatment, and under cell cycle-arresting treatments. The highest percentages of cumulus cells in the G0 + G1 stage were observed in uncultured, frozen/thawed cells originating from immature oocytes (79.8 +/- 2.2%), fresh and frozen/thawed cells from in vitro matured oocytes (84.1 +/- 6.2 and 77.8 +/- 5.7%, respectively), and in cycling cells (72.7 +/- 16.3 and 78.4 +/- 11.2%, respectively for cumulus cells from immature and in vitro matured oocytes). Serum starvation of cumulus cultures markedly decreased percentages of cells in G0 + G1, and prolonged starvation significantly increased (P < 0.05) percentages of cells in G2 + M phase. Culture of cumulus cells to confluency did not increase percentages of cells in G0 + G1. Contrary to findings in cumulus cells, significantly higher percentages of cells in G0 + G1 were apparent when fibroblast cells were cultured to confluency or serum starved, and significantly increased (P < 0.01) as the starvation period was prolonged. It is concluded that for particular cell types specific strategies should be used to attain improvements in the efficiency of cloning procedures.

  10. Whole-exome sequencing identifies a somatic missense mutation of NBN in clear cell sarcoma of the salivary gland.

    PubMed

    Zhang, Lei; Jia, Zhen; Mao, Fengbiao; Shi, Yueyi; Bu, Rong Fa; Zhang, Baorong

    2016-06-01

    Clear cell sarcoma (CCS) is a rare, low-grade carcinoma commonly located in the distal extremities of young adults involving tendons and aponeuroses. CCS is characterized by its poor prognosis due to late diagnosis, multiple local recurrence, propensity to late metastases, and a high rate of tumor-related mortality. The genetic cause for CCS is thought to be EWSR1 gene translocation. However, CCS lacking a translocation may have other, as yet uncharacterized, genetic mutations that can cause the same pathological effect. A combination of whole‑exome sequencing and Sanger sequencing of cancer tissue and venous blood from a patient diagnosed with CCS of the salivary gland revealed a somatic missense mutation, c.1061C>T (p.P354L), in exon 9 of the Nibrin gene (NBN). This somatic missense mutation led to the conversion of proline to leucine (p.P354L), resulting in deleterious effects for the NBN protein. Multiple-sequence alignments showed that codon 354, where the mutation (c.1061C>T) occurs, is located within a phylogenetically conserved region. In conclusion, we here report a somatic missense mutation c.1061C>T (p.P354L) in the NBN gene in a patient with CCS lacking an EWSR1-ATF1 fusion. Our findings broaden the genotypic spectrum of CCS and provide new molecular insight that should prove useful in the future clinical genetic diagnosis of CCS. PMID:27109316

  11. Guidelines for monitoring bulk tank milk somatic cell and bacterial counts.

    PubMed

    Jayarao, B M; Pillai, S R; Sawant, A A; Wolfgang, D R; Hegde, N V

    2004-10-01

    This study was conducted to establish guidelines for monitoring bulk tank milk somatic cell count and bacterial counts, and to understand the relationship between different bacterial groups that occur in bulk tank milk. One hundred twenty-six dairy farms in 14 counties of Pennsylvania participated, each providing one bulk tank milk sample every 15 d for 2 mo. The 4 bulk tank milk samples from each farm were examined for bulk tank somatic cell count and bacterial counts including standard plate count, preliminary incubation count, laboratory pasteurization count, coagulase-negative staphylococcal count, environmental streptococcal count, coliform count, and gram-negative noncoliform count. The milk samples were also examined for presence of Staphylococcus aureus, Streptococcus agalactiae, and Mycoplasma. The bacterial counts of 4 bulk tank milk samples examined over an 8-wk period were averaged and expressed as mean bacterial count per milliliter. The study revealed that an increase in the frequency of isolation of Staphylococcus aureus and Streptococcus agalactiae was significantly associated with an increased bulk tank somatic cell count. Paired correlation analysis showed that there was low correlation between different bacterial counts. Bulk tank milk with low (<5000 cfu/mL) standard plate count also had a significantly low level of mean bulk tank somatic cell count (<200,000 cells/mL), preliminary incubation count (<10,000 cfu/mL), laboratory pasteurization count (<100 cfu/mL), coagulase-negative staphylococci and environmental streptococcal counts (<500 cfu/mL), and noncoliform count (<200 cfu/mL). Coliform count was less likely to be associated with somatic cell or other bacterial counts. Herd size and farm management practices had considerable influence on somatic cell and bacterial counts in bulk tank milk. Dairy herds that used automatic milking detachers, sand as bedding material, dip cups for teat dipping instead of spraying, and practiced pre

  12. Single-Cell Genetic Analysis Using Automated Microfluidics to Resolve Somatic Mosaicism

    PubMed Central

    Wang, Jing; Weaver, Lesley S.; Gonzales, Michael L.; Sun, Gang; Unger, Marc A.; Ramakrishnan, Ramesh

    2015-01-01

    Somatic mosaicism occurs throughout normal development and contributes to numerous disease etiologies, including tumorigenesis and neurological disorders. Intratumor genetic heterogeneity is inherent to many cancers, creating challenges for effective treatments. Unfortunately, analysis of bulk DNA masks subclonal phylogenetic architectures created by the acquisition and distribution of somatic mutations amongst cells. As a result, single-cell genetic analysis is becoming recognized as vital for accurately characterizing cancers. Despite this, methods for single-cell genetics are lacking. Here we present an automated microfluidic workflow enabling efficient cell capture, lysis, and whole genome amplification (WGA). We find that ~90% of the genome is accessible in single cells with improved uniformity relative to current single-cell WGA methods. Allelic dropout (ADO) rates were limited to 13.75% and variant false discovery rates (SNV FDR) were 4.11x10-6, on average. Application to ER-/PR-/HER2+ breast cancer cells and matched normal controls identified novel mutations that arose in a subpopulation of cells and effectively resolved the segregation of known cancer-related mutations with single-cell resolution. Finally, we demonstrate effective cell classification using mutation profiles with 10X average exome coverage depth per cell. Our data demonstrate an efficient automated microfluidic platform for single-cell WGA that enables the resolution of somatic mutation patterns in single cells. PMID:26302375

  13. A XEN-like State Bridges Somatic Cells to Pluripotency during Chemical Reprogramming.

    PubMed

    Zhao, Yang; Zhao, Ting; Guan, Jingyang; Zhang, Xu; Fu, Yao; Ye, Junqing; Zhu, Jialiang; Meng, Gaofan; Ge, Jian; Yang, Susu; Cheng, Lin; Du, Yaqin; Zhao, Chaoran; Wang, Ting; Su, Linlin; Yang, Weifeng; Deng, Hongkui

    2015-12-17

    Somatic cells can be reprogrammed into pluripotent stem cells (PSCs) by using pure chemicals, providing a different paradigm to study somatic reprogramming. However, the cell fate dynamics and molecular events that occur during the chemical reprogramming process remain unclear. We now show that the chemical reprogramming process requires the early formation of extra-embryonic endoderm (XEN)-like cells and a late transition from XEN-like cells to chemically-induced (Ci)PSCs, a unique route that fundamentally differs from the pathway of transcription factor-induced reprogramming. Moreover, precise manipulation of the cell fate transition in a step-wise manner through the XEN-like state allows us to identify small-molecule boosters and establish a robust chemical reprogramming system with a yield up to 1,000-fold greater than that of the previously reported protocol. These findings demonstrate that chemical reprogramming is a promising approach to manipulate cell fates.

  14. Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors.

    PubMed

    Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R; Greenleaf, William J; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

    2015-07-01

    Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.

  15. mTOR-regulated senescence and autophagy during reprogramming of somatic cells to pluripotency: a roadmap from energy metabolism to stem cell renewal and aging.

    PubMed

    Menendez, Javier A; Vellon, Luciano; Oliveras-Ferraros, Cristina; Cufí, Sílvia; Vazquez-Martin, Alejandro

    2011-11-01

    Molecular controllers of the number and function of tissue stem cells may share common regulatory pathways for the nuclear reprogramming of somatic cells to become induced Pluripotent Stem Cells (iPSCs). If this hypothesis is true, testing the ability of longevity-promoting chemicals to improve reprogramming efficiency may provide a proof-of-concept validation tool for pivotal housekeeping pathways that limit the numerical and/or functional decline of adult stem cells. Reprogramming is a slow, stochastic process due to the complex and apparently unrelated cellular processes that are involved. First, forced expression of the Yamanaka cocktail of stemness factors, OSKM, is a stressful process that activates apoptosis and cellular senescence, which are the two primary barriers to cancer development and somatic reprogramming. Second, the a priori energetic infrastructure of somatic cells appears to be a crucial stochastic feature for optimal successful routing to pluripotency. If longevity-promoting compounds can ablate the drivers and effectors of cellular senescence while concurrently enhancing a bioenergetic shift from somatic oxidative mitochondria toward an alternative ATP-generating glycolytic metabotype, they could maximize the efficiency of somatic reprogramming to pluripotency. Support for this hypothesis is evidenced by recent findings that well-characterized mTOR inhibitors and autophagy activators (e.g., PP242, rapamycin and resveratrol) notably improve the speed and efficiency of iPSC generation. This article reviews the existing research evidence that the most established mTOR inhibitors can notably decelerate the cellular senescence that is imposed by DNA damage-like responses, which are somewhat equivalent to the responses caused by reprogramming factors. These data suggest that fine-tuning mTOR signaling can impact mitochondrial dynamics to segregate mitochondria that are destined for clearance through autophagy, which results in the loss of

  16. A chemical genetics approach for specific differentiation of stem cells to somatic cells: a new promising therapeutical approach.

    PubMed

    Sachinidis, Agapios; Sotiriadou, Isaia; Seelig, Bianca; Berkessel, Albrecht; Hescheler, Jürgen

    2008-01-01

    Cell replacement therapy of severe degenerative diseases such as diabetes, myocardial infarction and Parkinson's disease through transplantation of somatic cells generated from embryonic stem (ES) cells is currently receiving considerable attention for the therapeutic applications. ES cells harvested from the inner cell mass (ICM) of the early embryo, can proliferate indefinitely in vitro while retaining the ability to differentiate into all somatic cells thereby providing an unlimited renewable source of somatic cells. In this context, identifying soluble factors, in particular chemically synthesized small molecules, and signal cascades involved in specific differentiation processes toward a defined tissue specific cell type are crucial for optimizing the generation of somatic cells in vitro for therapeutic approaches. However, experimental models are required allowing rapid and "easy-to-handle" parallel screening of chemical libraries to achieve this goal. Recently, the forward chemical genetic screening strategy has been postulated to screen small molecules in cellular systems for a specific desired phenotypic effect. The current review is focused on the progress of ES cell research in the context of the chemical genetics to identify small molecules promoting specific differentiation of ES cells to desired cell phenotype. Chemical genetics in the context of the cell ES-based cell replacement therapy remains a challenge for the near future for several scientific fields including chemistry, molecular biology, medicinal physics and robotic technologies.

  17. [Mammalian DNA methylation and its roles during the induced re-programming of somatic cells].

    PubMed

    Hongwei, Song; Tiezhu, An; Shanhua, Piao; Chunsheng, Wang

    2014-05-01

    The technology of induced pluripotent stem cell (iPS) provides the possibility to reverse the terminal differentiated cells to pluripotent stem cells, and is therefore of great importance in both the theoretical research of stem cells and regenerative medicine. However, the efficiency of current induced reprogramming methods is extremely low, and the incomplete reprogramming often happens. It has been reported that some epigenetic memory of the somatic cells exists in these incomplete reprogrammed iPS cells, and DNA methylation, as a relative long-term and stable epigenetic modification, is one of the important factors that influence the efficiency of reprogramming and differentiative capacity of iPS cells. Mammalian DNA methylation, which normally appears on the CpG sites, occurs on the fifth carbon atom of the cytosine ring. DNA methylation can modulate the expression of somatic cell specific genes, and pluripotent genes; hence, it plays important roles in the processes of mammalian gene regulation, embryonic development and cell reprogramming. In addition, it has also been found that abnormal DNA methylation may lead to the disorder of genetic imprinting and the inactivation of X chromosome in iPS cells. Therefore, in order to provide a concise guidance of DNA methylation studies in iPS, we mainly review the mechanism, the distribution features of DNA methylation, and its roles in induced reprogramming of somatic cells. PMID:24846992

  18. Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2.

    PubMed

    Doege, Claudia A; Inoue, Keiichi; Yamashita, Toru; Rhee, David B; Travis, Skylar; Fujita, Ryousuke; Guarnieri, Paolo; Bhagat, Govind; Vanti, William B; Shih, Alan; Levine, Ross L; Nik, Sara; Chen, Emily I; Abeliovich, Asa

    2012-08-30

    Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by using the pluripotency factors Oct4, Sox2, Klf4 and c-Myc (together referred to as OSKM). iPSC reprogramming erases somatic epigenetic signatures—as typified by DNA methylation or histone modification at silent pluripotency loci—and establishes alternative epigenetic marks of embryonic stem cells (ESCs). Here we describe an early and essential stage of somatic cell reprogramming, preceding the induction of transcription at endogenous pluripotency loci such as Nanog and Esrrb. By day 4 after transduction with OSKM, two epigenetic modification factors necessary for iPSC generation, namely poly(ADP-ribose) polymerase-1 (Parp1) and ten-eleven translocation-2 (Tet2), are recruited to the Nanog and Esrrb loci. These epigenetic modification factors seem to have complementary roles in the establishment of early epigenetic marks during somatic cell reprogramming: Parp1 functions in the regulation of 5-methylcytosine (5mC) modification, whereas Tet2 is essential for the early generation of 5-hydroxymethylcytosine (5hmC) by the oxidation of 5mC (refs 3,4). Although 5hmC has been proposed to serve primarily as an intermediate in 5mC demethylation to cytosine in certain contexts, our data, and also studies of Tet2-mutant human tumour cells, argue in favour of a role for 5hmC as an epigenetic mark distinct from 5mC. Consistent with this, Parp1 and Tet2 are each needed for the early establishment of histone modifications that typify an activated chromatin state at pluripotency loci, whereas Parp1 induction further promotes accessibility to the Oct4 reprogramming factor. These findings suggest that Parp1 and Tet2 contribute to an epigenetic program that directs subsequent transcriptional induction at pluripotency loci during somatic cell reprogramming. PMID:22902501

  19. A Comparative View on Human Somatic Cell Sources for iPSC Generation

    PubMed Central

    2014-01-01

    The breakthrough of reprogramming human somatic cells was achieved in 2006 by the work of Yamanaka and Takahashi. From this point, fibroblasts are the most commonly used primary somatic cell type for the generation of induced pluripotent stem cells (iPSCs). Various characteristics of fibroblasts supported their utilization for the groundbreaking experiments of iPSC generation. One major advantage is the high availability of fibroblasts which can be easily isolated from skin biopsies. Furthermore, their cultivation, propagation, and cryoconservation properties are uncomplicated with respect to nutritional requirements and viability in culture. However, the required skin biopsy remains an invasive approach, representing a major drawback for using fibroblasts as the starting material. More and more studies appeared over the last years, describing the reprogramming of other human somatic cell types. Cells isolated from blood samples or urine, as well as more unexpected cell types, like pancreatic islet beta cells, synovial cells, or mesenchymal stromal cells from wisdom teeth, show promising characteristics for a reprogramming strategy. Here, we want to highlight the advantages of keratinocytes from human plucked hair as a widely usable, noninvasive harvesting method for primary material in comparison with other commonly used cell types. PMID:25431601

  20. Health status and productive performance of somatic cell cloned cattle and their offspring produced in Japan.

    PubMed

    Watanabe, Shinya; Nagai, Takashi

    2008-02-01

    Since the first somatic cell cloned calves were born in Japan in 1998, more than 500 cloned cattle have been produced by somatic cell nuclear transfer and many studies concerning cloned cattle and their offspring have been conducted in this country. However, most of the results have been published in Japanese; thus, the data produced in this country is not well utilized by researchers throughout the world. This article reviews the 65 reports produced by Japanese researchers (62 written in Japanese and 3 written in English), which employed 171 clones and 32 offspring, and categorizes them according to the following 7 categories: (1) genetic similarities and muzzle prints, (2) hematology and clinical chemistry findings, (3) pathology, (4) growth performance, (5) reproductive performance, (6) meat production performance and (7) milk production performance. No remarkable differences in health status or reproductive performance were found among conventionally bred cattle, somatic cell cloned cattle surviving to adulthood and offspring of somatic cell cloned cattle. Similarities in growth performance and meat quality were observed between nuclear donor cattle and their clones. The growth curves of the offspring resembled those of their full siblings.

  1. Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos

    SciTech Connect

    Fang, Zhen F.; Gai, Hui; Huang, You Z.; Li, Shan G.; Chen, Xue J.; Shi, Jian J.; Wu, Li; Liu, Ailian; Xu, Ping; Sheng, Hui Z. . E-mail: hzsheng2003@yahoo.com

    2006-11-01

    Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ES cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines.

  2. Gravity separation of fat, somatic cells, and bacteria in raw and pasteurized milks.

    PubMed

    Caplan, Z; Melilli, C; Barbano, D M

    2013-04-01

    The objective of experiment 1 was to determine if the extent of gravity separation of milk fat, bacteria, and somatic cells is influenced by the time and temperature of gravity separation or the level of contaminating bacteria present in the raw milk. The objective of experiment 2 was to determine if different temperatures of milk heat treatment affected the gravity separation of milk fat, bacteria, and somatic cells. In raw milk, fat, bacteria, and somatic cells rose to the top of columns during gravity separation. About 50 to 80% of the fat and bacteria were present in the top 8% of the milk after gravity separation of raw milk. Gravity separation for 7h at 12°C or for 22h at 4°C produced equivalent separation of fat, bacteria, and somatic cells. The completeness of gravity separation of fat was influenced by the level of bacteria in the milk before separation. Milk with a high bacterial count had less (about 50 to 55%) gravity separation of fat than milk with low bacteria count (about 80%) in 22h at 4°C. Gravity separation caused fat, bacteria, and somatic cells to rise to the top of columns for raw whole milk and high temperature, short-time pasteurized (72.6°C, 25s) whole milk. Pasteurization at ≥76.9°C for 25s prevented all 3 components from rising, possibly due to denaturation of native bovine immunoglobulins that normally associate with fat, bacteria, and somatic cells during gravity separation. Gravity separation can be used to produce reduced-fat milk with decreased bacterial and somatic cell counts, and may be a critical factor in the history of safe and unique traditional Italian hard cheeses produced from gravity-separated raw milk. A better understanding of the mechanism of this natural process could lead to the development of new nonthermal thermal technology (that does not involve heating the milk to high temperatures) to remove bacteria and spores from milk or other liquids.

  3. Gene transfer into mammalian somatic cells in vivo.

    PubMed

    Yang, N S

    1992-01-01

    Direct gene transfer into mammalian somatic tissues in vivo is a developing technology with potential application for human gene therapy. During the past 2 years, extensive progress and numerous breakthroughs have been made in this area of research. Genetically engineered retroviral vectors have been used successfully to infect live animals, effecting foreign gene expression in liver, blood vessels, and mammary tissues. Recombinant adenovirus and herpes simplex virus vectors have been utilized effectively for in vivo gene transfer into lung and brain tissues, respectively. Direct injection or particle bombardment of DNA has been demonstrated to provide a physical means for in situ gene transfer, while carrier-mediated DNA delivery techniques have been extended to target specific organs for gene expression. These technological developments in conjunction with the initiation of the NIH human gene therapy trials have marked a milestone in developing new medical treatments for various genetic diseases and cancer. Various in vivo gene transfer techniques should also provide new tools for basic research in molecular and developmental genetics.

  4. Induced DNA damage by dental resin monomers in somatic cells.

    PubMed

    Arossi, Guilherme Anziliero; Lehmann, Mauricio; Dihl, Rafael Rodrigues; Reguly, Maria Luiza; de Andrade, Heloisa Helena Rodrigues

    2010-02-01

    The present in vivo study investigated the genotoxicity of four dental resin monomers: triethyleneglycoldimethacrylate (TEGDMA), hydroxyethylmethacrylate (HEMA), urethanedimethacrylate (UDMA) and bisphenol A-glycidylmethacrylate (BisGMA). The Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster was applied to analyse their genotoxicity expressed as homologous mitotic recombination, point and chromosomal mutation. SMART detects the loss of heterozygosity of marker genes expressed phenotypically on the fly's wings. This fruit fly has an extensive genetic homology to mammalians, which makes it a suitable model organism for genotoxic investigations. The present findings provide evidence that the mechanistic basis underlying the genotoxicity of UDMA and TEGDMA is related to homologous recombination and gene/chromosomal mutation. A genotoxic pattern can correspondingly be discerned for both UDMA and TEGDMA: their genotoxicity is attributed respectively to 49% and 44% of mitotic recombination, as well as 51% and 56% of mutational events, including point and chromosomal alterations. The monomer UDMA is 1.6 times more active than TEGDMA to induce mutant clones per treatment unit. BisGMA and HEMA had no statistically significant effect on total spot frequencies - suggesting no genotoxic action in the SMART assay. The clinical significance of these observations has to be interpreted for data obtained in other bioassays.

  5. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    SciTech Connect

    Watanabe, Masahito; Umeyama, Kazuhiro; Matsunari, Hitomi; Takayanagi, Shuko; Haruyama, Erika; Nakano, Kazuaki; Fujiwara, Tsukasa; Ikezawa, Yuka; Nakauchi, Hiromitsu; and others

    2010-11-05

    Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  6. Calcineurin-NFAT Signaling Controls Somatic Cell Reprogramming in a Stage-Dependent Manner.

    PubMed

    Sun, Ming; Liao, Bing; Tao, Yu; Chen, Hao; Xiao, Feng; Gu, Junjie; Gao, Shaorong; Jin, Ying

    2016-05-01

    Calcineurin-NFAT signaling is critical for early lineage specification of mouse embryonic stem cells and early embryos. However, its roles in somatic cell reprogramming remain unknown. Here, we report that calcineurin-NFAT signaling has a dynamic activity and plays diverse roles at different stages of reprogramming. At the early stage, calcineurin-NFAT signaling is transiently activated and its activation is required for successful reprogramming. However, at the late stage of reprogramming, activation of calcineurin-NFAT signaling becomes a barrier for reprogramming and its inactivation is critical for successful induction of pluripotency. Mechanistically, calcineurin-NFAT signaling contributes to the reprogramming through regulating multiple early events during reprogramming, including mesenchymal to epithelial transition (MET), cell adhesion and emergence of SSEA1(+) intermediate cells. Collectively, this study reveals for the first time the important roles of calcineurin-NFAT signaling during somatic cell reprogramming and provides new insights into the molecular regulation of reprogramming.

  7. Nonimmunogenic radiation-induced lymphoma: immunity induction by a somatic cell hybrid

    SciTech Connect

    Yefenof, E.; Goldapfel, M.; Ber, R.

    1982-05-01

    The cell line designated PIR-2 is a nonimmunogenic X-ray-induced thymoma of C57BL/6 origin that is unable to induce antitumor immunity in syngeneic lymphocytes in vitro and in mice in vivo. Fusion of PIR-2 with an allogeneic universal fuser A9HT (clone 3c) resulted in the establishment of a somatic cell hybrid designated A9/PIR. C57BL/6 lymphocytes sensitized in vitro with A9/PIR could lyse parental PIR-2 cells, as well as other syngeneic tumors. However, immunization of mice with the hybrid significantly enhanced PIR-2 tumor takes while it partially protected the animals against a challenge with unrelated syngeneic tumors. The results imply that somatic cell hybridization can increase the immunogenicity of an otherwise nonimmunogenic tumor. However, in view of the enhancing effects of hybrid preimmunization on parental tumor cell growth, the possible application of this approach for immunotherapy is questionable.

  8. Induced malignant genome reprogramming in somatic cells by testis-specific factors.

    PubMed

    Wang, Jin; Emadali, Anouk; Le Bescont, Aurore; Callanan, Mary; Rousseaux, Sophie; Khochbin, Saadi

    2011-01-01

    Germline cell differentiation is controlled by a specific set of genes whose expression is tightly locked into the repressed state in somatic cells. Large-scale epigenome alterations, now evidenced in nearly all cancers, lead to aberrant activation of these normally silenced genes, as attested by the many reports describing the expression of testis-specific factors, known as cancer-testis genes, in various cancer cells. Here, based on the literature, we argue that off-context activity of some of the testis-specific epigenome regulators can reprogram the somatic cell epigenome toward a malignant state by favoring self-renewal and sustaining cell proliferation under stressful conditions, thereby constituting a major oncogenic mechanism.

  9. Reprogramming adult cells during organ regeneration in forest species

    PubMed Central

    Abarca, Dolores

    2009-01-01

    The possibility of regenerating whole plants from somatic differentiated cells emphasizes the plasticity of plant development. Cell-type respecification during regeneration can be induced in adult tissues as a consequence of injuries, changes in external or internal stimuli or changes in positional information. However, in many plant species, switching the developmental program of adult cells prior to organ regeneration is difficult, especially in forest species. Besides its impact on forest productivity, basic information on the flexibility of cell differentiation is necessary for a comprehensive understanding of the epigenetic control of cell differentiation and plant development. Studies of reprogramming adult cells in terms of regulative expression changes of selected genes will be of great interest to unveil basic mechanisms regulating cellular plasticity. PMID:19820297

  10. The evolutionary origin of somatic cells under the dirty work hypothesis.

    PubMed

    Goldsby, Heather J; Knoester, David B; Ofria, Charles; Kerr, Benjamin

    2014-05-01

    Reproductive division of labor is a hallmark of multicellular organisms. However, the evolutionary pressures that give rise to delineated germ and somatic cells remain unclear. Here we propose a hypothesis that the mutagenic consequences associated with performing metabolic work favor such differentiation. We present evidence in support of this hypothesis gathered using a computational form of experimental evolution. Our digital organisms begin each experiment as undifferentiated multicellular individuals, and can evolve computational functions that improve their rate of reproduction. When such functions are associated with moderate mutagenic effects, we observe the evolution of reproductive division of labor within our multicellular organisms. Specifically, a fraction of the cells remove themselves from consideration as propagules for multicellular offspring, while simultaneously performing a disproportionately large amount of mutagenic work, and are thus classified as soma. As a consequence, other cells are able to take on the role of germ, remaining quiescent and thus protecting their genetic information. We analyze the lineages of multicellular organisms that successfully differentiate and discover that they display unforeseen evolutionary trajectories: cells first exhibit developmental patterns that concentrate metabolic work into a subset of germ cells (which we call "pseudo-somatic cells") and later evolve to eliminate the reproductive potential of these cells and thus convert them to actual soma. We also demonstrate that the evolution of somatic cells enables phenotypic strategies that are otherwise not easily accessible to undifferentiated organisms, though expression of these new phenotypic traits typically includes negative side effects such as aging.

  11. Differential nanoreprotoxicity of silver nanoparticles in male somatic cells and spermatogonial stem cells

    PubMed Central

    Zhang, Xi-Feng; Choi, Yun-Jung; Han, Jae Woong; Kim, Eunsu; Park, Jung Hyun; Gurunathan, Sangiliyandi; Kim, Jin-Hoi

    2015-01-01

    Background Silver nanoparticles (AgNPs) possess unique physical, chemical, and biological properties. AgNPs have been increasingly used as anticancer, antiangiogenic, and antibacterial agents for the treatment of bacterial infections in open wounds as well as in ointments, bandages, and wound dressings. The present study aimed to investigate the effects of two different sizes of AgNPs (10 nm and 20 nm) in male somatic Leydig (TM3) and Sertoli (TM4) cells and spermatogonial stem cells (SSCs). Methods Here, we demonstrate a green and simple method for the synthesis of AgNPs using Bacillus cereus culture supernatants. The synthesized AgNPs were characterized using ultraviolet and visible absorption spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy (TEM). The toxicity of the synthesized AgNPs was evaluated by the effects on cell viability, metabolic activity, oxidative stress, apoptosis, and expression of genes encoding steroidogenic and tight junction proteins. Results AgNPs inhibited the viability and proliferation of TM3 and TM4 cells in a dose- and size-dependent manner by damaging cell membranes and inducing the generation of reactive oxygen species, which in turn affected SSC growth on TM3 and TM4 as feeder cells. Small AgNPs (10 nm) were more cytotoxic than medium-sized nanoparticles (20 nm). TEM revealed the presence of AgNPs in the cell cytoplasm and nucleus, and detected mitochondrial damage and enhanced formation of autosomes and autolysosomes in the AgNP-treated cells. Flow cytometry analysis using Annexin V/propidium iodide staining showed massive cell death by apoptosis or necrosis. Real-time polymerase chain reaction and western blot analyses indicated that in TM3 and TM4 cells, AgNPs activated the p53, p38, and pErk1/2 signaling pathways and significantly downregulated the expression of genes related to testosterone synthesis (TM3) and tight junctions (TM4). Furthermore, the exposure of TM3

  12. Culture and selection of somatic hybrids using an auxotrophic cell line.

    PubMed

    Hein, T; Przewoźny, T; Schieder, O

    1983-01-01

    Protoplast fusions between Nicotiana tabacum and N. paniculata and between N. tabacum and N. sylvestris were obtained by polyethylene glycol and Ca(NO3)2 treatment. The protoplasts of one parent originated from cell suspensions, while the protoplasts of the other originated from leaf mesophyll. The heterokaryons were detectable by their intermediate phenotype, namely the green chloroplasts from mesophyll and the dense cytoplasm from suspension cells. They were isolated with micropipettes immediately after fusion using a micromanipulator and were transferred into a protoplast suspension of an auxotrophic cell line serving as a nursery. This mutant is not able to utilize nitrate and had to be supplemented with amino acids. The somatic hybrids were selected by a stepwise reduction of the supplements, which caused the death of the mutant cell colonies, while the autotrophic somatic hybrids continued to grow. The hybrid character of the selected colonies was confirmed by isoenzyme investigations.

  13. Analytical evaluation for somatic mutation detection in circulating tumor cells isolated using a lateral magnetophoretic microseparator.

    PubMed

    Cho, Hyungseok; Kim, Jinho; Han, Song-I; Han, Ki-Ho

    2016-10-01

    CTCs are currently in the spotlight because provide comprehensive genetic information that enables monitoring of the evolution of cancer and selection of appropriate therapeutic strategies that cannot be obtained from a single-site tumor biopsy. Despite their importance, current techniques for isolating CTCs are limited in terms of their ability to yield high-quality CTCs from peripheral blood for use in profiling cancer genetic mutations by DNA sequencing technologies. This paper introduces a lateral magnetophoretic microseparator (the 'CTC-μChip') for isolating highly pure CTCs from blood, which facilitates the detection of somatic mutations in isolated CTCs. To isolate CTCs from peripheral blood, nucleated cells were first prepared by red blood cell lysis. Then, CTCs were isolated from nucleated cells within 30 min using the CTC-μChip. Analytical evaluation using 5 mL blood samples spiked with 5-50 MCF7 breast cancer cells demonstrated that the average recovery rate of the CTC-μChip was 99.08 %. The average number of residual white blood cells (WBCs) in isolated samples was 53, meaning that the WBC depletion rate is 472,000-fold (5.67 log), assuming that blood contains 5 × 10(6) WBCs per milliliter. The isolated MCF7 cells had a purity of 6.9 - 67.9 %, depending on the spiked MCF7 concentration. Using next-generation sequencing technology, heterozygous somatic mutations (PIK3CA and APC) of MCF7 cells were evaluated in the isolated samples. The results showed that somatic mutations could be detected in as few as two MCF7 cells per milliliter of blood, indicating that the CTC-μChip facilitates the detection of somatic variants in CTCs. PMID:27628059

  14. Magnetofection of human somatic cells with magnetite and cobalt ferrospinel nanoparticles.

    PubMed

    Sukoyan, M A; Khrapov, E A; Voronina, E N; Boyarskikh, U A; Gubanov, A I; Itin, V I; Magaeva, A A; Nayden, E P; Terekhova, O G; Filipenko, M L

    2013-03-01

    Superparamagnetic nanoparticles varying by their chemical composition and synthesis method were used to transfer DNA into somatic cells under the influence of constant magnetic field (method of magnetofection). Magnetite particles obtained by mechanochemical synthesis ensured higher expression of the marker gene GFP (evaluated by fluorescence intensity of the cell lysate) then particles of ferric oxide obtained by chemical co-precipitation and cobalt ferrospinel particles obtained by the mechanochemical method.

  15. Somatic embryogenesis in Arabidopsis thaliana is facilitated by mutations in genes repressing meristematic cell divisions.

    PubMed Central

    Mordhorst, A P; Voerman, K J; Hartog, M V; Meijer, E A; van Went, J; Koornneef, M; de Vries, S C

    1998-01-01

    Embryogenesis in plants can commence from cells other than the fertilized egg cell. Embryogenesis initiated from somatic cells in vitro is an attractive system for studying early embryonic stages when they are accessible to experimental manipulation. Somatic embryogenesis in Arabidopsis offers the additional advantage that many zygotic embryo mutants can be studied under in vitro conditions. Two systems are available. The first employs immature zygotic embryos as starting material, yielding continuously growing embryogenic cultures in liquid medium. This is possible in at least 11 ecotypes. A second, more efficient and reproducible system, employing the primordia timing mutant (pt allelic to hpt, cop2, and amp1), was established. A significant advantage of the pt mutant is that intact seeds, germinated in 2,4-dichlorophenoxyacetic acid (2, 4-D) containing liquid medium, give rise to stable embryonic cell cultures, circumventing tedious hand dissection of immature zygotic embryos. pt zygotic embryos are first distinguishable from wild type at early heart stage by a broader embryonic shoot apical meristem (SAM). In culture, embryogenic clusters originate from the enlarged SAMs. pt somatic embryos had all characteristic embryo pattern elements seen in zygotic embryos, but with higher and more variable numbers of cells. Embryogenic cell cultures were also established from seedling, of other mutants with enlarged SAMs, such as clavata (clv). pt clv double mutants showed additive effects on SAM size and an even higher frequency of seedlings producing embryogenic cell lines. pt clv double mutant plants had very short fasciated inflorescence stems and additive effects on the number of rosette leaves. This suggests that the PT and CLV genes act in independent pathways that control SAM size. An increased population of noncommitted SAM cells may be responsible for facilitated establishment of somatic embryogenesis in Arabidopsis. PMID:9611173

  16. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

    SciTech Connect

    Yue, Xiao-shan; Fujishiro, Masako; Toyoda, Masashi; Akaike, Toshihiro; Ito, Yoshihiro

    2010-04-16

    In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.

  17. Significant clinical, neuropathological and behavioural recovery from acute spinal cord trauma by transplantation of a well-defined somatic stem cell from human umbilical cord blood.

    PubMed

    Schira, Jessica; Gasis, Marcia; Estrada, Veronica; Hendricks, Marion; Schmitz, Christine; Trapp, Thorsten; Kruse, Fabian; Kögler, Gesine; Wernet, Peter; Hartung, Hans-Peter; Müller, Hans Werner

    2012-02-01

    Stem cell therapy is a potential treatment for spinal cord injury and different stem cell types have been grafted into animal models and humans suffering from spinal trauma. Due to inconsistent results, it is still an important and clinically relevant question which stem cell type will prove to be therapeutically effective. Thus far, stem cells of human sources grafted into spinal cord mostly included barely defined heterogeneous mesenchymal stem cell populations derived from bone marrow or umbilical cord blood. Here, we have transplanted a well-defined unrestricted somatic stem cell isolated from human umbilical cord blood into an acute traumatic spinal cord injury of adult immune suppressed rat. Grafting of unrestricted somatic stem cells into the vicinity of a dorsal hemisection injury at thoracic level eight resulted in hepatocyte growth factor-directed migration and accumulation within the lesion area, reduction in lesion size and augmented tissue sparing, enhanced axon regrowth and significant functional locomotor improvement as revealed by three behavioural tasks (open field Basso-Beattie-Bresnahan locomotor score, horizontal ladder walking test and CatWalk gait analysis). To accomplish the beneficial effects, neither neural differentiation nor long-lasting persistence of the grafted human stem cells appears to be required. The secretion of neurite outgrowth-promoting factors in vitro further suggests a paracrine function of unrestricted somatic stem cells in spinal cord injury. Given the highly supportive functional characteristics in spinal cord injury, production in virtually unlimited quantities at GMP grade and lack of ethical concerns, unrestricted somatic stem cells appear to be a highly suitable human stem cell source for clinical application in central nervous system injuries. PMID:21903726

  18. Molecular analysis of chromosomal rearrangements using pulsed field gel electrophoresis and somatic cell hybrids

    SciTech Connect

    Davis, L.M. )

    1991-01-01

    Many human genetic diseases, including some cancers, are characterized by consistent chromosome abnormalities, such as deletions and translocations. Analyses of these mutations often prove crucial to the eventual cloning and characterization of the gene(s) responsible for the disease. Two methods for analyzing these chromosome abnormalities have been developed in recent years: somatic cell hybridization and pulsed field gel electrophoresis (PFGE). Somatic cell hybridization is a technique for segregating an aberrant chromosome from its normal homologue in a cell derived from an unrelated species, which is usually a rodent. Demonstrations of these analytic techniques are presented, using as an example chromosomal abnormalities involving human chromosome band 11p13, the locus for the Wilms' tumor, aniridia, genitourinary abnormality, and mental retardation (WAGR) syndrome.

  19. Observations on intramammary infection and somatic cell counts in cows treated with recombinant bovine somatotropin.

    PubMed Central

    Lissemore, K D; Leslie, K E; McBride, B W; Burton, J H; Willan, A R; Bateman, K G

    1991-01-01

    Data were collected on udder health variables as part of a study of the effects of recombinant bovine somatotropin on production in lactating dairy cows. Milk samples, obtained at three intervals during the study, were assessed for their somatic cell count and bacteriological culture result. There was an increase in the prevalence of infection at mid-lactation in the 20.6 and 41.2 mg per day treatment groups as compared to the controls. There was no difference detected in the mean cell count between groups from the samples collected pretrial, mid-lactation, or late lactation. However, analysis of the individual cow Dairy Herd Improvement somatic cell count data showed a difference between groups which was most evident in mid-lactation. PMID:1884302

  20. Psychic and Somatic Symptoms of Depression among Young Adults, Institutionalized Aged and Noninstitutionalized Aged.

    ERIC Educational Resources Information Center

    Zemore, Robert; Eames, Nancy

    1979-01-01

    Tested hypothesis that the institutional nature of old-age homes increases depression in the elderly. Results provided no support that the aged are more depressed. Somatic complaints can be indicators of depression in the elderly if normative differences between young and old are taken into account. (Author)

  1. A molecular roadmap of reprogramming somatic cells into iPS cells.

    PubMed

    Polo, Jose M; Anderssen, Endre; Walsh, Ryan M; Schwarz, Benjamin A; Nefzger, Christian M; Lim, Sue Mei; Borkent, Marti; Apostolou, Effie; Alaei, Sara; Cloutier, Jennifer; Bar-Nur, Ori; Cheloufi, Sihem; Stadtfeld, Matthias; Figueroa, Maria Eugenia; Robinton, Daisy; Natesan, Sridaran; Melnick, Ari; Zhu, Jinfang; Ramaswamy, Sridhar; Hochedlinger, Konrad

    2012-12-21

    Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming. PMID:23260147

  2. Counting human somatic cell replications: methylation mirrors endometrial stem cell divisions.

    PubMed

    Kim, Jung Yeon; Tavaré, Simon; Shibata, Darryl

    2005-12-01

    Cell proliferation may be altered in many diseases, but it is uncertain exactly how to measure total numbers of divisions. Although it is impossible to count every division directly, potentially total numbers of stem cell divisions since birth may be inferred from numbers of somatic errors. The idea is that divisions are surreptitiously recorded by random errors that occur during replication. To test this "molecular clock" hypothesis, epigenetic errors encoded in certain methylation patterns were counted in glands from 30 uteri. Endometrial divisions can differ among women because of differences in estrogen exposures or numbers of menstrual cycles. Consistent with an association between mitotic age and methylation, there was an age-related increase in methylation with stable levels after menopause, and significantly less methylation was observed in lean or older multiparous women. Methylation patterns were diverse and more consistent with niche rather than immortal stem cell lineages. There was no evidence for decreased stem cell survival with aging. An ability to count lifetime numbers of stem cell divisions covertly recorded by random replication errors provides new opportunities to link cell proliferation with aging and cancer. PMID:16314580

  3. Interpretation of reprogramming to predict the success of somatic cell cloning.

    PubMed

    Eckardt, Sigrid; McLaughlin, K John

    2004-07-01

    In the context of mammalian somatic cell cloning, the term reprogramming refers to the processes that enable a somatic cell nucleus to adopt the role of a zygotic nucleus. Gene re-expression is one measure of reprogramming if correlated with subsequent developmental potential. This paper describes several experiments utilizing pre-implantation gene expression to evaluate reprogramming and clone viability. We have established a direct correlation between Oct4 expression in mouse clones at the blastocyst stage and their potential to maintain pluripotent embryonic cells essential for post-implantation development. Furthermore, the quality of gene expression in clones dramatically improves when genetically identical clones are combined in clone-clone aggregate chimeras. Clone--clone aggregates exhibit a higher developmental potential than single clones both in vitro and in vivo. This could be mediated by complementation between blastomeres from epigenetically different clones within the aggregate rather than by the increase in cell number resulting from aggregation. We also discuss the use of tetraploid embryos as a model to evaluate reprogramming using gene expression and demonstrate that somatic cell nuclei can be reprogrammed by blastomeres to re-express embryonic specific genes but not to contribute to post-implantation development.

  4. Critical POU domain residues confer Oct4 uniqueness in somatic cell reprogramming.

    PubMed

    Jin, Wensong; Wang, Lei; Zhu, Fei; Tan, Weiqi; Lin, Wei; Chen, Dahua; Sun, Qinmiao; Xia, Zongping

    2016-01-01

    The POU domain transcription factor Oct4 plays critical roles in self-renewal and pluripotency of embryonic stem cells (ESCs). Together with Sox2, Klf4 and c-Myc, Oct4 can reprogram any other cell types to pluripotency, in which Oct4 is the only factor that cannot be functionally replaced by other POU family members. To investigate the determinant elements of Oct4 uniqueness, we performed Ala scan on all Ser, Thr, Tyr, Lys and Arg of murine Oct4 by testing their capability in somatic cell reprogramming. We uncovered a series of residues that are important for Oct4 functionality, in which almost all of these key residues are within the POU domains making direct interaction with DNA. The Oct4 N- and C-terminal transactivation domains (TADs) are not unique and could be replaced by the Yes-associated protein (YAP) TAD domain to support reprogramming. More importantly, we uncovered two important residues that confer Oct4 uniqueness in somatic cell reprogramming. Our systematic structure-function analyses bring novel mechanistic insight into the molecular basis of how critical residues function together to confer Oct4 uniqueness among POU family for somatic cell reprogramming. PMID:26877091

  5. Reversal of informational entropy and the acquisition of germ-like immortality by somatic cells.

    PubMed

    Kyriazis, Marios

    2014-01-01

    We live within an increasingly technological, information-laden environment for the first time in human evolution. This subjects us (and will continue to subject us in an accelerating fashion) to an unremitting exposure to 'meaningful information that requires action'. Directly dependent upon this new environment are novel evolutionary pressures, which can modify existing resource allocation mechanisms and may eventually favour the survival of somatic cells (particularly neurons) at the expense of germ line cells. In this theoretical paper I argue that persistent, structured information-sharing in both virtual and real domains, leads to increased biological complexity and functionality, which reflects upon human survival characteristics. Certain biological immortalisation mechanisms currently employed by germ cells may thus need to be downgraded in order to enable somatic cells to manage these new energy demands placed by our modern environment. Relevant concepts from a variety of disciplines such as the evolution of complex adaptive systems, information theory, digital hyper-connectivity, and cell immortalisation will be reviewed. Using logical, though sometimes speculative arguments, I will attempt to describe a new biology. A biology not driven by sex and reproduction but by information and somatic longevity. PMID:24852017

  6. Somatic mutation and cell differentiation in neoplastic transformation

    SciTech Connect

    Huberman, E.; Collart, F.R.

    1987-01-01

    In brief, the authors suggest that tumor formation may result from continuous expression of growth facilitating genes that, as a result of irreversible changes during the initiation step, are placed under the control of genes expressed during normal differentiation. Thus, to understand carcinogenesis, we must decipher the processes that lead to the acquisition of a mature phenotype in both normal and tumor cells and characterize the growth dependency of tumor cells to inducers of cell differentiation. Furthermore, the growth of a variety of tumors may be controlled through the use of inducers of maturation that activate genes located beyond the gene that is altered during tumor initiation. 22 refs., 3 figs.

  7. Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells.

    PubMed

    Chung, H J; Hassan, M M; Park, J O; Kim, H J; Hong, S T

    2015-05-01

    Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery.

  8. Bovine viral diarrhea virus (BVDV) in cell lines used for somatic cell cloning.

    PubMed

    Stringfellow, David A; Riddell, Kay P; Givens, M Daniel; Galik, Patricia K; Sullivan, Eddie; Dykstra, Christine C; Robl, James; Kasinathan, Poothapillai

    2005-03-01

    Culture of cell lines from fetuses or postnatal animals is an essential part of somatic cell cloning. Fetal bovine serum (FBS) is commonly used in media for propagation of these cells. Unfortunately, bovine fetuses and postnatal animals as well as FBS are all possible sources of non-cytopathic bovine viral diarrhea virus (BVDV) which is widely distributed among cattle. This study was prompted when screening of samples sent to veterinary diagnostic labs revealed that 15 of 39 fetal fibroblast cell lines used in cloning research were positive for BVDV as determined by various assays including reverse transcription-polymerase chain reaction (RT-PCR). Goals of the research were to use both virus isolation and reverse transcription-nested polymerase chain reaction (RT-nPCR) to confirm which of the cell lines were actually infected with BVDV and to assay samples of media, FBS and the earliest available passages of each cell line in an attempt to determine the source of the viral infections. Sequence analysis of amplified cDNA from all isolates was performed to provide a definitive link between possible sources of virus and infected cell lines. Only 5 of the 39 cell lines were actually infected with BVDV. Three of these five lines were not infected at the earliest cryopreserved passage, leading to the conclusion that they likely became infected after culture in media containing contaminated FBS. In fact, sequence comparison of the amplified cDNA from one lot of FBS confirmed that it was the source of infection for one of these cell lines. Since BVDV was isolated from the remaining two cell lines at the earliest available passage, the fetuses from which they were established could not be ruled out as the source of the virus.

  9. Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells.

    PubMed

    Cieslak, Jakub; Mackowski, Mariusz; Czyzak-Runowska, Grazyna; Wojtowski, Jacek; Puppel, Kamila; Kuczynska, Beata; Pawlak, Piotr

    2015-01-01

    Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse's milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Polish Warmblood Horse) we aimed to investigate the utility of equine milk somatic cells as a source of mRNA and to screen the best reference genes for RT-qPCR using geNorm and NormFinder algorithms. The results showed that despite relatively low somatic cell counts in mare's milk, the amount and the quality of the extracted RNA are sufficient for gene expression studies. The analysis of the utility of 7 potential reference genes for RT-qPCR experiments for the normalization of equine milk somatic cells revealed some differences between the outcomes of the applied algorithms, although in both cases the KRT8 and TOP2B genes were pointed as the most stable. Analysis by geNorm showed that the combination of 4 reference genes (ACTB, GAPDH, TOP2B and KRT8) is required for apropriate RT-qPCR experiments normalization, whereas NormFinder algorithm pointed the combination of KRT8 and RPS9 genes as the most suitable. The trial study of the relative transcript abundance of the beta-casein gene with the use of various types and numbers of internal control genes confirmed once again that the selection of proper reference gene combinations is crucial for the final results of each real-time PCR experiment. PMID:26437076

  10. Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells.

    PubMed

    Cieslak, Jakub; Mackowski, Mariusz; Czyzak-Runowska, Grazyna; Wojtowski, Jacek; Puppel, Kamila; Kuczynska, Beata; Pawlak, Piotr

    2015-01-01

    Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse's milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Polish Warmblood Horse) we aimed to investigate the utility of equine milk somatic cells as a source of mRNA and to screen the best reference genes for RT-qPCR using geNorm and NormFinder algorithms. The results showed that despite relatively low somatic cell counts in mare's milk, the amount and the quality of the extracted RNA are sufficient for gene expression studies. The analysis of the utility of 7 potential reference genes for RT-qPCR experiments for the normalization of equine milk somatic cells revealed some differences between the outcomes of the applied algorithms, although in both cases the KRT8 and TOP2B genes were pointed as the most stable. Analysis by geNorm showed that the combination of 4 reference genes (ACTB, GAPDH, TOP2B and KRT8) is required for apropriate RT-qPCR experiments normalization, whereas NormFinder algorithm pointed the combination of KRT8 and RPS9 genes as the most suitable. The trial study of the relative transcript abundance of the beta-casein gene with the use of various types and numbers of internal control genes confirmed once again that the selection of proper reference gene combinations is crucial for the final results of each real-time PCR experiment.

  11. Reprogrammed Transcriptome in Rhesus-Bovine Interspecies Somatic Cell Nuclear Transfer Embryos

    PubMed Central

    Wang, Kai; Otu, Hasan H.; Chen, Ying; Lee, Young; Latham, Keith; Cibelli, Jose B.

    2011-01-01

    Background Global activation of the embryonic genome (EGA), one of the most critical steps in early mammalian embryo development, is recognized as the time when interspecies somatic cell nuclear transfer (iSCNT) embryos fail to thrive. Methodology/Principal Findings In this study, we analyzed the EGA-related transcriptome of rhesus-bovine iSCNT 8- to 16-cell embryos and dissected the reprogramming process in terms of embryonic gene activation, somatic gene silencing, and maternal RNA degradation. Compared with fibroblast donor cells, two thousand and seven genes were activated in iSCNT embryos, one quarter of them reaching expression levels comparable to those found in in vitro fertilized (IVF) rhesus embryos. This suggested that EGA in iSCNT embryos had partially recapitulated rhesus embryonic development. Eight hundred and sixty somatic genes were not silenced properly and continued to be expressed in iSCNT embryos, which indicated incomplete nuclear reprogramming. We compared maternal RNA degradation in bovine oocytes between bovine-bovine SCNT and iSCNT embryos. While maternal RNA degradation occurred in both SCNT and iSCNT embryos, we saw more limited overall degradation of maternal RNA in iSCNT embryos than in SCNT embryos. Several important maternal RNAs, like GPF9, were not properly processed in SCNT embryos. Conclusions/Significance Our data suggested that iSCNT embryos are capable of triggering EGA, while a portion of somatic cell-associated genes maintain their expression. Maternal RNA degradation seems to be impaired in iSCNT embryos. Further understanding of the biological roles of these genes, networks, and pathways revealed by iSCNT may expand our knowledge about cell reprogramming, pluripotency, and differentiation. PMID:21799794

  12. Transposable DNA elements and life history traits: II. Transposition of P DNA elements in somatic cells reduces fitness, mating activity, and locomotion of Drosophila melanogaster.

    PubMed

    Woodruff, R C; Thompson, J N; Barker, J S; Huai, H

    1999-01-01

    Some transposable DNA elements in higher organisms are active in somatic cells, as well as in germinal cells. What effect does the movement of DNA elements in somatic cells have on life history traits? It has previously been reported that somatically active P and mariner elements in Drosophila induce genetic damage and significantly reduce lifespan. In this study, we report that the movement of P elements in somatic cells also significantly reduces fitness, mating activity, and locomotion of Drosophila melanogaster. If other elements cause similar changes in life history traits, it is doubtful if transposable DNA elements remain active for long in somatic cells in natural populations.

  13. Optimization of procedures for cloning by somatic cell nuclear transfer in mice.

    PubMed

    Chung, Young Gie; Gao, Shaorong; Latham, Keith E

    2006-01-01

    Cloning by somatic cell nuclear transfer is a complex procedure that is dependent on correct interactions between oocyte and donor cell genome. These interactions require minimal insult to either the oocyte or the transplanted nucleus. Available data also indicate that reprogramming the donor cell genome may be slow, so that the cloned embryo expresses genes typical of the donor cell, and thus has different characteristics from normal embryos. Procedures that minimize damage to the donor genome and that address the unique characteristics of the cloned construct should enhance the efficacy of the method.

  14. Adult Stem and Progenitor Cells

    NASA Astrophysics Data System (ADS)

    Geraerts, Martine; Verfaillie, Catherine M.

    The discovery of adult stem cells in most adult tissues is the basis of a number of clinical studies that are carried out, with therapeutic use of hematopoietic stem cells as a prime example. Intense scientific debate is still ongoing as to whether adult stem cells may have a greater plasticity than previously thought. Although cells with some features of embryonic stem cells that, among others, express Oct4, Nanog and SSEA1 are isolated from fresh tissue, it is not clear if the greater differentiation potential is acquired during cell culture. Moreover, adult more pluripotent cells do not have all pluripotent characteristics typical for embryonic stem cells. Recently, some elegant studies were published in which adult cells could be completely reprogrammed to embryonic stem cell-like cells by overexpression of some key transcription factors for pluripotency (Oct4, Sox2, Klf4 and c-Myc). It will be interesting for the future to investigate the exact mechanisms underlying this reprogramming and whether similar transcription factor pathways are present and/or can be activated in adult more pluripotent stem cells.

  15. Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets.

    PubMed

    Rao, Shengbin; Fujimura, Tatsuya; Matsunari, Hitomi; Sakuma, Tetsushi; Nakano, Kazuaki; Watanabe, Masahito; Asano, Yoshinori; Kitagawa, Eri; Yamamoto, Takashi; Nagashima, Hiroshi

    2016-01-01

    Myostatin (MSTN) is a negative regulator of myogenesis, and disruption of its function causes increased muscle mass in various species. Here, we report the generation of MSTN-knockout (KO) pigs using genome editing technology combined with somatic-cell nuclear transfer (SCNT). Transcription activator-like effector nuclease (TALEN) with non-repeat-variable di-residue variations, called Platinum TALEN, was highly efficient in modifying genes in porcine somatic cells, which were then used for SCNT to create MSTN KO piglets. These piglets exhibited a double-muscled phenotype, possessing a higher body weight and longissimus muscle mass measuring 170% that of wild-type piglets, with double the number of muscle fibers. These results demonstrate that loss of MSTN increases muscle mass in pigs, which may help increase pork production for consumption in the future.

  16. Clinical study report on milk production in the offspring of a somatic cell cloned Holstein cow.

    PubMed

    Takahashi, Masahiro; Tsuchiya, Hideki; Hamano, Seizo; Inaba, Toshio; Kawate, Noritoshi; Tamada, Hiromichi

    2013-12-17

    This study examined two female offspring of a somatic cell cloned Holstein cow that had reproduction problems and milk production performance issues. The two offspring heifers, which showed healthy appearances and normal reproductive characteristics, calved on two separate occasions. The mean milk yields of the heifers in the first lactation period were 9,037 kg and 7,228 kg. The relative mean milk yields of these cows were 111.2% and 88.9%, respectively, when compared with that of the control group. No particular clinical abnormalities were revealed in milk yields and milk composition rate [e.g., fat, protein and solids-not-fat (SNF)], and reproductive characteristics of the offspring of the somatic cell cloned Holstein cow suggested that the cloned offspring had normal milk production.

  17. Clinical Study Report on Milk Production in the Offspring of a Somatic Cell Cloned Holstein Cow

    PubMed Central

    TAKAHASHI, Masahiro; TSUCHIYA, Hideki; HAMANO, Seizo; INABA, Toshio; KAWATE, Noritoshi; TAMADA, Hiromichi

    2013-01-01

    Abstract This study examined two female offspring of a somatic cell cloned Holstein cow that had reproduction problems and milk production performance issues. The two offspring heifers, which showed healthy appearances and normal reproductive characteristics, calved on two separate occasions. The mean milk yields of the heifers in the first lactation period were 9,037 kg and 7,228 kg. The relative mean milk yields of these cows were 111.2% and 88.9%, respectively, when compared with that of the control group. No particular clinical abnormalities were revealed in milk yields and milk composition rate [e.g., fat, protein and solids-not-fat (SNF)], and reproductive characteristics of the offspring of the somatic cell cloned Holstein cow suggested that the cloned offspring had normal milk production. PMID:23955271

  18. Effect of somatic cell count and mastitis pathogens on milk composition in Gyr cows

    PubMed Central

    2013-01-01

    Background Gyr cows are well adapted to tropical conditions, resistant to some tropical diseases and have satisfactory milk production. However, Gyr dairy herds have a high prevalence of subclinical mastitis, which negatively affects their milk yield and composition. The objectives of this study were (i) to evaluate the effects of seasonality, mammary quarter location (rear x front), mastitis-causing pathogen species, and somatic cell count (SCC) on milk composition in Gyr cows with mammary quarters as the experimental units and (ii) to evaluate the effects of seasonality and somatic cell count (SCC) on milk composition in Gyr cows with cows as the experimental units. A total of 221 lactating Gyr cows from three commercial dairy farms were selected for this study. Individual foremilk quarter samples and composite milk samples were collected once a month over one year from all lactating cows for analysis of SCC, milk composition, and bacteriological culture. Results Subclinical mastitis reduced lactose, nonfat solids and total solids content, but no difference was found in the protein and fat content between infected and uninfected quarters. Seasonality influenced milk composition both in mammary quarters and composite milk samples. Nevertheless, there was no effect of mammary quarter position on milk composition. Mastitis-causing pathogens affected protein, lactose, nonfat solids, and total solids content, but not milk fat content. Somatic cell count levels affected milk composition in both mammary quarters and composite samples of milk. Conclusions Intramammary infections in Gyr cows alter milk composition; however, the degree of change depends on the mastitis-causing pathogen. Somatic cell count is negatively associated with reduced lactose and nonfat solids content in milk. Seasonality significantly affects milk composition, in which the concentration of lactose, fat, protein, nonfat solids and total solids differs between dry and wet seasons in Gyr cows. PMID

  19. Potential of embryonic and adult stem cells in vitro.

    PubMed

    Czyz, Jaroslaw; Wiese, Cornelia; Rolletschek, Alexandra; Blyszczuk, Przemyslaw; Cross, Michael; Wobus, Anna M

    2003-01-01

    Recent developments in the field of stem cell research indicate their enormous potential as a source of tissue for regenerative therapies. The success of such applications will depend on the precise properties and potentials of stem cells isolated either from embryonic, fetal or adult tissues. Embryonic stem cells established from the inner cell mass of early mouse embryos are characterized by nearly unlimited proliferation, and the capacity to differentiate into derivatives of essentially all lineages. The recent isolation and culture of human embryonic stem cell lines presents new opportunities for reconstructive medicine. However, important problems remain; first, the derivation of human embryonic stem cells from in vitro fertilized blastocysts creates ethical problems, and second, the current techniques for the directed differentiation into somatic cell populations yield impure products with tumorigenic potential. Recent studies have also suggested an unexpectedly wide developmental potential of adult tissue-specific stem cells. Here too, many questions remain concerning the nature and status of adult stem cells both in vivo and in vitro and their proliferation and differentiation/transdifferentiation capacity. This review focuses on those issues of embryonic and adult stem cell biology most relevant to their in vitro propagation and differentiation. Questions and problems related to the use of human embryonic and adult stem cells in tissue regeneration and transplantation are discussed.

  20. Reprogramming of mouse somatic cells into pluripotent stem-like cells using a combination of small molecules.

    PubMed

    Kang, Phil Jun; Moon, Jai-Hee; Yoon, Byung Sun; Hyeon, Solji; Jun, Eun Kyoung; Park, Gyuman; Yun, Wonjin; Park, Jiyong; Park, Minji; Kim, Aeree; Whang, Kwang Youn; Koh, Gou Young; Oh, Sejong; You, Seungkwon

    2014-08-01

    Somatic cells can be reprogrammed to generate induced pluripotent stem cells (iPSCs) by overexpression of four transcription factors, Oct4, Klf4, Sox2, and c-Myc. However, exogenous expression of pluripotency factors raised concerns for clinical applications. Here, we show that iPS-like cells (iPSLCs) were generated from mouse somatic cells in two steps with small molecule compounds. In the first step, stable intermediate cells were generated from mouse astrocytes by Bmi1. These cells called induced epiblast stem cell (EpiSC)-like cells (iEpiSCLCs) are similar to EpiSCs in terms of expression of specific markers, epigenetic state, and ability to differentiate into three germ layers. In the second step, treatment with MEK/ERK and GSK3 pathway inhibitors in the presence of leukemia inhibitory factor resulted in conversion of iEpiSCLCs into iPSLCs that were similar to mESCs, suggesting that Bmi1 is sufficient to reprogram astrocytes to partially reprogrammed pluripotency. Next, Bmi1 function was replaced with Shh activators (oxysterol and purmorphamine), which demonstrating that combinations of small molecules can compensate for reprogramming factors and are sufficient to directly reprogram mouse somatic cells into iPSLCs. The chemically induced pluripotent stem cell-like cells (ciPSLCs) showed similar gene expression profiles, epigenetic status, and differentiation potentials to mESCs.

  1. Development of porcine tetraploid somatic cell nuclear transfer embryos is influenced by oocyte nuclei.

    PubMed

    Fu, Bo; Liu, Di; Ma, Hong; Guo, Zhen-Hua; Wang, Liang; Li, Zhong-Qiu; Peng, Fu-Gang; Bai, Jing

    2016-02-01

    Cloning efficiency in mammalian systems remains low because reprogramming of donor cells is frequently incomplete. Nuclear factors in the oocyte are removed by enucleation, and this removal may adversely affect reprogramming efficiency. Here, we investigated the role of porcine oocyte nuclear factors during reprogramming. We introduced somatic cell nuclei into intact MII oocytes to establish tetraploid somatic cell nuclear transfer (SCNT) embryos containing both somatic nuclei and oocyte nuclei. We then examined the influence of the oocyte nucleus on tetraploid SCNT embryo development by assessing characteristics including pronucleus formation, cleavage rate, and blastocyst formation. Overall, tetraploid SCNT embryos have a higher developmental competence than do standard diploid SCNT embryos. Therefore, we have established an embryonic model in which a fetal fibroblast nucleus and an oocyte metaphase II plate coexist. Tetraploid SCNT represents a new research platform that is potentially useful for examining interactions between donor nuclei and oocyte nuclei. This platform should facilitate further understanding of the roles played by nuclear factors during reprogramming.

  2. Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins

    PubMed Central

    Park, Ki-Eun; Park, Chi-Hun; Powell, Anne; Martin, Jessica; Donovan, David M.; Telugu, Bhanu P.

    2016-01-01

    The pig is an ideal large animal model for genetic engineering applications. A relatively short gestation interval and large litter size makes the pig a conducive model for generating and propagating genetic modifications. The domestic pig also shares close similarity in anatomy, physiology, size, and life expectancy, making it an ideal animal for modeling human diseases. Often, however, the technical difficulties in generating desired genetic modifications such as targeted knockin of short stretches of sequences or transgenes have impeded progress in this field. In this study, we have investigated and compared the relative efficiency of CRISPR/Cas ribonucleoproteins in engineering targeted knockin of pseudo attP sites downstream of a ubiquitously expressed COL1A gene in porcine somatic cells and generated live fetuses by somatic cell nuclear transfer (SCNT). By leveraging these knockin pseudo attP sites, we have demonstrated subsequent phiC31 integrase mediated integration of green fluorescent protein (GFP) transgene into the site. This work for the first time created an optimized protocol for CRISPR/Cas mediated knockin in porcine somatic cells, while simultaneously creating a stable platform for future transgene integration and generating transgenic animals. PMID:27240344

  3. Phenotypes of Aging Postovulatory Oocytes After Somatic Cell Nuclear Transfer in Mice.

    PubMed

    Lee, Ah Reum; Shimoike, Takashi; Wakayama, Teruhiko; Kishigami, Satoshi

    2016-06-01

    Oocytes rapidly lose their developmental potential after ovulation, termed postovulatory oocyte aging, and often exhibit characteristic phenotypes, such as cytofragmentation, abnormal spindle shapes, and chromosome misalignments. Here, we reconstructed mouse oocytes using somatic cell nuclear transfer (SCNT) to reveal the effect of somatic cell-derived nuclei on oocyte physiology during aging. Normal oocytes started undergoing cytofragmentation 24 hours after oocyte collection; however, this occurred earlier in SCNT oocytes and was more severe at 48 hours, suggesting that the transferred somatic cell nuclei affected oocyte physiology. We found no difference in the status of acetylated α-tubulin (Ac-Tub) and α-tubulin (Tub) between normal and SCNT aging oocytes, but unlike normal oocytes, aging SCNT oocytes did not have astral microtubules. Interestingly, aging SCNT oocytes displayed more severely scattered chromosomes or irregularly shaped spindles. Observations of the microfilaments showed that, in normal oocytes, there was a clear actin ring beneath the plasma membrane and condensed microfilaments around the spindle (the actin cap) at 0 hours, and the actin filaments started degenerating at 1 hour, becoming completely disrupted and distributed to the cytoplasm at 24 hours. By contrast, in SCNT oocytes, an actin cap formed around the transplanted nuclei within 1 hour of SCNT, which was still present at 24 hours. Thus, SCNT oocytes age in a similar but distinct way, suggesting that they not only contain nuclei with abnormal epigenetics but are also physiologically different. PMID:27253626

  4. Mutagenic effects of thiram in mammalian somatic cells.

    PubMed

    Paschin YuV; Bakhitova, L M

    1985-03-01

    The dimethylthiocarbamate fungicide thiram has been found to be a potent and direct inducer of point mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster cells in vitro. It also increased the incidence of micronuclei in polychromatic erythrocytes in the bone marrow of mice given a single ip dose of 100 mg/kg. Both the in vitro and the in vivo mutagenic responses were observed with doses of thiram that were cytotoxic.

  5. Somatic mosaicism and disease.

    PubMed

    Frank, Steven A

    2014-06-16

    The large number of cell divisions required to make a human body inevitably leads to the accumulation of somatic mutations. Such mutations cause individuals to be somatic mosaics. Recent advances in genomic technology now allow measurement of somatic diversity. Initial studies confirmed the expected high levels of somatic mutations within individuals. Going forward, the big questions concern the degree to which those somatic mutations influence disease. Theory predicts that the frequency of mutant cells should vary greatly between individuals. Such somatic mutational variability between individuals could explain much of the diversity in the risk of disease. But how variable is mosaicism between individuals in reality? What is the relation between the fraction of cells carrying a predisposing mutation and the risk of disease? What kinds of heritable somatic change lead to disease besides classical DNA mutations? What molecular processes connect a predisposing somatic change to disease? We know that predisposing somatic mutations strongly influence the onset of cancer. Likewise, neurodegenerative diseases may often begin from somatically mutated cells. If so, both neurodegeneration and cancer may be diseases of later life for which much of the risk may be set by early life somatic mutations.

  6. Somatic Embryogenesis in Peach Palm Using the Thin Cell Layer Technique: Induction, Morpho-histological Aspects and AFLP Analysis of Somaclonal Variation

    PubMed Central

    Steinmacher, D. A.; Krohn, N. G.; Dantas, A. C. M.; Stefenon, V. M.; Clement, C. R.; Guerra, M. P.

    2007-01-01

    Background and Aims The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm. Methods TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0–600 µm Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets. Key Results Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150–600 µm Picloram (83–97 %, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43 % embryogenic callus production from shoot meristem TCL on 300 µm Picloram. In maturation conditions, 34 ± 4 somatic embryos per embryogenic callus were obtained, and 45·0 ± 3·4 % of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80 % survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92 % of the regenerated plantlets were true to type. The use of TCL explants considerably improves the

  7. Potential of Herpesvirus Saimiri-Based Vectors To Reprogram a Somatic Ewing's Sarcoma Family Tumor Cell Line

    PubMed Central

    Brown, Hannah F.; Unger, Christian

    2013-01-01

    Herpesvirus saimiri (HVS) infects a range of human cell types with high efficiency. Upon infection, the viral genome can persist as high-copy-number, circular, nonintegrated episomes that segregate to progeny cells upon division. This allows HVS-based vectors to stably transduce a dividing cell population and provide sustained transgene expression in vitro and in vivo. Moreover, the HVS episome is able to persist and provide prolonged transgene expression during in vitro differentiation of mouse and human hemopoietic progenitor cells. Together, these properties are advantageous for induced pluripotent stem cell (iPSC) technology, whereby stem cell-like cells are generated from adult somatic cells by exogenous expression of specific reprogramming factors. Here we assess the potential of HVS-based vectors for the generation of induced pluripotent cancer stem-like cells (iPCs). We demonstrate that HVS-based exogenous delivery of Oct4, Nanog, and Lin28 can reprogram the Ewing's sarcoma family tumor cell line A673 to produce stem cell-like colonies that can grow under feeder-free stem cell culture conditions. Further analysis of the HVS-derived putative iPCs showed some degree of reprogramming into a stem cell-like state. Specifically, the putative iPCs had a number of embryonic stem cell characteristics, staining positive for alkaline phosphatase and SSEA4, in addition to expressing elevated levels of pluripotent marker genes involved in proliferation and self-renewal. However, differentiation trials suggest that although the HVS-derived putative iPCs are capable of differentiation toward the ectodermal lineage, they do not exhibit pluripotency. Therefore, they are hereby termed induced multipotent cancer cells. PMID:23596304

  8. The glycophorin A assay for somatic cell mutations in humans

    SciTech Connect

    Langlois, R.G.; Bigbee, W.L.; Jensen, R.H.

    1989-08-18

    In this report we briefly review our past experience and some new developments with the GPA assay. Particular emphasis will be placed on two areas that affect the utility of the GPA assay for human population monitoring. The first is our efforts to simplify the GPA assay to make it more generally available for large population studies. The second is to begin to understand some of the characteristics of human hemopoiesis which affect the accumulation and expression of mutant phenotype cells. 11 refs., 4 figs.

  9. Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig

    PubMed Central

    2012-01-01

    Background Somatic cell nuclear transfer (SCNT) is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research. However, the efficiency of this approach is crucially dependent on the source of nuclear donor cells. In this study, we evaluate the potential of primary porcine kidney cells (PKCs) as cell source for SCNT, including their proliferation capacity, transfection efficiency, and capacity to support full term development of SCNT embryos after additive gene transfer or homologous recombination. Results PKCs could be maintained in culture with stable karyotype for up to 71 passages, whereas porcine fetal fibroblasts (PFFs) and porcine ear fibroblasts (PEFs) could be hardly passaged more than 20 times. Compared with PFFs and PEFs, PKCs exhibited a higher proliferation rate and resulted in a 2-fold higher blastocyst rate after SCNT and in vitro cultivation. Among the four transfection methods tested with a GFP expression plasmid, best results were obtained with the NucleofectorTM technology, resulting in transfection efficiencies of 70% to 89% with high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of cell stress. Usage of genetically modified PKCs in SCNT resulted in approximately 150 piglets carrying at least one of 18 different transgenes. Several of those pigs originated from PKCs that underwent homologous recombination and antibiotic selection before SCNT. Conclusion The high proliferation capacity of PKCs facilitates the introduction of precise and complex genetic modifications in vitro. PKCs are thus a valuable cell source for the generation of porcine biomedical models by SCNT. PMID:23140586

  10. Mammary Stem Cell Based Somatic Mouse Models Reveal Breast Cancer Drivers Causing Cell Fate Dysregulation

    PubMed Central

    Zhang, Zheng; Christin, John R.; Wang, Chunhui; Ge, Kai; Oktay, Maja H.; Guo, Wenjun

    2016-01-01

    SUMMARY Cancer genomics have provided an unprecedented opportunity for understanding genetic causes of human cancer. However, distinguishing which mutations are functionally relevant to cancer pathogenesis remains a major challenge. We describe here a mammary stem cell (MaSC) organoid-based approach for rapid generation of somatic GEMMs (genetically engineered mouse models). By using RNAi and CRISPR-mediated genome engineering in MaSC-GEMMs, we have discovered that inactivation of Ptpn22 or Mll3, two genes mutated in human breast cancer, greatly accelerated PI3K-driven mammary tumorigenesis. Using these tumor models, we have also identified genetic alterations promoting tumor metastasis and causing resistance to PI3K-targeted therapy. Both Ptpn22 and Mll3 inactivation resulted in disruption of mammary gland differentiation and an increase in stem cell activity. Mechanistically, Mll3 deletion enhanced stem cell activity through activation of the HIF pathway. Thus, our study established a robust in vivo platform for functional cancer genomics and discovered functional breast cancer mutations. PMID:27653681

  11. Mammary-Stem-Cell-Based Somatic Mouse Models Reveal Breast Cancer Drivers Causing Cell Fate Dysregulation.

    PubMed

    Zhang, Zheng; Christin, John R; Wang, Chunhui; Ge, Kai; Oktay, Maja H; Guo, Wenjun

    2016-09-20

    Cancer genomics has provided an unprecedented opportunity for understanding genetic causes of human cancer. However, distinguishing which mutations are functionally relevant to cancer pathogenesis remains a major challenge. We describe here a mammary stem cell (MaSC) organoid-based approach for rapid generation of somatic genetically engineered mouse models (GEMMs). By using RNAi and CRISPR-mediated genome engineering in MaSC-GEMMs, we have discovered that inactivation of Ptpn22 or Mll3, two genes mutated in human breast cancer, greatly accelerated PI3K-driven mammary tumorigenesis. Using these tumor models, we have also identified genetic alterations promoting tumor metastasis and causing resistance to PI3K-targeted therapy. Both Ptpn22 and Mll3 inactivation resulted in disruption of mammary gland differentiation and an increase in stem cell activity. Mechanistically, Mll3 deletion enhanced stem cell activity through activation of the HIF pathway. Thus, our study has established a robust in vivo platform for functional cancer genomics and has discovered functional breast cancer mutations. PMID:27653681

  12. Effect of season on milk temperature, milk growth hormone, prolactin, and somatic cell counts of lactating cattle

    NASA Astrophysics Data System (ADS)

    Igono, M. O.; Johnson, H. D.; Steevens, B. J.; Hainen, W. A.; Shanklin, M. D.

    1988-09-01

    Monthly fluctuations in milk temperature, somatic cell counts, milk growth hormone and prolactin of lactating cows were measured in milk samples over a 1 year period. The seasonal patterns in milk temperature, somatic cell count and milk prolactin concentration showed a positive trend with increasing environmental temperatures. Milk growth hormone concentration increased with lactation level and declined significantly during summer heat. Milk temperature and the measured hormonal levels may serve as indicators of the impact of the climatic environment on lactating cattle.

  13. Direct exposure of chromosomes to nonactivated ovum cytoplasm is effective for bovine somatic cell nucleus reprogramming.

    PubMed

    Tani, T; Kato, Y; Tsunoda, Y

    2001-01-01

    We examined the in vitro developmental potential of nonactivated and activated enucleated ova receiving cumulus cells at various stages of the cell cycle. Eleven to 29% of activated ova receiving donor cells stopped developing at the 8-cell stage but 21% to 50% of nonactivated ova receiving donor cells at either the G(0), G(1), G(2), or M phase, or cycling cells developed into blastocysts. One normal calf was born after transferring five blastocysts that had developed from ova receiving donor cells at the M phase. The present study demonstrated that direct exposure of donor chromosomes to nonactivated ovum cytoplasm is effective for somatic cell nucleus reprogramming, and activated ovum cytoplasm does not reprogram the nucleus.

  14. Spatial separation of parental genomes in hybrids of somatic plant cells

    PubMed Central

    Gleba, Yuri Yu.; Parokonny, A.; Kotov, V.; Negrutiu, I.; Momot, V.

    1987-01-01

    Chromosome spatial arrangements on metaphase plates of intergeneric intertribal cell hybrids of Nicotiana chinensis and Atropa belladonna as well as interspecific somatic hybrid plants of Nicotiana plumbaginifolia and Nicotiana sylvestris were analyzed. In the metaphases of the first divisions of protoplast fusion products, chromosomes of the two parents were spatially separated (segmented metaphase). In long-term cultured somatic hybrids, the topology of genome separation pattern in both callus cells and plants showed changes in form from “segmental” to “radial.” Growing the hybrid cells in the presence of colchicine resulted in random chromosome arrangement both in cells directly exposed to different colchicine concentrations and in colchicine-treated cells grown in colchicine-free media. The degree of genome separation calculated for different cell clones remained constant during in vitro propagation of cells but was significantly lower for subclones derived from colchicine-treated cells. Therefore, it is concluded that spatial chromosome arrangement in metaphase is epigenetically controlled. Images PMID:16593838

  15. Similar GABAA receptor subunit composition in somatic and axon initial segment synapses of hippocampal pyramidal cells

    PubMed Central

    Kerti-Szigeti, Katalin; Nusser, Zoltan

    2016-01-01

    Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in α1 and α2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the α1, α2, β1, β2, β3 and γ2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the α1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The α2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content. DOI: http://dx.doi.org/10.7554/eLife.18426.001 PMID:27537197

  16. Somatic Embryogenesis of Date Palm (Phoenix dactylifera L.) Through Cell Suspension Culture.

    PubMed

    Naik, Poornananda M; Al-Khayri, Jameel M

    2016-01-01

    Date palm (Phoenix dactylifera L.) is the oldest and most economically important plant species distributed in the hot arid regions of the world. Propagation of date palm by seeds produces heterogeneous offspring with inferior field performance and poor fruit quality. Traditionally, date palm is propagated by offshoots, but this method is inefficient for mass propagation because of limited availability of offshoots. Plant regeneration through tissue culture is able to provide technologies for the large-scale propagation of healthy true-to-type plants. The most commonly used technology approach is somatic embryogenesis which presents a great potential for the rapid propagation and genetic resource preservation of this species. Significant progress has been made in the development and optimization of this regeneration pathway through the establishment of embryogenic suspension cultures. This chapter focuses on the methods employed for the induction of callus from shoot tip explants, establishment of cell suspension culture, and subsequent somatic embryogenesis and plant regeneration. PMID:27108330

  17. Effect of Hypoxia on Ldh-c Expression in Somatic Cells of Plateau Pika.

    PubMed

    Wei, Dengbang; Wei, Linna; Li, Xiao; Wang, Yang; Wei, Lian

    2016-08-01

    Sperm specific lactate dehydrogenases (LDH-C₄) is a lactate dehydrogenase that catalyzes the conversion of pyruvate to lactate. In mammals, Ldh-c was originally thought to be expressed only in testes and spermatozoa. Plateau pika (Ochotona curzoniae), which belongs to the genus Ochotona of the Ochotonidea family, is a hypoxia-tolerant mammal living 3000-5000 m above sea level on the Qinghai-Tibet Plateau, an environment which is strongly hypoxic. Ldh-c is expressed not only in testes and sperm, but also in the somatic tissues of plateau pika. To reveal the effect of hypoxia on pika Ldh-c expression, we investigated the mRNA and protein level of Ldh-c as well as the biochemical index of anaerobic glycolysis in pika somatic tissues at the altitudes of 2200 m, 3200 m and 3900 m. Our results showed that mRNA and protein expression levels of Ldh-c in the tissues of pika's heart, liver, brain and skeletal muscle were increased significantly from 2200 m to 3200 m, but had no difference from 3200 m to 3900 m; the activities of LDH and the contents of lactate showed no difference from 2200 m to 3200 m, but were increased significantly from 3200 m to 3900 m. Hypoxia up-regulated and maintained the expression levels of Ldh-c in the pika somatic cells. Under the hypoxia condition, plateau pikas increased anaerobic glycolysis in somatic cells by LDH-C₄, and that may have reduced their dependence on oxygen and enhanced their adaptation to the hypoxic environment.

  18. Effect of Hypoxia on Ldh-c Expression in Somatic Cells of Plateau Pika.

    PubMed

    Wei, Dengbang; Wei, Linna; Li, Xiao; Wang, Yang; Wei, Lian

    2016-01-01

    Sperm specific lactate dehydrogenases (LDH-C₄) is a lactate dehydrogenase that catalyzes the conversion of pyruvate to lactate. In mammals, Ldh-c was originally thought to be expressed only in testes and spermatozoa. Plateau pika (Ochotona curzoniae), which belongs to the genus Ochotona of the Ochotonidea family, is a hypoxia-tolerant mammal living 3000-5000 m above sea level on the Qinghai-Tibet Plateau, an environment which is strongly hypoxic. Ldh-c is expressed not only in testes and sperm, but also in the somatic tissues of plateau pika. To reveal the effect of hypoxia on pika Ldh-c expression, we investigated the mRNA and protein level of Ldh-c as well as the biochemical index of anaerobic glycolysis in pika somatic tissues at the altitudes of 2200 m, 3200 m and 3900 m. Our results showed that mRNA and protein expression levels of Ldh-c in the tissues of pika's heart, liver, brain and skeletal muscle were increased significantly from 2200 m to 3200 m, but had no difference from 3200 m to 3900 m; the activities of LDH and the contents of lactate showed no difference from 2200 m to 3200 m, but were increased significantly from 3200 m to 3900 m. Hypoxia up-regulated and maintained the expression levels of Ldh-c in the pika somatic cells. Under the hypoxia condition, plateau pikas increased anaerobic glycolysis in somatic cells by LDH-C₄, and that may have reduced their dependence on oxygen and enhanced their adaptation to the hypoxic environment. PMID:27490559

  19. Effect of Hypoxia on Ldh-c Expression in Somatic Cells of Plateau Pika

    PubMed Central

    Wei, Dengbang; Wei, Linna; Li, Xiao; Wang, Yang; Wei, Lian

    2016-01-01

    Sperm specific lactate dehydrogenases (LDH-C4) is a lactate dehydrogenase that catalyzes the conversion of pyruvate to lactate. In mammals, Ldh-c was originally thought to be expressed only in testes and spermatozoa. Plateau pika (Ochotona curzoniae), which belongs to the genus Ochotona of the Ochotonidea family, is a hypoxia-tolerant mammal living 3000–5000 m above sea level on the Qinghai-Tibet Plateau, an environment which is strongly hypoxic. Ldh-c is expressed not only in testes and sperm, but also in the somatic tissues of plateau pika. To reveal the effect of hypoxia on pika Ldh-c expression, we investigated the mRNA and protein level of Ldh-c as well as the biochemical index of anaerobic glycolysis in pika somatic tissues at the altitudes of 2200 m, 3200 m and 3900 m. Our results showed that mRNA and protein expression levels of Ldh-c in the tissues of pika’s heart, liver, brain and skeletal muscle were increased significantly from 2200 m to 3200 m, but had no difference from 3200 m to 3900 m; the activities of LDH and the contents of lactate showed no difference from 2200 m to 3200 m, but were increased significantly from 3200 m to 3900 m. Hypoxia up-regulated and maintained the expression levels of Ldh-c in the pika somatic cells. Under the hypoxia condition, plateau pikas increased anaerobic glycolysis in somatic cells by LDH-C4, and that may have reduced their dependence on oxygen and enhanced their adaptation to the hypoxic environment. PMID:27490559

  20. Transient Acquisition of Pluripotency During Somatic Cell Transdifferentiation with iPSC Reprogramming Factors

    PubMed Central

    Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D.; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R.; Greenleaf, William J.; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H.

    2015-01-01

    Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors1,2. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation3–6. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced transdifferentiation pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by different methods. PMID:26098448

  1. Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata

    NASA Technical Reports Server (NTRS)

    Vasilenko, A.; McDaniel, J. K.; Conger, B. V.

    2000-01-01

    Somatic embryos initiate and develop directly from single mesophyll cells in in vitro-cultured leaf segments of orchardgrass (Dactylis glomerata L.). Embryogenic cells establish themselves in the predivision stage by formation of thicker cell walls and dense cytoplasm. Electron microscopy observations for embryos ranging from the pre-cell-division stage to 20-cell proembryos confirm previous light microscopy studies showing a single cell origin. They also confirm that the first division is predominantly periclinal and that this division plane is important in establishing embryo polarity and in determining the embryo axis. If the first division is anticlinal or if divisions are in random planes after the first division, divisions may not continue to produce an embryo. This result may produce an embryogenic cell mass, callus formation, or no structure at all. Grant numbers: NAGW-3141, NAG10-0221.

  2. Molecular evidence that homologous recombination occurs in proliferating human somatic cells.

    PubMed Central

    Groden, J; Nakamura, Y; German, J

    1990-01-01

    A strategy has been developed to detect and characterize certain heritable genomic alterations that occur as cells proliferate in vitro. Multiple subclones of cells were isolated from two clonal lymphoblastoid cell lines--one from a boy with Bloom's syndrome (BS), a cancer-predisposing condition known to feature excessive somatic mutation, the other from a normal man. The DNAs from the cell lines were hybridized to a panel of probes that can detect restriction fragment length polymorphisms, and the patterns of polymorphism in the primary clones were compared with that in each of the secondary clones. In one of the BS secondary clones three loci, positioned distally on the long arm of chromosome 3 and that are heterozygous in the donor and all other cell lines derived from the primary clone, had lost heterozygosity and apparently had become homozygous; in contrast, heterozygous loci more proximal on 3q had retained their heterozygosity, as had those on 3p. Taking into account the pattern of chromosome instability uniquely characteristic of BS, the most plausible explanation for the alterations in the altered clone is that somatic recombination had occurred in vitro, via homologous chromatid interchange. Such spontaneous recombinational events in nonneoplastic, nonmutagenized cells may contribute to the high cancer incidence in BS and, by analogy, to cancer that arises in the general population. Images PMID:1971948

  3. Factors Determining the Efficiency of Porcine Somatic Cell Nuclear Transfer: Data Analysis with Over 200,000 Reconstructed Embryos.

    PubMed

    Liu, Tianbin; Dou, Hongwei; Xiang, Xi; Li, Lin; Li, Yong; Lin, Lin; Pang, Xinzhi; Zhang, Yijie; Chen, Yu; Luan, Jing; Xu, Ying; Yang, Zhenzhen; Yang, Wenxian; Liu, Huan; Li, Feida; Wang, Hui; Yang, Huanming; Bolund, Lars; Vajta, Gabor; Du, Yutao

    2015-12-01

    Data analysis in somatic cell nuclear transfer (SCNT) research is usually limited to several hundreds or thousands of reconstructed embryos. Here, we report mass results obtained with an established and consistent porcine SCNT system (handmade cloning [HMC]). During the experimental period, 228,230 reconstructed embryos and 82,969 blastocysts were produced. After being transferred into 656 recipients, 1070 piglets were obtained. First, the effects of different types of donor cells, including fetal fibroblasts (FFs), adult fibroblasts (AFs), adult preadipocytes (APs), and adult blood mesenchymal (BM) cells, were investigated on the further in vitro and in vivo development. Compared to adult donor cells (AFs, APs, BM cells, respectively), FF cells resulted in a lower blastocyst/reconstructed embryo rate (30.38% vs. 37.94%, 34.65%, and 34.87%, respectively), but a higher overall efficiency on the number of piglets born alive per total blastocysts transferred (1.50% vs. 0.86%, 1.03%, and 0.91%, respectively) and a lower rate of developmental abnormalities (10.87% vs. 56.57%, 24.39%, and 51.85%, respectively). Second, recloning was performed with cloned adult fibroblasts (CAFs) and cloned fetal fibroblasts (CFFs). When CAFs were used as the nuclear donor, fewer developmental abnormalities and higher overall efficiency were observed compared to AFs (56.57% vs. 28.13% and 0.86% vs. 1.59%, respectively). However, CFFs had an opposite effect on these parameters when compared with CAFs (94.12% vs. 10.87% and 0.31% vs. 1.50%, respectively). Third, effects of genetic modification on the efficiency of SCNT were investigated with transgenic fetal fibroblasts (TFFs) and gene knockout fetal fibroblasts (KOFFs). Genetic modification of FFs increased developmental abnormalities (38.96% and 25.24% vs. 10.87% for KOFFs, TFFs, and FFs, respectively). KOFFs resulted in lower overall efficiency compared to TFFs and FFs (0.68% vs. 1.62% and 1.50%, respectively). In conclusion, this is the

  4. Heterogeneous nuclear ribonucleoprotein complexes from Xenopus laevis oocytes and somatic cells.

    PubMed

    Marcu, A; Bassit, B; Perez, R; Piñol-Roma, S

    2001-09-01

    HnRNP proteins have been implicated in most stages of cellular mRNA metabolism, including processing, nucleocytoplasmic transport, stability, and localization. Several hnRNP proteins are also known to participate in key early developmental decisions. In order to facilitate functional studies of these pre-mRNA- and mRNA-binding proteins in a vertebrate organism amenable to developmental studies and experimental manipulation, we identified and purified the major hnRNP proteins and isolated the hnRNP complex from Xenopus laevis oocytes and somatic cells. Using affinity chromatography and immunological methods, we isolated a family of >15 abundant single-stranded nucleic acid-binding proteins, which range in apparent molecular weight from approximately 20 kDa to >150 kDa, and with isoelectric points from <5 to >8. Monoclonal antibodies revealed that a subset of these proteins are major hnRNP proteins in both oocytes and somatic cells in culture, and include proteins related to human hnRNP A2/B1/B2 and hnRNP K. UV crosslinking in living cells demonstrated that these proteins bind poly(A)+ RNA in vivo. Immunopurification using a monoclonal antibodyto X. aevishnRNPA2 resulted in the isolation of RNP complexes that contain a specific subset of single-stranded nucleic acid-binding proteins. The protein composition of complexes isolated from somatic cells and from oocyte germinal vesicles was similar, suggesting that the overall properties and functions of hnRNP proteins in these two cell types are comparable. These findings, together with the novel probes generated here, will also facilitate studies of the function of vertebrate RNA-binding proteins using the well characterized X. laevis oocyte and early embryo as experimental systems. PMID:11669376

  5. Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

    SciTech Connect

    Fan, Yong; Chen, Xinjie; Luo, Yumei; Chen, Xiaolin; Li, Shaoying; Huang, Yulin; Sun, Xiaofang

    2009-04-24

    The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.

  6. Somatically Acquired LINE-1 Insertions in Normal Esophagus Undergo Clonal Expansion in Esophageal Squamous Cell Carcinoma.

    PubMed

    Doucet-O'Hare, Tara T; Sharma, Reema; Rodić, Nemanja; Anders, Robert A; Burns, Kathleen H; Kazazian, Haig H

    2016-09-01

    Squamous cell carcinoma of the esophagus (SCC) is the most common form of esophageal cancer in the world and is typically diagnosed at an advanced stage when successful treatment is challenging. Understanding the mutational profile of this cancer may identify new treatment strategies. Because somatic retrotransposition has been shown in tumors of the gastrointestinal system, we focused on LINE-1 (L1) mobilization as a source of genetic instability in this cancer. We hypothesized that retrotransposition is ongoing in SCC patients. The expression of L1 encoded proteins is necessary for retrotransposition to occur; therefore, we evaluated the expression of L1 open reading frame 1 protein (ORF1p). Using immunohistochemistry, we detected ORF1p expression in all four SCC cases evaluated. Using L1-seq, we identified and validated 74 somatic insertions in eight tumors of the nine evaluated. Of these, 12 insertions appeared to be somatic, not genetically inherited, and sub-clonal (i.e., present in less than one copy per genome equivalent) in the adjacent normal esophagus (NE), while clonal in the tumor. Our results indicate that L1 retrotransposition is active in SCC of the esophagus and that insertion events are present in histologically NE that expands clonally in the subsequent tumor. PMID:27319353

  7. Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation.

    PubMed

    Yang, Feikun; Hao, Ru; Kessler, Barbara; Brem, Gottfried; Wolf, Eckhard; Zakhartchenko, Valeri

    2007-01-01

    The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres from in vivo fertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF, P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that in in vivo fertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo derived embryos improved development to blastocyst, but no cloned

  8. Generation of biallelic knock-out sheep via gene-editing and somatic cell nuclear transfer

    PubMed Central

    Li, Honghui; Wang, Gui; Hao, Zhiqiang; Zhang, Guozhong; Qing, Yubo; Liu, Shuanghui; Qing, Lili; Pan, Weirong; Chen, Lei; Liu, Guichun; Zhao, Ruoping; Jia, Baoyu; Zeng, Luyao; Guo, Jianxiong; Zhao, Lixiao; Zhao, Heng; Lv, Chaoxiang; Xu, Kaixiang; Cheng, Wenmin; Li, Hushan; Zhao, Hong-Ye; Wang, Wen; Wei, Hong-Jiang

    2016-01-01

    Transgenic sheep can be used to achieve genetic improvements in breeds and as an important large-animal model for biomedical research. In this study, we generated a TALEN plasmid specific for ovine MSTN and transfected it into fetal fibroblast cells of STH sheep. MSTN biallelic-KO somatic cells were selected as nuclear donor cells for SCNT. In total, cloned embryos were transferred into 37 recipient gilts, 28 (75.7%) becoming pregnant and 15 delivering, resulting in 23 lambs, 12 of which were alive. Mutations in the lambs were verified via sequencing and T7EI assay, and the gene mutation site was consistent with that in the donor cells. Off-target analysis was performed, and no off-target mutations were detected. MSTN KO affected the mRNA expression of MSTN relative genes. The growth curve for the resulting sheep suggested that MSTN KO caused a remarkable increase in body weight compared with those of wild-type sheep. Histological analyses revealed that MSTN KO resulted in muscle fiber hypertrophy. These findings demonstrate the successful generation of MSTN biallelic-KO STH sheep via gene editing in somatic cells using TALEN technology and SCNT. These MSTN mutant sheep developed and grew normally, and exhibited increased body weight and muscle growth. PMID:27654750

  9. MicroRNAs in regulation of pluripotency and somatic cell reprogramming

    PubMed Central

    Wang, Tian; Shi, San-bao; Sha, Hong-ying

    2013-01-01

    MicroRNAs (miRNAs), a group of small non-coding RNAs, have emerged as significant modulators in the establishment and generation of pluripotency, a developmental process that consists of complex cell-fate arrangements. The finding of embryonic stem cell (ESC) cycle-specific miRNAs reveals an important regulation scheme of pluripotency. Subsequent studies showed the ESC-enriched or ESC-depleted miRNAs can regulate induced pluripotent stem cells(iPSC). Moreover, miRNA profiling of iPSC and ESC may distinguish them from one another and facilitate the complex of regulatory network. The accumulative effects of miRNA action enable using miRNA alone to generate iPSCs. Despite the robustness of iPSC studies, further investigations are needed since miRNA may have more impact on induced pluripotency, and the roles of miRNAs in somatic cell nuclear transfer (SCNT), another approach toward cellular reprogramming, remains unclear. This point-of-view article will discuss miRNAs and their impact on the normal and induced pluripotency, as well as bring new insights on somatic cell reprogramming. PMID:23921205

  10. Developmental autonomy and somatic niche construction promotes robust cell fate decisions.

    PubMed

    Bershad, Anya K; Fuentes, Miguel A; Krakauer, David C

    2008-09-21

    During the course of development cells undergo division producing a variety of cell types. Proliferation and differentiation are dependent on both genetic programs, encoded by the cellular genome, and environmental cues produced by the local cellular environment imposing local selection pressures on cells. We explore the role that cellular signals play over a large range of potential parameter regimes, in minimizing developmental error: errors in differentiation where an inappropriate proportion of differentiated daughter cells are generated. We find that trophic factors produced by the population of dividing cells can compensate for increased error rates when signals act through a form of positive feedback--survival signals. We operationalize these signals as the somatic niche and refer to their production as somatic niche construction. We find that tissue development switches to an autonomous state, independent of cellular signals, when errors are unmanageably high or density regulation is very strong. A signal-selective regime--strong niche dependence--is favored at low to intermediate error, assuming compartmentalized density dependence.

  11. Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells

    PubMed Central

    Panula, Sarita; Medrano, Jose V.; Kee, Kehkooi; Bergström, Rosita; Nguyen, Ha Nam; Byers, Blake; Wilson, Kitchener D.; Wu, Joseph C.; Simon, Carlos; Hovatta, Outi; Reijo Pera, Renee A.

    2011-01-01

    Historically, our understanding of molecular genetic aspects of human germ cell development has been limited, at least in part due to inaccessibility of early stages of human development to experimentation. However, the derivation of pluripotent stem cells may provide the necessary human genetic system to study germ cell development. In this study, we compared the potential of human induced pluripotent stem cells (iPSCs), derived from adult and fetal somatic cells to form primordial and meiotic germ cells, relative to human embryonic stem cells. We found that ∼5% of human iPSCs differentiated to primordial germ cells (PGCs) following induction with bone morphogenetic proteins. Furthermore, we observed that PGCs expressed green fluorescent protein from a germ cell-specific reporter and were enriched for the expression of endogenous germ cell-specific proteins and mRNAs. In response to the overexpression of intrinsic regulators, we also observed that iPSCs formed meiotic cells with extensive synaptonemal complexes and post-meiotic haploid cells with a similar pattern of ACROSIN staining as observed in human spermatids. These results indicate that human iPSCs derived from reprogramming of adult somatic cells can form germline cells. This system may provide a useful model for molecular genetic studies of human germline formation and pathology and a novel platform for clinical studies and potential therapeutical applications. PMID:21131292

  12. Induction of differentiation by down-regulation of Nanog and Rex-1 in cord blood derived unrestricted somatic stem cells.

    PubMed

    Langroudi, Lida; Forouzandeh, Mehdi; Soleimani, Masoud; Atashi, Amir; Golestaneh, Azadeh Fahim

    2013-07-01

    Stem cells with high self-renewal and tissue regeneration potentials are the core components of regenerative medicine. Adult stem cells with many available sources, high repairing ability, and also possessing no ethical issues are popular candidates in the clinical field. In this study we looked upon the effects of two transcription factors Nanog and Rex-1 in self-renewal and differentiation abilities of a subpopulation of cord blood stem cells known as unrestricted somatic stem cells (USSCs). USSCs were expanded and transfected in vitro with siRNAs targeting either Nanog, Rex-1, and in combination. Gene suppressions were achieved at both transcript and proteome level. Differentiations were evaluated by specific Real time PCR and differentiating staining. Nanog knock down revealed a significant increase in osteogenic markers, Osteocalcin and Osteopontin expression as well as a positive Alizarin Red staining, which proposes Osteogenesis. This treatment also became positive for Oil Red staining, implying adipogenic differentiation as well. In contrast, Rex-1 knock down showed an increase in MAP II and Nestin expression, which is a hall mark of neural differentiation. Surprisingly, treatment with both siRNAs did not express any changes in any of the assessed markers. Therefore, our results indicated a bilateral mesenchymal differentiation for Nanog and a neural lineage fate for Rex-1 suppression. Considering that both transcription factors are core activators of self-renewal and also are orchestrating with other factors, our results imply a positive feedback in response to changes in the regulatory network of self-renewal.

  13. Genomic Stability of Lyophilized Sheep Somatic Cells before and after Nuclear Transfer

    PubMed Central

    Iuso, Domenico; Czernik, Marta; Di Egidio, Fiorella; Sampino, Silvestre; Zacchini, Federica; Bochenek, Michal; Smorag, Zdzislaw; Modlinski, Jacek A.; Ptak, Grazyna; Loi, Pasqualino

    2013-01-01

    The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT. PMID:23308098

  14. Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer.

    PubMed

    Wu, Zhenfang; Xu, Zhiqian; Zou, Xian; Zeng, Fang; Shi, Junsong; Liu, Dewu; Urschitz, Johann; Moisyadi, Stefan; Li, Zicong

    2013-12-01

    The production of animals by somatic cell nuclear transfer (SCNT) is inefficient, with approximately 2% of micromanipulated oocytes going to term and resulting in live births. However, it is the most commonly used method for the generation of cloned transgenic livestock as it facilitates the attainment of transgenic animals once the nuclear donor cells are stably transfected and more importantly as alternatives methods of transgenesis in farm animals have proven even less efficient. Here we describe piggyBac-mediated transposition of a transgene into porcine primary cells and use of these genetically modified cells as nuclear donors for the generation of transgenic pigs by SCNT. Gene transfer by piggyBac transposition serves to provide an alternative approach for the transfection of nuclear donor cells used in SCNT.

  15. De novo generation of HSCs from somatic and pluripotent stem cell sources

    PubMed Central

    Vo, Linda T.

    2015-01-01

    Generating human hematopoietic stem cells (HSCs) from autologous tissues, when coupled with genome editing technologies, is a promising approach for cellular transplantation therapy and for in vitro disease modeling, drug discovery, and toxicology studies. Human pluripotent stem cells (hPSCs) represent a potentially inexhaustible supply of autologous tissue; however, to date, directed differentiation from hPSCs has yielded hematopoietic cells that lack robust and sustained multilineage potential. Cellular reprogramming technologies represent an alternative platform for the de novo generation of HSCs via direct conversion from heterologous cell types. In this review, we discuss the latest advancements in HSC generation by directed differentiation from hPSCs or direct conversion from somatic cells, and highlight their applications in research and prospects for therapy. PMID:25762177

  16. Inhibition of GSK-3β enhances neural differentiation in unrestricted somatic stem cells.

    PubMed

    Dastjerdi, Fatemeh Vahid; Zeynali, Bahman; Tafreshi, Azita Parvaneh; Shahraz, Anahita; Chavoshi, Mahin Sadat; Najafabadi, Irandokht Khaki; Vardanjani, Marzieh Mowlavi; Atashi, Amir; Soleimani, Masoud

    2012-11-01

    GSK-3β is a key molecule in several signalling pathways, including the Wnt/β-catenin signalling pathway. There is increasing evidence suggesting Wnt/β-catenin signalling is involved in the neural differentiation of embryonic, somatic and neural stem cells. However, a large body of evidence indicates that this pathway maintains stem cells in a proliferative state. To address this controversy, we have investigated whether the Wnt/β-catenin pathway is present and involved in the neural differentiation of newly introduced USSCs (unrestricted somatic stem cells). Our results indicate that the components of Wnt/β-catenin signalling are present in undifferentiated USSCs. We also show that the treatment of neurally induced USSCs with BIO (6-bromoindirubin-3'-oxime), a specific GSK-3β inhibitor and Wnt activator, for 5 and 10 days results in increased expression of a general neuronal marker (β-tubulin III). Moreover, the expression of pGSK-3β and stabilized β-catenin increased by BIO in neurally induced USSCs, indicates that the Wnt pathway is activated and functional in these cells. Thus, inhibition of GSK-3β in USSCs enhances their neural differentiation, which suggests a positive role of the Wnt/β-catenin signalling pathway towards neural fate.

  17. Dynamic and static maintenance of epigenetic memory in pluripotent and somatic cells.

    PubMed

    Shipony, Zohar; Mukamel, Zohar; Cohen, Netta Mendelson; Landan, Gilad; Chomsky, Elad; Zeliger, Shlomit Reich; Fried, Yael Chagit; Ainbinder, Elena; Friedman, Nir; Tanay, Amos

    2014-09-01

    Stable maintenance of gene regulatory programs is essential for normal function in multicellular organisms. Epigenetic mechanisms, and DNA methylation in particular, are hypothesized to facilitate such maintenance by creating cellular memory that can be written during embryonic development and then guide cell-type-specific gene expression. Here we develop new methods for quantitative inference of DNA methylation turnover rates, and show that human embryonic stem cells preserve their epigenetic state by balancing antagonistic processes that add and remove methylation marks rather than by copying epigenetic information from mother to daughter cells. In contrast, somatic cells transmit considerable epigenetic information to progenies. Paradoxically, the persistence of the somatic epigenome makes it more vulnerable to noise, since random epimutations can accumulate to massively perturb the epigenomic ground state. The rate of epigenetic perturbation depends on the genomic context, and, in particular, DNA methylation loss is coupled to late DNA replication dynamics. Epigenetic perturbation is not observed in the pluripotent state, because the rapid turnover-based equilibrium continuously reinforces the canonical state. This dynamic epigenetic equilibrium also explains how the epigenome can be reprogrammed quickly and to near perfection after induced pluripotency.

  18. Production efficiency and telomere length of the cloned pigs following serial somatic cell nuclear transfer.

    PubMed

    Kurome, Mayuko; Hisatomi, Hisashi; Matsumoto, Shirou; Tomii, Ryo; Ueno, Satoshi; Hiruma, Katsumi; Saito, Hitoshi; Nakamura, Kimitoshi; Okumura, Kenji; Matsumoto, Mitsuhito; Kaji, Yuji; Endo, Fumio; Nagashima, Hiroshi

    2008-08-01

    The aim of the present study was to examine the production efficiency of cloned pigs by serial somatic cell nuclear transfer (SCNT) and to ascertain any changes in the telomere lengths of multiple generations of pigs. Using fetal fibroblasts as the starting nuclear donor cells, porcine salivary gland progenitor cells were collected from the resultant first-generation cloned pigs to successively produce second- and third-generation clones, with no significant differences in production efficiency, which ranged from 1.4% (2/140) to 3.3% (13/391) among the 3 generations. The average telomere lengths (terminal restriction fragment values) for the first, second and third generation clones were 16.3, 18.1 and 20.5 kb, respectively, and were comparable to those in age-matched controls. These findings suggest that third-generation cloned pigs can be produced by serial somatic cell cloning without compromising production efficiency and that the telomere lengths of cloned pigs from the first to third generations are normal. PMID:18490858

  19. Oncogenic transformation of Drosophila somatic cells induces a functional piRNA pathway.

    PubMed

    Fagegaltier, Delphine; Falciatori, Ilaria; Czech, Benjamin; Castel, Stephane; Perrimon, Norbert; Simcox, Amanda; Hannon, Gregory J

    2016-07-15

    Germline genes often become re-expressed in soma-derived human cancers as "cancer/testis antigens" (CTAs), and piRNA (PIWI-interacting RNA) pathway proteins are found among CTAs. However, whether and how the piRNA pathway contributes to oncogenesis in human neoplasms remain poorly understood. We found that oncogenic Ras combined with loss of the Hippo tumor suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from annotated generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also altered expression of some protein-coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that, at least in this context, the piRNA pathway may play a functional role in cancer. PMID:27474441

  20. Molecular detection of chromosomal abnormalities in germ and somatic cells of aged male mice

    SciTech Connect

    Lowe, X.; Baulch, J.; Quintana, L.; Ramsey, M.; Breneman, J.; Tucker, J.; Wyrobek, A.; Collins, B.; Allen, J.; Holland, N.

    1994-12-31

    Three cytogenetic methods were applied to eight B6C3F1 male mice aged 22.5 - 30.5mo to determine if advanced age was associated with an elevated risk of producing chromosomally defective germinal and somatic cells; sperm aneuploidy analysis by multi-color fluorescence in situ hybridization for three chromosomes, spermatid micronucleus analysis with anti-kinetochore antibodies, and translocation analysis of somatic metaphases by {open_quotes}painting{close_quotes} for two chromosomes. Eight mice aged 2.4mo served as controls. Sperm aneuploidy was measured by multi-color fluorescence in situ co-hybridization with DNA probes specific for chromosomes X, Y and 8, scoring 10,000 cells per animal. The aged group showed significant 1.5 - 2.0 fold increases in the hyperhaploidy phenotypes X-X-8, Y-Y-8, 8-8-Y, and 8-8-X with the greater effects appearing in animals aged >29mo. The aged group also showed significantly increased frequencies of micronucleated spermatids (2.0 vs 0.4 per 1000; all were kinetochore negative). Analysis of metaphase chromosomes from blood by {open_quotes}painting{close_quotes} of chromosomes 2 and 8 yielded 4 translocation per 858 cell-equivalents in the aged group which was a non-significant elevation over 0/202 in controls. Although interpretation must be cautious due to the small number of animals analyzed, these findings suggest that advanced paternal age may be a risk factor for chromosomal abnormalities of reproductive and somatic importance.

  1. Microarray Analysis of Siberian Ginseng Cyclic Somatic Embryogenesis Culture Systems Provides Insight into Molecular Mechanisms of Embryogenic Cell Cluster Generation

    PubMed Central

    Zhou, Chenguang; Liu, Likun; Li, Chenghao

    2014-01-01

    Four systems of cyclic somatic embryogenesis of Siberian ginseng (Eleutherococcus senticosus Maxim) were used to study the mechanism of embryonic cell cluster generation. The first, direct somatic embryo induction (DSEI), generates secondary embryos directly from the primary somatic embryos; the second, direct embryogenic cell cluster induction (DEC)), induces embryogenic cell clusters directly from somatic embryos in agar medium. Subsequently, we found that when DEC-derived somatic embryos are transferred to suspension culture or a bioreactor culture, only somatic embryos are induced, and embryogenic cell clusters cannot form. Therefore, these new lines were named DEC cultured by liquid medium (ECS) and DEC cultured by bioreactor (ECB), respectively. Transmission electron microscopy showed that DEC epidermal cells contained a variety of inclusions, distinct from other lines. A cDNA library of DEC was constructed, and 1,948 gene clusters were obtained and used as probes. RNA was prepared from somatic embryos from each of the four lines and hybridized to a microarray. In DEC, 7 genes were specifically upregulated compared with the other three lines, and 4 genes were downregulated. EsXTH1 and EsPLT1, which were among the genes upregulated in DEC, were cloned using the rapid amplification of cDNA ends (RACE). Real-time quantitative PCR showed EsXTH1 was more highly expressed in DEC than in other lines throughout the culture cycle, and EsPLT1 expression in DEC increased as culture duration increased, but remained at a low expression level in other lines. These results suggest that EsXTH1 and EsPLT1 may be the essential genes that play important roles during the induction of embryogenic cell clusters. PMID:24743225

  2. Life histories have a history: effects of past and present conditions on adult somatic growth rates in wild Trinidadian guppies.

    PubMed

    Auer, Sonya K; Lopez-Sepulcre, Andrés; Heatherly, Thomas; Kohler, Tyler J; Bassar, Ronald D; Thomas, Steven A; Reznick, David N

    2012-07-01

    1. Environmental conditions in the present, more recent past and during the juvenile stage can have significant effects on adult performance and population dynamics, but their relative importance and potential interactions remain unexplored. 2. We examined the influence of food availability at the time of sampling, 2 months prior and during the juvenile stage on adult somatic growth rates in wild Trinidadian guppies (Poecilia reticulata). 3. We found that food availability during both the early and later parts of an individual's ontogeny had important consequences for adult growth strategies, but the direction of these effects differed among life stages and their magnitude, in some cases, depended on food levels experienced during other life stages. Current food levels and those 2 months prior to growth measurements had positive effects on adult growth rate; though, food levels 2 months prior had a greater effect on growth than current food levels. In contrast, the effects of food availability during the juvenile stage were higher in magnitude but opposite in direction to current food levels and those 2 months prior to growth rate measurements. Individuals recruiting under low food levels grew faster as adults than individuals recruiting during periods of high food availability. There was also a positive interaction between food levels experienced during the juvenile stage and 2 months prior such that the effects of juvenile food level diminished as the food level experienced 2 months prior increased. 4. These results suggest that the similar conditions occurring at different life stages can have different effects on short- and long-term growth strategies of individuals within a population. They also demonstrate that, while juvenile conditions can have lasting effects on adult performance, the strength of that effect can be dampened by environmental conditions experienced as an adult. 5. A simultaneous consideration of past events in both the

  3. Mapping a marsupial X chromosome using kangaroo-mouse somatic cell hybrids.

    PubMed

    Donald, J A; Hope, R M

    1981-01-01

    A series of marsupial-eutherian somatic cell hybrids was produced by fusion between lymphocytes from the red kangaroo (Macropus rufus) and HPRT-deficient mouse cells. The hybrids lost marsupial chromosomes and could therefore be used to map marsupial genes. Several of the hybrids contained a complete red kangaroo X chromosome and expressed the kangaroo form of the enzymes HPRT, G6PD, and PGKA. A number of HPRT-deficient revertant cell lines were derived from the hybrids. These possessed a variety of partially deleted X chromosomes. With these cell lines, it has been possible to establish the X-linkage of the genes for HPRT, G6PD, and PGKA in this marsupial and to localize these three genes to the terminal portion of the euchromatic arm of the red kangaroo X chromosome.

  4. Plant cell electrophysiology: applications in growth enhancement, somatic hybridisation and gene transfer.

    PubMed

    Ochatt, Sergio

    2013-12-01

    The use and exploitation of electrophysiology with plant cells have witnessed a slow but steady increase for a number of purposes in recent years. First envisaged only as a tool for the recovery of somatic hybrid plants following protoplast electrofusion, or for transient and/or stable genetic transformation following electroporation-mediated entry of foreign genes into protoplasts and cells, electrophysiological studies with plant cells and tissues have since spanned into other areas, and particularly for the assessment of the possible effects of electric and electromagnetic fields on the subsequent growth and differentiation competences of the electro-treated cells. This review will critically discuss these various applications of electrophysiology and will also aim at analysing the fundamental physiological and physico-chemical mechanisms underlying them. PMID:23562891

  5. Inducing somatic meiosis-like reduction at high frequency by caffeine in root-tip cells of Vicia faba.

    PubMed

    Chen, Y; Zhang, L; Zhou, Y; Geng, Y; Chen, Z

    2000-07-20

    Germinated seeds of Vicia faba were treated in caffeine solutions of different concentration for different durations to establish the inducing system of somatic meiosis-like reduction. The highest frequency of somatic meiosis-like reduction could reach up to 54.0% by treating the root tips in 70 mmol/l caffeine solution for 2 h and restoring for 24 h. Two types of somatic meiosis-like reduction were observed. One was reductional grouping, in which the chromosomes in a cell usually separated into two groups, and the role of spindle fibers did not show. The other type was somatic meiosis, which was analogous to meiosis presenting in gametogenesis, and chromosome pairing and chiasmata were visualized.

  6. Stn1 is critical for telomere maintenance and long-term viability of somatic human cells.

    PubMed

    Boccardi, Virginia; Razdan, Neetu; Kaplunov, Jessica; Mundra, Jyoti J; Kimura, Masayuki; Aviv, Abraham; Herbig, Utz

    2015-06-01

    Disruption of telomere maintenance pathways leads to accelerated entry into cellular senescence, a stable proliferative arrest that promotes aging-associated disorders in some mammals. The budding yeast CST complex, comprising Cdc13, Stn1, and Ctc1, is critical for telomere replication, length regulation, and end protection. Although mammalian homologues of CST have been identified recently, their role and function for telomere maintenance in normal somatic human cells are still incompletely understood. Here, we characterize the function of human Stn1 in cultured human fibroblasts and demonstrate its critical role in telomere replication, length regulation, and function. In the absence of high telomerase activity, shRNA-mediated knockdown of hStn1 resulted in aberrant and fragile telomeric structures, stochastic telomere attrition, increased telomere erosion rates, telomere dysfunction, and consequently accelerated entry into cellular senescence. Oxidative stress augmented the defects caused by Stn1 knockdown leading to almost immediate cessation of cell proliferation. In contrast, overexpression of hTERT suppressed some of the defects caused by hStn1 knockdown suggesting that telomerase can partially compensate for hStn1 loss. Our findings reveal a critical role for human Stn1 in telomere length maintenance and function, supporting the model that efficient replication of telomeric repeats is critical for long-term viability of normal somatic mammalian cells.

  7. Intermediate filaments promote nuclear mechanical constraints during somatic cell nuclear transfer in the mouse.

    PubMed

    Gall, Laurence; Brochard, Vincent; Ruffini, Sylvie; Laffont, Ludivine; Fleurot, Renaud; Lavin, Tiphaine Aguirre; Jouneau, Alice; Beaujean, Nathalie

    2012-12-01

    The somatic cell nuclear transfer (SCNT) procedure requires nuclear remodeling to return differentiated somatic nuclei to the totipotent undifferentiated stage. We hypothesize that mechanical constraints might occur upon SCNT and thereby affect nuclear remodeling. Therefore, we analyzed the nuclear structures upon SCNT using as donors either wild-type fibroblasts with a dense vimentin network or vimentin-deprived cells [embryonic stem cells (ESCs) and fibroblasts invalidated for vimetin]. We demonstrated that following nuclear transfer of wild-type fibroblasts, vimentin intermediate filaments (IFs) persisted around the transplanted nuclei and 88% of them presented severe distortions. We also showed that the presence of vimentin filaments in the reconstructed embryos was correlated with DNA damage, as evidenced by γH2A.X foci. On the other hand, when ESCs or vimentin-null (Vim(-/-)) fibroblasts devoid of IFs were used as nuclear donors, no nuclear distortion and less DNA damage were observed. Altogether we believe that the introduction of vimentin into recipient oocytes during SCNT induces a mechanical constraint on the transplanted nucleus that is responsible for nuclear distortions and DNA damage. This could lead to incomplete reprogramming that would be detrimental to further embryonic development.

  8. Bioinformatic analyses of sense and antisense expression from terminal inverted repeat transposons in Drosophila somatic cells.

    PubMed

    Harrington, Andrew W; Steiniger, Mindy

    2016-01-01

    Understanding regulation of transposon movement in somatic cells is important as mobile elements can cause detrimental genomic rearrangements. Generally, transposons move via one of 2 mechanisms; retrotransposons utilize an RNA intermediate, therefore copying themselves and amplifying throughout the genome, while terminal inverted repeat transposons (TIR Tns) excise DNA sequences from the genome and integrate into a new location. Our recently published work indicates that retrotransposons in Drosophila tissue culture cells are actively transcribed in the antisense direction. Our data support a model in which convergent transcription of retrotransposons from intra element transcription start sites results in complementary RNAs that hybridize to form substrates for Dicer-2, the endogenous small interfering (esi)RNA generating enzyme. Here, we extend our previous analysis to TIR Tns. In contrast to retrotransposons, our data show that antisense TIR Tn RNAs result from transcription of intronic TIR Tns oriented antisense to their host genes. Also, disproportionately less esiRNAs are generated from TIR transcripts than from retrotransposons and transcription of very few individual TIR Tns could be confirmed. Collectively, these data support a model in which TIR Tns are regulated at the level of Transposase production while retrotransposons are regulated with esiRNA post-transcriptional mechanisms in Drosophila somatic cells. PMID:26986720

  9. Identification of key factors conquering developmental arrest of somatic cell cloned embryos by combining embryo biopsy and single-cell sequencing

    PubMed Central

    Liu, Wenqiang; Liu, Xiaoyu; Wang, Chenfei; Gao, Yawei; Gao, Rui; Kou, Xiaochen; Zhao, Yanhong; Li, Jingyi; Wu, You; Xiu, Wenchao; Wang, Su; Yin, Jiqing; Liu, Wei; Cai, Tao; Wang, Hong; Zhang, Yong; Gao, Shaorong

    2016-01-01

    Differentiated somatic cells can be reprogrammed into totipotent embryos through somatic cell nuclear transfer. However, most cloned embryos arrest at early stages and the underlying molecular mechanism remains largely unexplored. Here, we first developed a somatic cell nuclear transfer embryo biopsy system at two- or four-cell stage, which allows us to trace the developmental fate of the biopsied embryos precisely. Then, through single-cell transcriptome sequencing of somatic cell nuclear transfer embryos with different developmental fates, we identified that inactivation of Kdm4b, a histone H3 lysine 9 trimethylation demethylase, functions as a barrier for two-cell arrest of cloned embryos. Moreover, we discovered that inactivation of another histone demethylase Kdm5b accounts for the arrest of cloned embryos at the four-cell stage through single-cell analysis. Co-injection of Kdm4b and Kdm5b can restore transcriptional profiles of somatic cell nuclear transfer embryos and greatly improve the blastocyst development (over 95%) as well as the production of cloned mice. Our study therefore provides an effective approach to identify key factors responsible for the developmental arrest of somatic cell cloned embryos. PMID:27462457

  10. Identification of key factors conquering developmental arrest of somatic cell cloned embryos by combining embryo biopsy and single-cell sequencing.

    PubMed

    Liu, Wenqiang; Liu, Xiaoyu; Wang, Chenfei; Gao, Yawei; Gao, Rui; Kou, Xiaochen; Zhao, Yanhong; Li, Jingyi; Wu, You; Xiu, Wenchao; Wang, Su; Yin, Jiqing; Liu, Wei; Cai, Tao; Wang, Hong; Zhang, Yong; Gao, Shaorong

    2016-01-01

    Differentiated somatic cells can be reprogrammed into totipotent embryos through somatic cell nuclear transfer. However, most cloned embryos arrest at early stages and the underlying molecular mechanism remains largely unexplored. Here, we first developed a somatic cell nuclear transfer embryo biopsy system at two- or four-cell stage, which allows us to trace the developmental fate of the biopsied embryos precisely. Then, through single-cell transcriptome sequencing of somatic cell nuclear transfer embryos with different developmental fates, we identified that inactivation of Kdm4b, a histone H3 lysine 9 trimethylation demethylase, functions as a barrier for two-cell arrest of cloned embryos. Moreover, we discovered that inactivation of another histone demethylase Kdm5b accounts for the arrest of cloned embryos at the four-cell stage through single-cell analysis. Co-injection of Kdm4b and Kdm5b can restore transcriptional profiles of somatic cell nuclear transfer embryos and greatly improve the blastocyst development (over 95%) as well as the production of cloned mice. Our study therefore provides an effective approach to identify key factors responsible for the developmental arrest of somatic cell cloned embryos. PMID:27462457

  11. Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs.

    PubMed

    Kang, Eunju; Wang, Xinjian; Tippner-Hedges, Rebecca; Ma, Hong; Folmes, Clifford D L; Gutierrez, Nuria Marti; Lee, Yeonmi; Van Dyken, Crystal; Ahmed, Riffat; Li, Ying; Koski, Amy; Hayama, Tomonari; Luo, Shiyu; Harding, Cary O; Amato, Paula; Jensen, Jeffrey; Battaglia, David; Lee, David; Wu, Diana; Terzic, Andre; Wolf, Don P; Huang, Taosheng; Mitalipov, Shoukhrat

    2016-05-01

    The genetic integrity of iPSCs is an important consideration for therapeutic application. In this study, we examine the accumulation of somatic mitochondrial genome (mtDNA) mutations in skin fibroblasts, blood, and iPSCs derived from young and elderly subjects (24-72 years). We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but are not detectable in whole tissues. The frequency of mtDNA defects in iPSCs increased with age, and many mutations were non-synonymous or resided in RNA coding genes and thus can lead to respiratory defects. Our results highlight a need to monitor mtDNA mutations in iPSCs, especially those generated from older patients, and to examine the metabolic status of iPSCs destined for clinical applications. PMID:27151456

  12. Analysis of protein coding mutations in hiPSCs and their possible role during somatic cell reprogramming

    PubMed Central

    Ruiz, Sergio; Gore, Athurva; Li, Zhe; Panopoulos, Athanasia D.; Montserrat, Nuria; Fung, Ho-Lim; Giorgetti, Alessandra; Bilic, Josipa; Batchelder, Erika M.; Zaehres, Holm; Schöler, Hans R.; Zhang, Kun; Belmonte, Juan Carlos Izpisua

    2013-01-01

    Recent studies indicate that human induced pluripotent stem cells (hiPSCs) contain genomic structural variations and point mutations in coding regions. However, these studies have focused on fibroblast-derived hiPSCs, and it is currently unknown whether the use of alternative somatic cell sources with varying reprogramming efficiencies would result in different levels of genetic alterations. Here we characterize the genomic integrity of eight hiPSC lines derived from five different non-fibroblast somatic cell types. We show that protein-coding mutations are a general feature of the hiPSC state and are independent of somatic cell source. Furthermore, we analyze a total of 17 point mutations found in hiPSCs and demonstrate that they do not generally facilitate the acquisition of pluripotency and thus are not likely to provide a selective advantage for reprogramming. PMID:23340422

  13. Transcription Factor Oct1 Is a Somatic and Cancer Stem Cell Determinant

    PubMed Central

    Shelton, Dawne; Andersen, Jared N.; Chidester, Stephanie; Kang, Jinsuk; Gligorich, Keith M.; Jones, David A.; Spangrude, Gerald J.; Welm, Bryan E.; Tantin, Dean

    2012-01-01

    Defining master transcription factors governing somatic and cancer stem cell identity is an important goal. Here we show that the Oct4 paralog Oct1, a transcription factor implicated in stress responses, metabolic control, and poised transcription states, regulates normal and pathologic stem cell function. Oct1HI cells in the colon and small intestine co-express known stem cell markers. In primary malignant tissue, high Oct1 protein but not mRNA levels strongly correlate with the frequency of CD24LOCD44HI cancer-initiating cells. Reducing Oct1 expression via RNAi reduces the proportion of ALDHHI and dye effluxHI cells, and increasing Oct1 increases the proportion of ALDHHI cells. Normal ALDHHI cells harbor elevated Oct1 protein but not mRNA levels. Functionally, we show that Oct1 promotes tumor engraftment frequency and promotes hematopoietic stem cell engraftment potential in competitive and serial transplants. In addition to previously described Oct1 transcriptional targets, we identify four Oct1 targets associated with the stem cell phenotype. Cumulatively, the data indicate that Oct1 regulates normal and cancer stem cell function. PMID:23144633

  14. [Regulation of somatic embryogenesis in Panax ginseng C. A. Meyer cell cultures by PgCDPK2DS1].

    PubMed

    Shumakova, O A; Kiselev, K V

    2014-06-01

    We isolated the full-length cDNA of PgCDPK2DS1 gene, the expression of which was significantly increased at early stages of embryo development in cell cultures of ginseng P. ginseng 2c3. Interest in this gene also was supported by its nonstandard structure: the amino acid sequence of the PgCDPK2DS1 gene contained only the N-terminal domain and 80% of the kinase domain. Overexpression of the PgCDPK2DS1 gene in nonembryonic calli 1c resulted in the appearance of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture 1c-2d. Also, expression of the plant embryogenesis marker genes WUS and SERK significantly increased in cell culture 1c-2d. The observed embryo-like structures were at early stages of embryo development; attempts to obtain an adult plant from these embryo-like structures were unsuccessful. Overexpression of PgCDPK2DS1 gene in the embryonic cell culture PG resulted in a decrease of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture PG-2d. Moreover, expression of the plant embryogenesis marker genes WUS and SERK and expression of the endogenous PgCDPK2DS1 significantly decreased in the cell culture PG-2d. Thus, for the first time it was shown that the PgCDPK2DS1 gene is involved in the regulation of somatic embryogenesis in P. ginseng cell cultures.

  15. Improved efficiency of bovine cloning by autologous somatic cell nuclear transfer.

    PubMed

    Yang, Xiao-Yu; Li, Hua; Ma, Qing-Wen; Yan, Jing-Bin; Zhao, Jiang-Guo; Li, Hua-Wei; Shen, Hai-Qing; Liu, Hai-Feng; Huang, Ying; Huang, Shu-Zhen; Zeng, Yi-Tao; Zeng, Fanyi

    2006-11-01

    Somatic cell nuclear transfer (SCNT) has been used for the cloning of various mammals. However, the rates of successful, healthy birth are generally poor. To improve cloning efficiency, we report the utilization of an 'autologous SCNT' cloning technique in which the somatic nucleus of a female bovine donor is transferred to its own enucleated oocyte recovered by ovum pick up, in contrast to the routine 'allogeneic SCNT' procedure using oocytes from unrelated females. Our results showed that embryos derived from autologous SCNThave significantly higher developmental competence than those derived from allogeneic SCNT, especiallyat the eight-cell (60 vs 44%), morula (45 vs 36%), and blastocyst (38 vs 23%) stages. The pregnancy and birth rates were also higher for the autologous (39 and 23%), compared to the allogeneic (22 and 6%) SCNT groups. Genome-wide histone3-lysine9 methylation profiles reveal that autologous SCNTembryos have less epigenetic defects than the allogeneic SCNTembryos. This study indicates that autologous SCNT can improve the efficiency of bovine cloning with less reprogramming deficiency.

  16. Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

    SciTech Connect

    Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Park, Sang-Min; Ryu, Junghyun; Ahn, Jin Seop; Heo, Soon Young; Kang, Jee Hyun; Choi, You Jung; Choi, Seong-Jun; Shim, Hosup

    2014-10-03

    Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.

  17. Spontaneous and induced chromosome damage in somatic cells of sporadic and familial Alzheimer's disease patients.

    PubMed

    Trippi, F; Botto, N; Scarpato, R; Petrozzi, L; Bonuccelli, U; Latorraca, S; Sorbi, S; Migliore, L

    2001-07-01

    Alzheimer's disease (AD) is a neurodegenerative disorder of the elderly with a complex etiology due to the interaction between genetic and environmental factors. At least 15% of cases are inherited as an autosomal dominant mutation, but the majority are sporadic. We evaluated cytogenetic alterations, both spontaneous and chemical-induced [aluminium (Al) and griseofulvin (GF)], by means of the micronucleus (MN) test in lymphocytes or skin fibroblasts of 14 patients with sporadic and eight with familial Alzheimer's disease (FAD), respectively. The spontaneous MN frequencies of sporadic (20.8 +/- 9.2) and familial (20.7 +/- 4.6) AD patients are significantly higher than those of the respective control groups (9.0 +/- 6.8 and 6.7 +/- 3.4). In all AD patients, GF significantly increased the spontaneous MN frequency of somatic cells to a lesser extent (P < 0.05) as compared with the control group. Al treatment did not induce MN in AD patients. The results of the present study indicate that different types of somatic cells from sporadic and familial AD patients show comparable levels of spontaneous cytogenetic anomalies, and MN induction is partially reduced or lacking according to the type of chemical treatments. PMID:11420400

  18. Aberrant expression of Xist in aborted porcine fetuses derived from somatic cell nuclear transfer embryos.

    PubMed

    Yuan, Lin; Wang, Anfeng; Yao, Chaogang; Huang, Yongye; Duan, Feifei; Lv, Qinyan; Wang, Dongxu; Ouyang, Hongsheng; Li, Zhanjun; Lai, Liangxue

    2014-01-01

    Cloned pigs generated by somatic cell nuclear transfer (SCNT) show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR) and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP). q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos. PMID:25429426

  19. Many LINE1 elements contribute to the transcriptome of human somatic cells

    PubMed Central

    Rangwala, Sanjida H; Zhang, Lili; Kazazian, Haig H

    2009-01-01

    Background While LINE1 (L1) retroelements comprise nearly 20% of the human genome, the majority are thought to have been rendered transcriptionally inactive, due to either mutation or epigenetic suppression. How many L1 elements 'escape' these forms of repression and contribute to the transcriptome of human somatic cells? We have cloned out expressed sequence tags corresponding to the 5' and 3' flanks of L1 elements in order to characterize the population of elements that are being actively transcribed. We also examined expression of a select number of elements in different individuals. Results We isolated expressed sequence tags from human lymphoblastoid cell lines corresponding to 692 distinct L1 element sites, including 410 full-length elements. Four of the expression tagged sites corresponding to full-length elements from the human specific L1Hs subfamily were examined in European-American individuals and found to be differentially expressed in different family members. Conclusions A large number of different L1 element sites are expressed in human somatic tissues, and this expression varies among different individuals. Paradoxically, few elements were tagged at high frequency, indicating that the majority of expressed L1s are transcribed at low levels. Based on our preliminary expression studies of a limited number of elements in a single family, we predict a significant degree of inter-individual transcript-level polymorphism in this class of sequence. PMID:19772661

  20. Transgenic chicken, mice, cattle, and pig embryos by somatic cell nuclear transfer into pig oocytes.

    PubMed

    Gupta, Mukesh Kumar; Das, Ziban Chandra; Heo, Young Tae; Joo, Jin Young; Chung, Hak-Jae; Song, Hyuk; Kim, Jae-Hwan; Kim, Nam-Hyung; Lee, Hoon Taek; Ko, Dae Hwan; Uhm, Sang Jun

    2013-08-01

    This study explored the possibility of producing transgenic cloned embryos by interspecies somatic cell nuclear transfer (iSCNT) of cattle, mice, and chicken donor cells into enucleated pig oocytes. Enhanced green florescent protein (EGFP)-expressing donor cells were used for the nuclear transfer. Results showed that the occurrence of first cleavage did not differ significantly when pig, cattle, mice, or chicken cells were used as donor nuclei (p>0.05). However, the rate of blastocyst formation was significantly higher in pig (14.9±2.1%; p<0.05) SCNT embryos than in cattle (6.3±2.5%), mice (4.2±1.4%), or chicken (5.1±2.4%) iSCNT embryos. The iSCNT embryos also contained a significantly less number of cells per blastocyst than those of SCNT pig embryos (p<0.05). All (100%) iSCNT embryos expressed the EGFP gene, as evidenced by the green florescence under ultraviolet (UV) illumination. Microinjection of purified mitochondria from cattle somatic cells into pig oocytes did not have any adverse effect on their postfertilization in vitro development and embryo quality (p>0.05). Moreover, NCSU23 medium, which was designed for in vitro culture of pig embryos, was able to support the in vitro development of cattle, mice, and chicken iSCNT embryos up to the blastocyst stage. Taken together, these data suggest that enucleated pig oocytes may be used as a universal cytoplast for production of transgenic cattle, mice, and chicken embryos by iSCNT. Furthermore, xenogenic transfer of mitochondria to the recipient cytoplast may not be the cause for poor embryonic development of cattle-pig iSCNT embryos.

  1. Kinetochore fiber formation in animal somatic cells: dueling mechanisms come to a draw

    PubMed Central

    2008-01-01

    The attachment to and movement of a chromosome on the mitotic spindle are mediated by the formation of a bundle of microtubules (MTs) that tethers the kinetochore on the chromosome to a spindle pole. The origin of these “kinetochore fibers” (K fibers) has been investigated for over 125 years. As noted in 1944 by Schrader [Mitosis, Columbia University Press, New York, 110 pp.], there are three possible ways to form a K fiber: (a) it grows from the pole until it contacts the kinetochore, (b) it grows directly from the kinetochore, or (c) it forms as a result of an interaction between the pole and the chromosome. Since Schrader's time, it has been firmly established that K fibers in centrosome-containing animal somatic cells form as kinetochores capture MTs growing from the spindle pole (route a). It is now similarly clear that in cells lacking centrosomes, including higher plants and many animal oocytes, K fibers “self-assemble” from MTs generated by the chromosomes (route b). Can animal somatic cells form K fibers in the absence of centrosomes by the “self-assembly” pathway? In 2000, the answer to this question was shown to be a resounding “yes.” With this result, the next question became whether the presence of a centrosome normally suppresses K fiber self-assembly or if this route works concurrently with centrosome-mediated K-fiber formation. This question, too, has recently been answered: observations on untreated live animal cells expressing green fluorescent protein-tagged tubulin clearly show that kinetochores can nucleate the formation of their associated MTs in a unique manner in the presence of functional centrosomes. The concurrent operation of these two “dueling” routes for forming K fibers in animal cells helps explain why the attachment of kinetochores and the maturation of K fibers occur as quickly as they do on all chromosomes within a cell. PMID:16270218

  2. Induction of somatic embryogenesis in explants of shoot cultures established from adult Eucalyptus globulus and E. saligna × E. maidenii trees.

    PubMed

    Corredoira, E; Ballester, A; Ibarra, M; Vieitez, A M

    2015-06-01

    A reproducible procedure for induction of somatic embryogenesis (SE) from adult trees of Eucalyptus globulus Labill. and the hybrid E. saligna Smith × E. maidenii has been developed for the first time. Somatic embryos were obtained from both shoot apex and leaf explants of all three genotypes evaluated, although embryogenic frequencies were significantly influenced by the species/genotype, auxin and explant type. Picloram was more efficient for somatic embryo induction than naphthaleneacetic acid (NAA), with the highest frequency of induction being obtained in Murashige and Skoog medium containing 40 µM picloram and 40 mg l(-1) gum Arabic, in which 64% of the shoot apex explants and 68.8% of the leaf explants yielded somatic embryos. The embryogenic response of the hybrid was higher than that of the E. globulus, especially when NAA was used. The cultures initiated on picloram-containing medium consisted of nodular embryogenic structures surrounded by a mucilaginous coating layer that emerged from a watery callus developed from the initial explants. Cotyledonary somatic embryos were differentiated after subculture of these nodular embryogenic structures on a medium lacking plant growth regulators. Histological analysis confirmed the bipolar organization of the somatic embryos, with shoot and root meristems and closed procambial tissue that bifurcated into small cotyledons. The root pole was more differentiated than the shoot pole, which appeared to be formed by a few meristematic layers. Maintenance of the embryogenic lines by secondary SE was attained by subculturing individual cotyledonary embryos or small clusters of globular and torpedo embryos on medium with 16.11 µM NAA at 4- to 5-week intervals. Somatic embryos converted into plantlets after being transferred to liquid germination medium although plant regeneration remained poor. PMID:25877768

  3. Cognitive and somatic anxiety.

    PubMed

    Steptoe, A; Kearsley, N

    1990-01-01

    Three hundred and forty adults (including sports players, recreational exercisers, mediators and sedentary controls) completed three inventories purporting to measure cognitive and somatic aspects of anxiety. These were the Cognitive-Somatic Anxiety Questionnaire (CSAQ) devised by Schwartz, Davidson & Goleman (Psychosomatic Medicine, 40, 321-328, 1978), the Worry-Emotionality Scale (WES, Morris, Davis & Hutchens, Journal of Educational Psychology, 73, 541-555, 1981) and the Lehrer-Woolfolk (1982) Anxiety Symptom Questionnaire (LWASQ). Factor analysis of the CSAQ and WES identified distinct cognitive and somatic anxiety factors in both inventories. Higher somatic than cognitive ratings were recorded on the CSAQ and WES, while the pattern was reversed on the LWASQ. The CSAQ can tentatively be recommended as a useful measure of these two anxiety components. We were unable to confirm an observation made previously in the literature that practice of meditation is associated with reduced cognitive anxiety, or that exercise is linked with lower somatic anxiety.

  4. Stochastic modeling indicates that aging and somatic evolution in the hematopoetic system are driven by non-cell-autonomous processes.

    PubMed

    Rozhok, Andrii I; Salstrom, Jennifer L; DeGregori, James

    2014-12-01

    Age-dependent tissue decline and increased cancer incidence are widely accepted to be rate-limited by the accumulation of somatic mutations over time. Current models of carcinogenesis are dominated by the assumption that oncogenic mutations have defined advantageous fitness effects on recipient stem and progenitor cells, promoting and rate-limiting somatic evolution. However, this assumption is markedly discrepant with evolutionary theory, whereby fitness is a dynamic property of a phenotype imposed upon and widely modulated by environment. We computationally modeled dynamic microenvironment-dependent fitness alterations in hematopoietic stem cells (HSC) within the Sprengel-Liebig system known to govern evolution at the population level. Our model for the first time integrates real data on age-dependent dynamics of HSC division rates, pool size, and accumulation of genetic changes and demonstrates that somatic evolution is not rate-limited by the occurrence of mutations, but instead results from aged microenvironment-driven alterations in the selective/fitness value of previously accumulated genetic changes. Our results are also consistent with evolutionary models of aging and thus oppose both somatic mutation-centric paradigms of carcinogenesis and tissue functional decline. In total, we demonstrate that aging directly promotes HSC fitness decline and somatic evolution via non-cell-autonomous mechanisms.

  5. Activities of indigenous proteolytic enzymes in caprine milk of different somatic cell counts.

    PubMed

    Albenzio, M; Santillo, A; Kelly, A L; Caroprese, M; Marino, R; Sevi, A

    2015-11-01

    Individual caprine milk with different somatic cell counts (SCC) were studied with the aim of investigating the percentage distribution of leukocyte cell types and the activities of indigenous proteolytic enzymes; proteolysis of casein was also studied in relation to cell type following recovery from milk. The experiment was conducted on 5 intensively managed dairy flocks of Garganica goats; on the basis of SCC, the experimental groups were denoted low (L-SCC; <700,000 cells/mL), medium (M-SCC; from 701,000 to 1,500,000 cells/mL), and high (H-SCC; >1,501,000 cells/mL) SCC. Leukocyte distribution differed between groups; polymorphonuclear neutrophilic leukocytes were higher in M-SCC and H-SCC milk samples, the percentage macrophages was the highest in H-SCC, and levels of nonviable cells significantly decreased with increasing SCC. Activities of all the main proteolytic enzymes were affected by SCC; plasmin activity was the highest in H-SCC milk and the lowest in L-SCC, and elastase and cathepsin D activities were the highest in M-SCC. Somatic cell count influenced casein hydrolysis patterns, with less intact α- and β-casein in H-SCC milk. Higher levels of low electrophoretic mobility peptides were detected in sodium caseinate incubated with leukocytes isolated from L-SCC milk, independent of cell type, whereas among cells recovered from M-SCC milk, macrophages yielded the highest levels of low electrophoretic mobility peptides from sodium caseinate. The level of high electrophoretic mobility peptides was higher in sodium caseinate incubated with polymorphonuclear neutrophilic leukocytes and macrophages isolated from M-SCC, whereas the same fraction of peptides was always the highest, independent of leukocyte type, for cells recovered from H-SCC milk. In caprine milk, a level of 700,000 cells/mL represented the threshold for changes in leukocyte distribution, which is presumably related to the immune status of the mammary gland. Differences in the profile of

  6. MicroRNA-34c Expression in Donor Cells Influences the Early Development of Somatic Cell Nuclear Transfer Bovine Embryos

    PubMed Central

    Wang, Bo; Wang, Yongsheng; Zhang, Man; Du, Yue; Zhang, Yijun; Xing, Xupeng; Zhang, Lei; Su, JianMin

    2014-01-01

    Abstract The essence of the reprogramming activity of somatic cell nuclear transfer (SCNT) embryos is to produce normal fertilized embryos. However, reprogramming of somatic cells is not as efficient as the reprogramming of sperm. In this report, we describe the effect of an inducible, specific miR-34 microRNA expression in donor cells that enables a similar level of sperm:transgene expression on the early development of SCNT embryos. Our results showed that donor cells with doxycycline (dox)-induced miR-34c expression for the preparation of SCNT embryos resulted in altered developmental rates, histone modification (H3K9ac and H3K4me3), and extent of apoptosis. The cleavage rate and blastocyst formation of the induced nuclear transfer (NT) group were significantly increased. The immunofluorescence signal of H3K9ac in embryos in the induced NT group significantly increased in two-cell- and eight-cell-stage embryos; that of H3K4me3 increased significantly in eight-cell-stage embryos. Although significant differences in staining signals of apoptosis were not detected between groups, lower apoptosis levels were observed in the induced NT group. In conclusion, miR-34c expression induced by dox treatment enhances the developmental potential of SCNT embryos, modifies the epigenetic status, and changes blastocyst quality. PMID:25437869

  7. Incomplete DNA methylation underlies a transcriptional memory of the somatic cell in human iPS cells

    PubMed Central

    Ohi, Yuki; Qin, Han; Hong, Chibo; Blouin, Laure; Polo, Jose M.; Guo, Tingxia; Qi, Zhongxia; Downey, Sara L.; Manos, Philip D.; Rossi, Derrick J.; Yu, Jingwei; Hebrok, Matthias; Hochedlinger, Konrad; Costello, Joseph F.; Song, Jun S.; Ramalho-Santos, Miguel

    2013-01-01

    Human induced pluripotent stem (iPS) cells are remarkably similar to embryonic stem (ES) cells, but recent reports suggest that there may be important differences between them. We performed a systematic comparison of human iPS cells generated from hepatocytes (representative of endoderm), skin fibroblasts (mesoderm) and melanocytes (ectoderm). All low passage iPS cells analyzed retain a transcriptional memory of the original cells. The persistent expression of somatic genes can be partially explained by incomplete promoter DNA methylation. This epigenetic mechanism underlies a robust form of memory that can be found in iPS cells generated by multiple laboratories using different methods, including RNA transfection. Incompletely silenced genes tend to be isolated from other genes that are repressed during reprogramming, indicating that recruitment of the silencing machinery may be inefficient at isolated genes. Knockdown of the incompletely reprogrammed gene C9orf64 reduces the efficiency of human iPS cell generation, suggesting that somatic memory genes may be functionally relevant during reprogramming. PMID:21499256

  8. Femtosecond laser based enucleation of porcine oocytes for somatic cell nuclear transfer

    NASA Astrophysics Data System (ADS)

    Kütemeyer, K.; Lucas-Hahn, A.; Petersen, B.; Hassel, P.; Lemme, E.; Niemann, H.; Heisterkamp, A.

    2009-07-01

    Cloning of several mammalian species has been achieved by somatic cell nuclear transfer (SCNT) in recent years. However, this method still results in very low efficiencies around 1% which originate from suboptimal culture conditions and highly invasive techniques for oocyte enucleation and injection of the donor cell using micromanipulators. In this paper, we present a new minimal invasive method for oocyte imaging and enucleation based on the application of femtosecond (fs) laser pulses. After imaging of the oocyte with multiphoton microscopy, ultrashort pulses are focused onto the metaphase plate of MII-oocytes in order to ablate the DNA molecules. We show that fs laser based enucleation of porcine oocytes completely inhibits the first mitotic cleavage after parthenogenetic activation while maintaining intact oocyte morphology in most cases. In contrast, control groups without previous irradiation of the metaphase plate are able to develop to the blastocyst stage. Further experiments have to clarify the suitability of fs laser based enucleated oocytes for SCNT.

  9. Lack of complementation in somatic cell hybrids between fibroblasts from patients with different forms of cystinosis

    SciTech Connect

    Pellett, O.L.; Smith, M.L.; Greene, A.A.; Schneider, J.A. )

    1988-05-01

    Cystinosis is an autosomal recessive disease in which three clinical forms are recognized: infantile nephropathic, with renal tubular damage by 1 year of age and progressive glomerular insufficiency; intermediate, with tubular and glomerular insufficiency beginning at a later age; benign, with no kidney damage. Skin fibroblasts cultured from patients with all types of cystinosis show increased intralysosomal free (nonprotein) cystine; however, fibroblasts from heterozygotes have normal free-cystine values. To determine whether genetic complementation occurs between the different forms, somatic cell hybrids were constructed between cells from a patient with infantile nephropathic cystinosis and cells from patients with other types of cystinosis. If complementation occurred, the hybrids would be expected to have normal cystine levels. To construct hybrid cells, a universal parent cell type (TG1-neo), which was hypoxanthine/aminopterin/thymidine (HAT) sensitive and G418 resistant was constructed from an infantile nephropathic cystinosis fibroblast strain. Polyethylene glycol fusion of TG1-neo with other cells that are not HAT sensitive or G418 resistant allowed for selection of hybrid cells in a medium containing HAT and the aminoglycoside G418. As indicated by elevated cystine levels, complementation did not occur between TG1-neo and two different benign cystinosis strains, an intermediate cystinosis strain, or another nephropathic cystinosis cell strain. When a normal fibroblast strain was fused with TG1-neo, all 15 hybrid clones studied contained normal amounts of intracellular free cystine.

  10. A mutation of cdc-25.1 causes defects in germ cells but not in somatic tissues in C. elegans.

    PubMed

    Kim, Jiyoung; Lee, Ah-Reum; Kawasaki, Ichiro; Strome, Susan; Shim, Yhong-Hee

    2009-07-31

    By screening C. elegans mutants for severe defects in germline proliferation, we isolated a new loss-of-function allele of cdc-25.1, bn115. bn115 and another previously identified loss-of-function allele nr2036 do not exhibit noticeable cell division defects in the somatic tissues but have reduced numbers of germ cells and are sterile, indicating that cdc-25.1 functions predominantly in the germ line during postembryonic development, and that cdc-25.1 activity is probably not required in somatic lineages during larval development. We analyzed cell division of germ cells and somatic tissues in bn115 homozygotes with germline-specific anti-PGL-1 immunofluorescence and GFP transgenes that express in intestinal cells, in distal tip cells, and in gonadal sheath cells, respectively. We also analyzed the expression pattern of cdc-25.1 with conventional and quantitative RT-PCR. In the presence of three other family members of cdc-25 in C. elegans defects are observed only in the germ line but not in the somatic tissues in cdc-25.1 single mutants, and cdc-25.1 is expressed predominantly, if not exclusively, in the germ line during postembryonic stages. Our findings indicate that the function of cdc-25.1 is unique in the germ line but likely redundant with other members in the soma.

  11. The sexual identity of adult intestinal stem cells controls organ size and plasticity.

    PubMed

    Hudry, Bruno; Khadayate, Sanjay; Miguel-Aliaga, Irene

    2016-02-18

    Sex differences in physiology and disease susceptibility are commonly attributed to developmental and/or hormonal factors, but there is increasing realization that cell-intrinsic mechanisms play important and persistent roles. Here we use the Drosophila melanogaster intestine to investigate the nature and importance of cellular sex in an adult somatic organ in vivo. We find that the adult intestinal epithelium is a cellular mosaic of different sex differentiation pathways, and displays extensive sex differences in expression of genes with roles in growth and metabolism. Cell-specific reversals of the sexual identity of adult intestinal stem cells uncovers the key role this identity has in controlling organ size, reproductive plasticity and response to genetically induced tumours. Unlike previous examples of sexually dimorphic somatic stem cell activity, the sex differences in intestinal stem cell behaviour arise from intrinsic mechanisms that control cell cycle duration and involve a new doublesex- and fruitless-independent branch of the sex differentiation pathway downstream of transformer. Together, our findings indicate that the plasticity of an adult somatic organ is reversibly controlled by its sexual identity, imparted by a new mechanism that may be active in more tissues than previously recognized. PMID:26887495

  12. The sexual identity of adult intestinal stem cells controls organ size and plasticity

    PubMed Central

    Hudry, Bruno; Khadayate, Sanjay; Miguel-Aliaga, Irene

    2016-01-01

    SUMMARY Sex differences in physiology and disease susceptibility are commonly attributed to developmental and/or hormonal factors, but there is increasing realisation that cell-intrinsic mechanisms play important and persistent roles1,2. Here we use the Drosophila melanogaster intestine to investigate the nature and significance of cellular sex in an adult somatic organ in vivo. We find that the adult intestinal epithelium is a cellular mosaic of different sex differentiation pathways, and displays extensive sex differences in expression of genes with roles in growth and metabolism. Cell-specific reversals of the sexual identity of adult intestinal stem cells uncover its key roles in controlling organ size, its reproductive plasticity and its response to genetically induced tumours. Unlike previous examples of sexually dimorphic somatic stem cell activity, the sex differences in intestinal stem cell behaviour arise from intrinsic mechanisms, which control cell cycle duration and involve a new doublesex- and fruitless-independent branch of the sex differentiation pathway downstream of transformer. Together, our findings indicate that the plasticity of an adult somatic organ is reversibly controlled by its sexual identity, imparted by a new mechanism that may be active in more tissues than previously recognised. PMID:26887495

  13. Evaluation of mutagenic, recombinogenic and carcinogenic potential of (+)-usnic acid in somatic cells of Drosophila melanogaster.

    PubMed

    Machado, Nayane Moreira; de Rezende, Alexandre Azenha Alves; Nepomuceno, Júlio César; Tavares, Denise Crispim; Cunha, Wilson Roberto; Spanó, Mário Antônio

    2016-10-01

    The main of this study was to evaluate the mutagenic and carcinogenic potential of (+) - usnic acid (UA), using Somatic Mutation and Recombination Test (SMART) and the test for detecting epithelial tumor clones (wts) in Drosophila melanogaster. Larvae from 72 ± 4 h from Drosophila were fed with UA (5.0, 10.0 or 20.0 mM); urethane (10.0 mM) (positive control); and solvent (Milli-Q water, 1% Tween-80 and 3% ethanol) (negative control). ST cross produced increase in total mutant spots in the individuals treated with 5.0, 10.0 or 20.0 mM of UA. HB cross produced spot frequencies in the concentration of 5.0 mM that were higher than the frequency for the same concentration in the ST cross. In the highest concentrations the result was negative, which means that the difference observed can be attributed, in part, to the high levels of P450, suggesting that increasing the metabolic capacity maximized the toxic effect of these doses. In the evaluation of carcinogenesis using the wts test, the results obtained for the same concentrations of UA show a positive result for the presence of tumors when compared to the negative control. We conclude that UA has recombinogenic, mutagenic and carcinogenic effects on somatic cells in D. melanogaster. PMID:27497765

  14. Genetic toxicology of dental composite resin extracts in somatic cells in vivo.

    PubMed

    Arossi, Guilherme Anziliero; Dihl, Rafael Rodrigues; Lehmann, Mauricio; Reguly, Maria Luiza; de Andrade, Heloísa Helena Rodrigues

    2010-07-01

    The aim of this study was to assess the potential genetic toxicity associated to nine aqueous extracts from dental composite resins (Charisma, Fill Magic, Fill Magic Flow, Durafill, TPH Spectrum, Concept, Natural Look, Filtek Z250 and Filtek P60) and one random extract. Homologous mitotic recombination, point and chromosomal mutation effects were determined in somatic proliferative cells of Drosophila melanogaster exposed to aqueous extracts of the clinically used composites. Reproducible increases in clone mutant spot frequencies induced by diluted extract of Fill Magic Flow were observed. These increments were exclusively associated to the induction of homologous recombination - a genetic phenomenon involved in the loss of heterozygosis. The other eight composite resins and the random extract had no statistically significant effect on total spot frequencies - suggesting that they are non-genotoxic in the somatic mutation and recombination test assay, which agrees with the applications they have in dentistry. These findings - supported by numerous studies showing a positive correlation between carcinogenicity in man and genotoxicity in the Drosophila wing spot test - point to the potential risks some composite resins pose to the health of patients and dentistry personnel.

  15. Influence of milk somatic cell content on Parmigiano-Reggiano cheese yield.

    PubMed

    Summer, Andrea; Franceschi, Piero; Formaggioni, Paolo; Malacarne, Massimo

    2015-05-01

    The aim of this study was to determine the influence of the somatic cell content (SCC) of milk on Parmigiano-Reggiano cheese yield, produced in commercial cheese factories under field conditions. The study was carried out following the production of 56 batches of Parmigiano-Reggiano in 13 commercial cheese factories by processing milk collected from Italian Friesian cattle herds. The vat-milk (V-milk) used for making each cheese batch was obtained by mixing evening milk (partially skimmed following spontaneous separation of fat overnight, natural creaming) and morning milk. The batches of cheese produced were divided into 5 classes according to the SCC value of the evening milk determined prior to natural creaming (class 1, from 0 to 200,000; 2, 201,000-300,000; 3, 301,000-400,000; 4, 401,000-500,000; 5, over 501,000 cells/ml). The cheese yield was calculated as the amount of 24-h cheese, expressed in kilograms, obtained from 100 kg of V-milk (24 h ACY). The values of fat, crude protein, true protein, casein and 24 h ACY of V-milk were negatively correlated with the somatic cell score (SCS) of the evening milk. Conversely, a positive correlation was observed between chloride and SCS. Fat, protein fractions (crude protein, casein and whey proteins), P and titratable acidity of V-milk were positively correlated with its 24 h ACY, while chloride, pH and SCS showed a negative correlation. A significant drop in 24 h ACY was observed in classes 3, 4 and 5, therefore when the SCC of the evening milk exceeded 300,000 cells/ml. Finally a lower recovery of milk fat in cheese was observed as SCC of evening milk increase.

  16. Condensin II Subunit dCAP-D3 Restricts Retrotransposon Mobilization in Drosophila Somatic Cells

    PubMed Central

    Schuster, Andrew T.; Sarvepalli, Kavitha; Murphy, Eain A.; Longworth, Michelle S.

    2013-01-01

    Retrotransposon sequences are positioned throughout the genome of almost every eukaryote that has been sequenced. As mobilization of these elements can have detrimental effects on the transcriptional regulation and stability of an organism's genome, most organisms have evolved mechanisms to repress their movement. Here, we identify a novel role for the Drosophila melanogaster Condensin II subunit, dCAP-D3 in preventing the mobilization of retrotransposons located in somatic cell euchromatin. dCAP-D3 regulates transcription of euchromatic gene clusters which contain or are proximal to retrotransposon sequence. ChIP experiments demonstrate that dCAP-D3 binds to these loci and is important for maintaining a repressed chromatin structure within the boundaries of the retrotransposon and for repressing retrotransposon transcription. We show that dCAP-D3 prevents accumulation of double stranded DNA breaks within retrotransposon sequence, and decreased dCAP-D3 levels leads to a precise loss of retrotransposon sequence at some dCAP-D3 regulated gene clusters and a gain of sequence elsewhere in the genome. Homologous chromosomes exhibit high levels of pairing in Drosophila somatic cells, and our FISH analyses demonstrate that retrotransposon-containing euchromatic loci are regions which are actually less paired than euchromatic regions devoid of retrotransposon sequences. Decreased dCAP-D3 expression increases pairing of homologous retrotransposon-containing loci in tissue culture cells. We propose that the combined effects of dCAP-D3 deficiency on double strand break levels, chromatin structure, transcription and pairing at retrotransposon-containing loci may lead to 1) higher levels of homologous recombination between repeats flanking retrotransposons in dCAP-D3 deficient cells and 2) increased retrotransposition. These findings identify a novel role for the anti-pairing activities of dCAP-D3/Condensin II and uncover a new way in which dCAP-D3/Condensin II influences local

  17. Condensin II subunit dCAP-D3 restricts retrotransposon mobilization in Drosophila somatic cells.

    PubMed

    Schuster, Andrew T; Sarvepalli, Kavitha; Murphy, Eain A; Longworth, Michelle S

    2013-10-01

    Retrotransposon sequences are positioned throughout the genome of almost every eukaryote that has been sequenced. As mobilization of these elements can have detrimental effects on the transcriptional regulation and stability of an organism's genome, most organisms have evolved mechanisms to repress their movement. Here, we identify a novel role for the Drosophila melanogaster Condensin II subunit, dCAP-D3 in preventing the mobilization of retrotransposons located in somatic cell euchromatin. dCAP-D3 regulates transcription of euchromatic gene clusters which contain or are proximal to retrotransposon sequence. ChIP experiments demonstrate that dCAP-D3 binds to these loci and is important for maintaining a repressed chromatin structure within the boundaries of the retrotransposon and for repressing retrotransposon transcription. We show that dCAP-D3 prevents accumulation of double stranded DNA breaks within retrotransposon sequence, and decreased dCAP-D3 levels leads to a precise loss of retrotransposon sequence at some dCAP-D3 regulated gene clusters and a gain of sequence elsewhere in the genome. Homologous chromosomes exhibit high levels of pairing in Drosophila somatic cells, and our FISH analyses demonstrate that retrotransposon-containing euchromatic loci are regions which are actually less paired than euchromatic regions devoid of retrotransposon sequences. Decreased dCAP-D3 expression increases pairing of homologous retrotransposon-containing loci in tissue culture cells. We propose that the combined effects of dCAP-D3 deficiency on double strand break levels, chromatin structure, transcription and pairing at retrotransposon-containing loci may lead to 1) higher levels of homologous recombination between repeats flanking retrotransposons in dCAP-D3 deficient cells and 2) increased retrotransposition. These findings identify a novel role for the anti-pairing activities of dCAP-D3/Condensin II and uncover a new way in which dCAP-D3/Condensin II influences local

  18. Genotoxicity evaluation of buprofezin, petroleum oil and profenofos in somatic and germ cells of male mice.

    PubMed

    Fahmy, M A; Abdalla, E F

    1998-01-01

    The two pest control agents, buprofezin and petroleum oil (Super Royal), were tested to evaluate their potential mutagenicity, in comparison with the organophosphorus insecticide profenofos. Chromosomal aberration analysis was used in both somatic and germ cells of male mice. Single oral treatment at three different dose levels (1/16, 1/8 and 1/4 LD50) for each insecticide induced an increase in the percentage of chromosomal aberrations in bone-marrow cells 24 h post-treatment, indicating a dose-dependent relationship. The percentage of chromosomal aberrations reached 23 +/- 0.73, 10.5 +/- 0.64 and 15 +/- 1.4 after treatment with the highest tested dose of profenofos, buprofezin and Super Royal, respectively. Such percentages did not exceed the corresponding value of the positive control, mitomycin C (29.2 +/- 0.69). The percentage of chromosomal aberrations induced by the different doses of profenofos was still highly significant even after excluding gaps. The same trend of results was noticed only at the highest tested dose of buprofezin and Super Royal. With respect to germ cells, profenofos is also a potent inducer of chromosomal aberrations in 1ry spermatocytes, giving percentages of 14 +/- 1.3 and 19 +/- 1.6 at the two higher doses of 4.25 and 8.5 mg kg(-1) body wt., respectively. Buprofezin and Super Royal had no significant effect on mouse spermatocytes at the tested concentrations. The various types of induced aberrations were examined and recorded in both somatic and germ cells. In conclusion, the present investigation indicates that the two pest control agents buprofezin and Super Royal are relatively much safer compounds than the conventional organophosphorus insecticides. PMID:9804428

  19. The SNF2 family ATPase LSH promotes cell-autonomous de novo DNA methylation in somatic cells

    PubMed Central

    Termanis, Ausma; Torrea, Natalia; Culley, Jayne; Kerr, Alastair; Ramsahoye, Bernard; Stancheva, Irina

    2016-01-01

    Methylation of DNA at carbon 5 of cytosine is essential for mammalian development and implicated in transcriptional repression of genes and transposons. New patterns of DNA methylation characteristic of lineage-committed cells are established at the exit from pluripotency by de novo DNA methyltransferases enzymes, DNMT3A and DNMT3B, which are regulated by developmental signaling and require access to chromatin-organized DNA. Whether or not the capacity for de novo DNA methylation of developmentally regulated loci is preserved in differentiated somatic cells and can occur in the absence of exogenous signals is currently unknown. Here, we demonstrate that fibroblasts derived from chromatin remodeling ATPase LSH (HELLS)-null mouse embryos, which lack DNA methylation from centromeric repeats, transposons and a number of gene promoters, are capable of reestablishing DNA methylation and silencing of misregulated genes upon re-expression of LSH. We also show that the ability of LSH to bind ATP and the cellular concentration of DNMT3B are critical for cell-autonomous de novo DNA methylation in somatic cells. These data suggest the existence of cellular memory that persists in differentiated cells through many cell generations and changes in transcriptional state. PMID:27179028

  20. Biomimetic extracellular matrix mediated somatic stem cell differentiation: applications in dental pulp tissue regeneration.

    PubMed

    Ravindran, Sriram; George, Anne

    2015-01-01

    Dental caries is one of the most widely prevalent infectious diseases in the world. It affects more than half of the world's population. The current treatment for necrotic dental pulp tissue arising from dental caries is root canal therapy. This treatment results in loss of tooth sensitivity and vitality making it prone for secondary infections. Over the past decade, several tissue-engineering approaches have attempted regeneration of the dental pulp tissue. Although several studies have highlighted the potential of dental stem cells, none have transitioned into a clinical setting owing to limited availability of dental stem cells and the need for growth factor delivery systems. Our strategy is to utilize the intact ECM of pulp cells to drive lineage specific differentiation of bone marrow derived mesenchymal stem cells. From a clinical perspective, pulp ECM scaffolds can be generated using cell lines and patient specific somatic stem cells can be used for regeneration. Our published results have shown the feasibility of using pulp ECM scaffolds for odontogenic differentiation of non-dental mesenchymal cells. This focused review discusses the issues surrounding dental pulp tissue regeneration and the potential of our strategy to overcome these issues.

  1. Biomimetic extracellular matrix mediated somatic stem cell differentiation: applications in dental pulp tissue regeneration

    PubMed Central

    Ravindran, Sriram; George, Anne

    2015-01-01

    Dental caries is one of the most widely prevalent infectious diseases in the world. It affects more than half of the world's population. The current treatment for necrotic dental pulp tissue arising from dental caries is root canal therapy. This treatment results in loss of tooth sensitivity and vitality making it prone for secondary infections. Over the past decade, several tissue-engineering approaches have attempted regeneration of the dental pulp tissue. Although several studies have highlighted the potential of dental stem cells, none have transitioned into a clinical setting owing to limited availability of dental stem cells and the need for growth factor delivery systems. Our strategy is to utilize the intact ECM of pulp cells to drive lineage specific differentiation of bone marrow derived mesenchymal stem cells. From a clinical perspective, pulp ECM scaffolds can be generated using cell lines and patient specific somatic stem cells can be used for regeneration. Our published results have shown the feasibility of using pulp ECM scaffolds for odontogenic differentiation of non-dental mesenchymal cells. This focused review discusses the issues surrounding dental pulp tissue regeneration and the potential of our strategy to overcome these issues. PMID:25954205

  2. BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei.

    PubMed

    Huang, Jiaojiao; Zhang, Hongyong; Yao, Jing; Qin, Guosong; Wang, Feng; Wang, Xianlong; Luo, Ailing; Zheng, Qiantao; Cao, Chunwei; Zhao, Jianguo

    2016-01-01

    Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P<0.05) and in vivo (cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression.

  3. Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer

    PubMed Central

    Degrelle, Séverine A.; Jaffrezic, Florence; Campion, Evelyne; Lê Cao, Kim-Anh; Le Bourhis, Daniel; Richard, Christophe; Rodde, Nathalie; Fleurot, Renaud; Everts, Robin E.; Lecardonnel, Jérôme; Heyman, Yvan; Vignon, Xavier; Tian, Xiuchun C.; Lewin, Harris A.; Renard, Jean-Paul; Hue, Isabelle

    2012-01-01

    Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular “uncoupling”. Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters

  4. Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

    SciTech Connect

    Ostrup, Olga; Hyttel, Poul; Klaerke, Dan A.; Collas, Philippe

    2011-09-02

    Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression. This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.

  5. Single nucleotide polymorphism and haplotype effects associated with somatic cell score in German Holstein cattle

    PubMed Central

    2014-01-01

    Background To better understand the genetic determination of udder health, we performed a genome-wide association study (GWAS) on a population of 2354 German Holstein bulls for which daughter yield deviations (DYD) for somatic cell score (SCS) were available. For this study, we used genetic information of 44 576 informative single nucleotide polymorphisms (SNPs) and 11 725 inferred haplotype blocks. Results When accounting for the sub-structure of the analyzed population, 16 SNPs and 10 haplotypes in six genomic regions were significant at the Bonferroni threshold of P ≤ 1.14 × 10-6. The size of the identified regions ranged from 0.05 to 5.62 Mb. Genomic regions on chromosomes 5, 6, 18 and 19 coincided with known QTL affecting SCS, while additional genomic regions were found on chromosomes 13 and X. Of particular interest is the region on chromosome 6 between 85 and 88 Mb, where QTL for mastitis traits and significant SNPs for SCS in different Holstein populations coincide with our results. In all identified regions, except for the region on chromosome X, significant SNPs were present in significant haplotypes. The minor alleles of identified SNPs on chromosomes 18 and 19, and the major alleles of SNPs on chromosomes 6 and X were favorable for a lower SCS. Differences in somatic cell count (SCC) between alternative SNP alleles reached 14 000 cells/mL. Conclusions The results support the polygenic nature of the genetic determination of SCS, confirm the importance of previously reported QTL, and provide evidence for the segregation of additional QTL for SCS in Holstein cattle. The small size of the regions identified here will facilitate the search for causal genetic variations that affect gene functions. PMID:24898131

  6. A somatic cell hybrid panel for localizing DNA segments near the Huntington's disease gene.

    PubMed

    MacDonald, M E; Anderson, M A; Gilliam, T C; Tranejaerg, L; Carpenter, N J; Magenis, E; Hayden, M R; Healey, S T; Bonner, T I; Gusella, J F

    1987-09-01

    Thirty-four random DNA probes from the terminal half of the human chromosome 4 short arm were further localized within 4pter----p15.1. A panel of somatic cell hybrid lines defining six chromosomal regions within 4pter----p15.1 was constructed using human cell lines containing translocation or deletion chromosomes. The vast majority of the DNA sequences, 32 of 34 or 94%, mapped to the three most proximal regions comprising 4p16.1----4p15.1. Only two probes were localized distal to 4p16.1: one in the region 4p16.3----4p16.1 and one in 4p16.3. D4S10, a polymorphic DNA marker linked to the Huntington's disease defect, has previously been mapped to the terminal region of 4p with conflicting assignments to 4p16.1 and 4p16.3. Analysis of restriction fragment length polymorphisms demonstrated hemizygosity for D4S10 in a patient with Wolf-Hirschhorn syndrome resulting from an unbalanced translocation t(4;8)(p16.3;p23.1), supporting the 4p16.3 localization. Our panel of somatic cell hybrids provides a rapid method for mapping new probes to the same vicinity as that of D4S10. However, the relative paucity of such DNA segments identified here suggests that a more directed approach may be required to generate additional markers near the HD gene.

  7. Intestinal stem cells in the adult Drosophila midgut

    SciTech Connect

    Jiang, Huaqi; Edgar, Bruce A.

    2011-11-15

    Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury. -- Highlights: Black-Right-Pointing-Pointer The homeostasis and regeneration of adult fly midguts are mediated by ISCs. Black-Right-Pointing-Pointer Damaged enterocytes induce the proliferation of intestinal stem cells (ISC). Black-Right-Pointing-Pointer EGFR and Jak/Stat signalings mediate compensatory ISC proliferation. Black-Right-Pointing-Pointer Notch signaling regulates ISC self-renewal and differentiation.

  8. Evidence for trans regulation of apoptosis in intertypic somatic cell hybrids.

    PubMed Central

    Gourdeau, H; Walker, P R

    1994-01-01

    The genetic components required for glucocorticoid induction of apoptosis were studied by using somatic cell hybridization. Intertypic whole-cell hybrids were generated by crossing the glucocorticoid-resistant rat liver cell line Fado-2 with the glucocorticoid-sensitive mouse thymoma cell line BW5147.3. Morphological and biochemical criteria were used to assess sensitivity or resistance to glucocorticoid-induced cell death. Both phenotypes were observed, and all of the hybrids retained a functional glucocorticoid receptor as judged by their abilities to induce the metallothionein gene in response to dexamethasone (Dex). Sensitivity to apoptosis did not correlate with morphological phenotype in that not all suspension cells were sensitive. The effect of glucocorticoids on the expression of apoptosis-linked genes was analyzed in a subset of Dex-sensitive and Dex-resistant hybrids. p53 and c-myc mRNAs were present in parental cells as well as sensitive and resistant hybrid cells, and their levels were not affected by glucocorticoid treatment. bcl-2 expression was restricted to the thymoma cell line and was also not affected by glucocorticoids. We did not detect any bcl-2 mRNA in the hepatoma cell line and the hybrids, suggesting that, as with most tissue-specific genes, bcl-2 is regulated in trans. Furthermore, while the majority of hybrids analyzed retained a full complement of mouse chromosomes, sensitive hybrids were missing some rat chromosomes (preferentially chromosomes 16 and 19), indicating that apoptosis is subject to trans repression. Resistant cells thus appear to repress the activity or synthesis of a nuclear factor that interacts with a glucocorticoid-dependent gene(s) to activate the cell death pathway. Images PMID:8065345

  9. Evidence for trans regulation of apoptosis in intertypic somatic cell hybrids.

    PubMed

    Gourdeau, H; Walker, P R

    1994-09-01

    The genetic components required for glucocorticoid induction of apoptosis were studied by using somatic cell hybridization. Intertypic whole-cell hybrids were generated by crossing the glucocorticoid-resistant rat liver cell line Fado-2 with the glucocorticoid-sensitive mouse thymoma cell line BW5147.3. Morphological and biochemical criteria were used to assess sensitivity or resistance to glucocorticoid-induced cell death. Both phenotypes were observed, and all of the hybrids retained a functional glucocorticoid receptor as judged by their abilities to induce the metallothionein gene in response to dexamethasone (Dex). Sensitivity to apoptosis did not correlate with morphological phenotype in that not all suspension cells were sensitive. The effect of glucocorticoids on the expression of apoptosis-linked genes was analyzed in a subset of Dex-sensitive and Dex-resistant hybrids. p53 and c-myc mRNAs were present in parental cells as well as sensitive and resistant hybrid cells, and their levels were not affected by glucocorticoid treatment. bcl-2 expression was restricted to the thymoma cell line and was also not affected by glucocorticoids. We did not detect any bcl-2 mRNA in the hepatoma cell line and the hybrids, suggesting that, as with most tissue-specific genes, bcl-2 is regulated in trans. Furthermore, while the majority of hybrids analyzed retained a full complement of mouse chromosomes, sensitive hybrids were missing some rat chromosomes (preferentially chromosomes 16 and 19), indicating that apoptosis is subject to trans repression. Resistant cells thus appear to repress the activity or synthesis of a nuclear factor that interacts with a glucocorticoid-dependent gene(s) to activate the cell death pathway.

  10. [Langerhans cell histiocytosis in adults].

    PubMed

    Néel, A; Artifoni, M; Donadieu, J; Lorillon, G; Hamidou, M; Tazi, A

    2015-10-01

    Langerhans cell histiocytosis (LCH) is a rare disease characterized by the infiltration of one or more organs by Langerhans cell-like dendritic cells, most often organized in granulomas. The disease has been initially described in children. The clinical picture of LCH is highly variable. Bone, skin, pituitary gland, lung, central nervous system, lymphoid organs are the main organs involved whereas liver and intestinal tract localizations are less frequently encountered. LCH course ranges from a fulminant multisystem disease to spontaneous resolution. Several randomized controlled trials have enable pediatricians to refine the management of children with LCH. Adult LCH has some specific features and poses distinct therapeutic challenges, knowing that data on these patients are limited. Herein, we will provide an overview of current knowledge regarding adult LCH and its management. We will also discuss recent advances in the understanding of the disease, (i.e. the role of BRAF oncogene) that opens the way toward targeted therapies.

  11. [Langerhans cell histiocytosis in adults].

    PubMed

    Néel, A; Artifoni, M; Donadieu, J; Lorillon, G; Hamidou, M; Tazi, A

    2015-10-01

    Langerhans cell histiocytosis (LCH) is a rare disease characterized by the infiltration of one or more organs by Langerhans cell-like dendritic cells, most often organized in granulomas. The disease has been initially described in children. The clinical picture of LCH is highly variable. Bone, skin, pituitary gland, lung, central nervous system, lymphoid organs are the main organs involved whereas liver and intestinal tract localizations are less frequently encountered. LCH course ranges from a fulminant multisystem disease to spontaneous resolution. Several randomized controlled trials have enable pediatricians to refine the management of children with LCH. Adult LCH has some specific features and poses distinct therapeutic challenges, knowing that data on these patients are limited. Herein, we will provide an overview of current knowledge regarding adult LCH and its management. We will also discuss recent advances in the understanding of the disease, (i.e. the role of BRAF oncogene) that opens the way toward targeted therapies. PMID:26150351

  12. Relative frequencies of homologous recombination between plasmids introduced into DNA repair-deficient and other mammalian somatic cell lines.

    PubMed

    Wahls, W P; Moore, P D

    1990-07-01

    Twelve mammalian somatic cell lines, some of them DNA damage-sensitive mutants paired with their respective wild-type parental lines, were assayed for their ability to catalyze extrachromosomal, intermolecular homologous recombination between pSV2neo plasmid recombination substrates. All of the somatic cell lines analyzed are capable of catalyzing homologous recombination; however, there is a wide range of efficiencies with which they do so. Five human cell lines display a fourfold range of recombination frequencies, and six hamster cell lines vary almost 20-fold. Linearizing one of the recombination substrates stimulates recombination in all but one of the cell lines. Two of the three paired mutant cell lines display a threefold reduction in their ability to catalyze homologous recombination when compared to their respective parental cell lines, indicating that the mutations that render them sensitive to DNA damaging agents might also play a role in homologous recombination. PMID:2218721

  13. Single cell analysis demonstrating somatic mosaicism involving 11p in a patient with paternal isodisomy and Beckwith-Wiedemann Syndrome

    SciTech Connect

    Bischoff, F.Z.; McCaskill, C.; Subramanian, S.

    1994-09-01

    Beckwith-Wiedemann Syndrome (BWS) is characterized by numerous growth abnormalities including exomphalos, macroglossia, gigantism, and hemihypertrophy or hemihyperplasia. The {open_quotes}BWS gene{close_quotes} appears to be maternally repressed and is suspected to function as a growth factor or regulator of somatic growth, since activation of this gene through a variety of mechanisms appears to result in somatic overgrowth and tumor development. Mosaic paternal isodisomy of 11p has been observed previously by others in patients with BWS by Southern blot analysis of genomic DNA. The interpretation of these results was primarily based on the intensities of the hybridization signals for the different alleles. In our study, we demonstrate somatic mosaicism directly through PCR and single cell analysis. Peripheral blood was obtained from a patient with BWS and initial genomic DNA analysis by PCR was suggestive of somatic mosaicism for paternal isodisomy of 11p. Through micromanipulation, single cells were isolated and subjected to primer extention preamplification. Locus-specific microsatellite marker analyses by PCR were performed to determine the chromosome 11 origins in the preamplified individual cells. Two populations of cells were detected, a population of cells with normal biparental inheritance and a population of cells with paternal isodisomy of 11p and biparental disomy of 11q. Using the powerful approach of single cell analysis, the detected somatic mosaicism provides evidence for a mitotic recombinational event that has resulted in loss of the maternal 11p region and gain of a second copy of paternal 11p in some cells. The direct demonstration of mosaicism may explain the variable phenotypes and hemihypertrophy often observed in BWS.

  14. Xist repression shows time-dependent effects on the reprogramming of female somatic cells to induced pluripotent stem cells.

    PubMed

    Chen, Qi; Gao, Shuai; He, Wenteng; Kou, Xiaochen; Zhao, Yanhong; Wang, Hong; Gao, Shaorong

    2014-10-01

    Although the reactivation of silenced X chromosomes has been observed as part of the process of reprogramming female somatic cells into induced pluripotent stem cells (iPSCs), it remains unknown whether repression of the X-inactive specific transcript (Xist) can greatly enhance female iPSC induction similar to that observed in somatic cell nuclear transfer studies. In this study, we discovered that the repression of Xist plays opposite roles in the early and late phases of female iPSCs induction. Our results demonstrate that the downregulation of Xist by an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible short hairpin RNA (shRNA) system can greatly impair the mesenchymal-to-epithelial transition (MET) in the early phase of iPSC induction but can significantly promote the transition of pre-iPSCs to iPSCs in the late phase. Furthermore, we demonstrate that although the knockdown of Xist did not affect the H3K27me3 modification on the X chromosome, macroH2A was released from the inactivated X chromosome (Xi). This enables the X chromosome silencing to be a reversible event. Moreover, we demonstrate that the supplementation of vitamin C (Vc) can augment and stabilize the reversible X chromosome by preventing the relocalization of macroH2A to the Xi. Therefore, our study reveals an opposite role of Xist repression in the early and late stages of reprogramming female somatic cells to pluripotency and demonstrates that the release of macroH2A by Xist repression enables the transition from pre-iPSCs to iPSCs.

  15. Interaction between genes Mos and mwh expressed in somatic cells of Drosophila melanogaster

    SciTech Connect

    Vaisman, N.Ya.; Zakharov, I.K.

    1995-07-01

    Gene Mosaic (Mos) of chromosome 3 of Drosophila melanogaster was located by means of dominant markers Ly, Sb, and Dr. This gene was shown to be located between Ly and Sb in the centromeric region (45-50 map units). An analysis of interaction between Mos and mwh genes in cis- and trans-heterozygotes showed a significant effect of the Mos gene on mutability (recombinogenesis) of chromosome mwh in somatic cells. In the cis heterozygote mwh Mos/++, the frequency of small mutant clones on wings of flies increased. In mwh/Mos heterozygotes, the Mos gene caused a significant reduction of dorsocentral and scutellar bristles (78% in mwh/Mos, 85% in mwh +/+ Mos, and 98% in mwh Mos/mwh +). 20 refs., 3 tabs.

  16. Somatic cell nuclear transfer and transgenesis in large animals: current and future insights.

    PubMed

    Galli, C; Lagutina, I; Perota, A; Colleoni, S; Duchi, R; Lucchini, F; Lazzari, G

    2012-06-01

    Somatic cell nuclear transfer (SCNT) was first developed in livestock for the purpose of accelerating the widespread use of superior genotypes. Although many problems still exist now after fifteen years of research owing to the limited understanding of genome reprogramming, SCNT has provided a powerful tool to make copies of selected individuals in different species, to study genome pluripotency and differentiation, opening new avenues of research in regenerative medicine and representing the main route for making transgenic livestock. Besides well-established methods to deliver transgenes, recent development in enzymatic engineering to edit the genome provides more precise and reproducible tools to target-specific genomic loci especially for producing knockout animals. The interest in generating transgenic livestock lies in the agricultural and biomedical areas and it is, in most cases, at the stage of research and development, with few exceptions that are making the way into practical applications.

  17. In vitro development of canine somatic cell nuclear transfer embryos in different culture media.

    PubMed

    Kim, Dong-Hoon; No, Jin-Gu; Choi, Mi-Kyung; Yeom, Dong-Hyeon; Kim, Dong-Kyo; Yang, Byoung-Chul; Yoo, Jae Gyu; Kim, Min Kyu; Kim, Hong-Tea

    2015-01-01

    The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.

  18. Communication between oocytes and somatic cells regulates volatile pheromone production in Caenorhabditis elegans

    PubMed Central

    Leighton, Daniel H. W.; Choe, Andrea; Wu, Shannon Y; Sternberg, Paul W.

    2014-01-01

    Males of the androdioecious species Caenorhabditis elegans are more likely to attempt to mate with and successfully inseminate C. elegans hermaphrodites that do not concurrently harbor sperm. Although a small number of genes have been implicated in this effect, the mechanism by which it arises remains unknown. In the context of the battle of the sexes, it is also unknown whether this effect is to the benefit of the male, the hermaphrodite, or both. We report that successful contact between mature sperm and oocyte in the C. elegans gonad at the start of fertilization causes the oocyte to release a signal that is transmitted to somatic cells in its mother, with the ultimate effect of reducing her attractiveness to males. Changes in hermaphrodite attractiveness are tied to the production of a volatile pheromone, the first such pheromone described in C. elegans. PMID:25453110

  19. Herd level approach to high bulk milk somatic cell count problems in dairy cattle.

    PubMed

    Barkema, Herman W; De Vliegher, Sarne; Piepers, Sofie; Zadoks, Ruth N

    2013-06-01

    Since the introduction of the standard mastitis prevention program in the late 1960s, enormous progress has been made in decreasing the average bulk milk somatic cell count (BMSCC). In many countries, reduction of BMSCC has been encouraged through premium payments or penalty systems. However, the success of the program depends heavily on consistent implementation of management practices. The approach to problem solving in a herd with high BMSCC must include the following elements: (1) problem definition using primary udder health parameters; (2) detection of cows causing the problem; (3) definition of short- and long-term goals; (4) formulation and implementation of a herd management plan; and (5) evaluation of the results. Findings and plans are recorded for use at follow-up visits. Every high BMSCC problem can be solved if farmers are sufficiently motivated, if farm advisors are sufficiently knowledgeable, and if farmer and advisors work together according to a jointly determined plan. PMID:23706026

  20. Conservation of the Sapsaree (Canis familiaris), a Korean Natural Monument, using somatic cell nuclear transfer.

    PubMed

    Jang, Goo; Hong, SoGun; Kang, JungTaek; Park, JungEun; Oh, HyunJu; Park, ChanKyu; Ha, JiHong; Kim, DaeYong; Kim, MinKyu; Lee, ByeongChun

    2009-09-01

    A recent emerging technology, somatic cell nuclear transfer (SCNT), has been considered for conserving threatened or endangered species. Sapsaree is a native breed in Korea and has been designated as a Natural Monument. The aim of this study was to produce a Sapsaree by SCNT for breed conservation. Donor fibroblasts from a 9-year-old male Sapsaree were placed into the perivitelline spaces of enucleated in vivo matured oocytes and fused electrically. A total of 309 cloned embryos were transferred into the oviducts of 15 naturally synchronized recipients. Two recipients were diagnosed as pregnant, and each delivered one cloned puppy, both of which weighed 530 g. Overall, this study demonstrated that an endangered canine breed can be conserved by SCNT.

  1. Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1

    PubMed Central

    Wang, Wei; Yang, Jian; Liu, Hui; Lu, Dong; Chen, Xiongfeng; Zenonos, Zenon; Campos, Lia S.; Rad, Roland; Guo, Ge; Zhang, Shujun; Bradley, Allan; Liu, Pentao

    2011-01-01

    Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expressing four transcription factors: Oct4, Sox2, Klf4, and c-Myc. Here we report that enhancing RA signaling by expressing RA receptors (RARs) or by RA agonists profoundly promoted reprogramming, but inhibiting it using a RAR-α dominant-negative form completely blocked it. Coexpressing Rarg (RAR-γ) and Lrh-1 (liver receptor homologue 1; Nr5a2) with the four factors greatly accelerated reprogramming so that reprogramming of mouse embryonic fibroblast cells to ground-state iPSCs requires only 4 d induction of these six factors. The six-factor combination readily reprogrammed primary human neonatal and adult fibroblast cells to exogenous factor-independent iPSCs, which resembled ground-state mouse ES cells in growth properties, gene expression, and signaling dependency. Our findings demonstrate that signaling through RARs has critical roles in molecular reprogramming and that the synergistic interaction between Rarg and Lrh1 directs reprogramming toward ground-state pluripotency. The human iPSCs described here should facilitate functional analysis of the human genome. PMID:21990348

  2. Repression of Germline RNAi Pathways in Somatic Cells by Retinoblastoma Pathway Chromatin Complexes

    PubMed Central

    Wu, Xiaoyun; Shi, Zhen; Cui, Mingxue; Han, Min; Ruvkun, Gary

    2012-01-01

    The retinoblastoma (Rb) tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene. PMID:22412383

  3. The effect of multiplex-PCR-assessed major pathogens causing subclinical mastitis on somatic cell profiles.

    PubMed

    Goli, Mohammad; Ezzatpanah, Hamid; Ghavami, Mehrdad; Chamani, Mohammad; Aminafshar, Mehdi; Toghiani, Majid; Eghbalsaied, Shahin

    2012-10-01

    The major pathogens causing mastitis were evaluated by multiplex-polymerase chain reaction (M-PCR) with self-designed primers in four quarters of the first, third, and fifth parities in industrial, semi-industrial, and traditional dairy cattle farms in Iran. With the incidence of infection in the quarters by Staphylococcus aureus and Streptococcus agalactiae, the mean log somatic cell count (log SCC) increased from 5.06 to 5.77. The smallest changes occurred with Escherichia coli. Contagious pathogens, when compared with environmental pathogens, were more prevalent and common and created more profound quantitative and qualitative changes in SCC profiles. The second part of the study surveyed the diversity of contaminating pathogens and their effect on quantitative and qualitative profiles of somatic cells. M-PCR was used to determine the absence (M-PCR(-)) and presence of one (M-PCR(+1)), two (M-PCR(+2)), and three (M-PCR(+3)) major pathogens in raw milk samples. Quarter log SCC increased from 5.06 (for M-PCR(-1)) to 5.5 (for M-PCR(+1)), 5.7 (for M-PCR(+2)), and 6 (for M-PCR(+3)). Percent changes in polymorphonuclears (PMNs) were not significant between different quarters and parities but were significant between different farms in terms of pathogen diversity (P < 0.05). Therefore, by increasing the number of types of major pathogens involved in subclinical mastitis, SCC of udder quarters and the proportion of PMNs significantly increased, whereas the proportion of lymphocytes significantly decreased. This subject is very important in increasing the shelf life of dairy products, because PMNs are introduced to the enzymatic pools. PMID:22535149

  4. Transmembrane voltage potential of somatic cells controls oncogene-mediated tumorigenesis at long-range

    PubMed Central

    Chernet, Brook T.; Levin, Michael

    2014-01-01

    The microenvironment is increasingly recognized as a crucial aspect of cancer. In contrast and complement to the field's focus on biochemical factors and extracellular matrix, we characterize a novel aspect of host:tumor interaction – endogenous bioelectric signals among non-excitable somatic cells. Extending prior work focused on the bioelectric state of cancer cells themselves, we show for the first time that the resting potentials of distant cells are critical for oncogene-dependent tumorigenesis. In the Xenopus laevis tadpole model, we used human oncogenes such as mutant KRAS to drive formation of tumor-like structures that exhibited overproliferation, increased nuclear size, hypoxia, acidity, and leukocyte attraction. Remarkably, misexpression of hyperpolarizing ion channels at distant sites within the tadpole significantly reduced the incidence of these tumors. The suppression of tumorigenesis could also be achieved by hyperpolarization using native CLIC1 chloride channels, suggesting a treatment modality not requiring gene therapy. Using a dominant negative approach, we implicate HDAC1 as the mechanism by which resting potential changes affect downstream cell behaviors. Based on published data on the voltage-mediated changes of butyrate flux through the SLC5A8 transporter, we present a model linking resting potentials of host cells to the ability of oncogenes to initiate tumorigenesis. Antibiotic data suggest that the relevant butyrate is generated by a native bacterial species, identifying a novel link between the microbiome and cancer that is mediated by alterations in bioelectric signaling. PMID:24830454

  5. Transmembrane voltage potential of somatic cells controls oncogene-mediated tumorigenesis at long-range.

    PubMed

    Chernet, Brook T; Levin, Michael

    2014-05-30

    The microenvironment is increasingly recognized as a crucial aspect of cancer. In contrast and complement to the field's focus on biochemical factors and extracellular matrix, we characterize a novel aspect of host:tumor interaction - endogenous bioelectric signals among non-excitable somatic cells. Extending prior work focused on the bioelectric state of cancer cells themselves, we show for the first time that the resting potentials of distant cells are critical for oncogene-dependent tumorigenesis. In the Xenopus laevis tadpole model, we used human oncogenes such as mutant KRAS to drive formation of tumor-like structures that exhibited overproliferation, increased nuclear size, hypoxia, acidity, and leukocyte attraction. Remarkably, misexpression of hyperpolarizing ion channels at distant sites within the tadpole significantly reduced the incidence of these tumors. The suppression of tumorigenesis could also be achieved by hyperpolarization using native CLIC1 chloride channels, suggesting a treatment modality not requiring gene therapy. Using a dominant negative approach, we implicate HDAC1 as the mechanism by which resting potential changes affect downstream cell behaviors. Based on published data on the voltage-mediated changes of butyrate flux through the SLC5A8 transporter, we present a model linking resting potentials of host cells to the ability of oncogenes to initiate tumorigenesis. Antibiotic data suggest that the relevant butyrate is generated by a native bacterial species, identifying a novel link between the microbiome and cancer that is mediated by alterations in bioelectric signaling. PMID:24830454

  6. Bcl6 Is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells.

    PubMed

    Williams, Alan M; Maman, Yaakov; Alinikula, Jukka; Schatz, David G

    2016-01-01

    The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID.

  7. Bcl6 Is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells

    PubMed Central

    Williams, Alan M.; Maman, Yaakov; Alinikula, Jukka; Schatz, David G.

    2016-01-01

    The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID. PMID:26900682

  8. Consequence of alternative standards for bulk tank somatic cell count of dairy herds in the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparison of dairy operations failing compliance with current US and European Union (EU) standards for bulk-tank somatic cell count (BTSCC) as well as BTSCC standards proposed by 3 national organizations were evaluated using 2 populations of US dairy herds: Dairy Herd Improvement Association (DHI) ...

  9. Factors associated with high milk test day somatic cell counts in large dairy herds in Brandenburg. I: Housing conditions.

    PubMed

    Köster, G; Tenhagen, B-A; Heuwieser, W

    2006-04-01

    The aim of this study was to examine influences of housing conditions on the udder health in 80 German dairy herds with a herd size between 100 and 1100 cows. Data were collected using a standardized questionnaire for the farm manager and a farm visit using a standardized data capture form on hygiene and management. The somatic cell counts of all lactating cows on each farm were collected monthly by the local dairy herd improvement association and analysed to assess udder health status. Factor analysis was used to analyse the variables describing the environmental hygiene. The values derived for the extracted components were classified into good, moderate and poor. The association of the categories was then analysed for their influence on log somatic cell count of the current month (CMSCC) and the year before the farm visit (YASCC) by a one-way anova. In comparison to other housing systems, free stalls with cubicles had the lowest geometric mean somatic cell count. Three components were derived from the factor analysis. Of those, acceptance of the cubicles by the cows and barn hygiene were determined as components influencing the CMSCC and YASCC significantly, while the association of hygiene of the milking parlour with somatic cell counts was only significant for YASCC. The results of the study show that the cow comfort and housing hygiene have a substantial impact on milk quality and should therefore become the focus of further research on the farm management practices.

  10. Obsessive-compulsive disorder (OCD): Practical strategies for pharmacological and somatic treatment in adults.

    PubMed

    Fineberg, Naomi A; Reghunandanan, Samar; Simpson, Helen B; Phillips, Katharine A; Richter, Margaret A; Matthews, Keith; Stein, Dan J; Sareen, Jitender; Brown, Angus; Sookman, Debbie

    2015-05-30

    This narrative review gathers together a range of international experts to critically appraise the existing trial-based evidence relating to the efficacy and tolerability of pharmacotherapy for obsessive compulsive disorder in adults. We discuss the diagnostic evaluation and clinical characteristics followed by treatment options suitable for the clinician working from primary through to specialist psychiatric care. Robust data supports the effectiveness of treatment with selective serotonin reuptake inhibitors (SSRIs) and clomipramine in the short-term and the longer-term treatment and for relapse prevention. Owing to better tolerability, SSRIs are acknowledged as the first-line pharmacological treatment of choice. For those patients for whom first line treatments have been ineffective, evidence supports the use of adjunctive antipsychotic medication, and some evidence supports the use of high-dose SSRIs. Novel compounds are also the subject of active investigation. Neurosurgical treatments, including ablative lesion neurosurgery and deep brain stimulation, are reserved for severely symptomatic individuals who have not experienced sustained response to both pharmacological and cognitive behavior therapies.

  11. Somatic Cell Fusions Reveal Extensive Heterogeneity in Basal-like Breast Cancer.

    PubMed

    Su, Ying; Subedee, Ashim; Bloushtain-Qimron, Noga; Savova, Virginia; Krzystanek, Marcin; Li, Lewyn; Marusyk, Andriy; Tabassum, Doris P; Zak, Alexander; Flacker, Mary Jo; Li, Mei; Lin, Jessica J; Sukumar, Saraswati; Suzuki, Hiromu; Long, Henry; Szallasi, Zoltan; Gimelbrant, Alexander; Maruyama, Reo; Polyak, Kornelia

    2015-06-16

    Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated genetic and epigenetic (DNA methylation and chromatin) profiling. We found that the basal-like trait is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but a high degree of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for luminal-basal fusions, and we identified EN1, TBX18, and TCF4 as candidate transcriptional regulators of the luminal-to-basal switch. Our findings highlight the remarkable epigenetic plasticity of breast cancer cells. PMID:26051943

  12. Production of Cloned Korean Native Pig by Somatic Cell Nuclear Transfer

    PubMed Central

    Hwang, In-Sul; Kwon, Dae-Jin; Oh, Keun Bong; Ock, Sun-A; Chung, Hak-Jae; Cho, In-Cheol; Lee, Jeong-Woong; Im, Gi-Sun; Hwang, Seongsoo

    2015-01-01

    The Korean native pig (KNP) have been considered as animal models for animal biotechnology research because of their relatively small body size and their presumably highly inbred status due to the closed breeding program. However, little is reported about the use of KNP for animal biotechnology researches. This study was performed to establish the somatic cell nuclear transfer (SCNT) protocol for the production of swine leukocyte antigens (SLA) homotype-defined SCNT KNP. The ear fibroblast cells originated from KNP were cultured and used as donor cell. After thawing, the donor cells were cultured for 1 hour with 15 μM roscovitine prior to the nuclear transfer. The numbers of reconstructed and parthenogenetic embryos transferred were 98 ± 35.2 and 145 ± 11.2, respectively. The pregnancy and delivery rate were 3/5 (60%) and 2/5 (40%). One healthy SLA homotype-defined SCNT KNP was successfully generated. The recipient-based individual cloning efficiency ranged from 0.65 to 1.08%. Taken together, it can be postulated that the methodological establishment of the production of SLA homotype-defined cloned KNP can be applied to the generation of transgenic cloned KNP as model animals for human disease and xenotransplantation researches. PMID:27004264

  13. Production of Cloned Korean Native Pig by Somatic Cell Nuclear Transfer.

    PubMed

    Hwang, In-Sul; Kwon, Dae-Jin; Oh, Keun Bong; Ock, Sun-A; Chung, Hak-Jae; Cho, In-Cheol; Lee, Jeong-Woong; Im, Gi-Sun; Hwang, Seongsoo

    2015-06-01

    The Korean native pig (KNP) have been considered as animal models for animal biotechnology research because of their relatively small body size and their presumably highly inbred status due to the closed breeding program. However, little is reported about the use of KNP for animal biotechnology researches. This study was performed to establish the somatic cell nuclear transfer (SCNT) protocol for the production of swine leukocyte antigens (SLA) homotype-defined SCNT KNP. The ear fibroblast cells originated from KNP were cultured and used as donor cell. After thawing, the donor cells were cultured for 1 hour with 15 μM roscovitine prior to the nuclear transfer. The numbers of reconstructed and parthenogenetic embryos transferred were 98 ± 35.2 and 145 ± 11.2, respectively. The pregnancy and delivery rate were 3/5 (60%) and 2/5 (40%). One healthy SLA homotype-defined SCNT KNP was successfully generated. The recipient-based individual cloning efficiency ranged from 0.65 to 1.08%. Taken together, it can be postulated that the methodological establishment of the production of SLA homotype-defined cloned KNP can be applied to the generation of transgenic cloned KNP as model animals for human disease and xenotransplantation researches.

  14. In vitro and in vivo genotoxic effects of somatic cell nuclear transfer cloned cattle meat.

    PubMed

    Lee, Nam-Jin; Yang, Byoung-Chul; Jung, Yu-Ri; Lee, Jung-Won; Im, Gi-Sun; Seong, Hwan-Hoo; Park, Jin-Ki; Kang, Jong-Koo; Hwang, Seongsoo

    2011-09-01

    Although the nutritional composition and health status after consumption of the meat and milk derived from both conventionally bred (normal) and somatic cell nuclear transferred (cloned) animals and their progeny are not different, little is known about their food safeties like genetic toxicity. This study is performed to examine both in vitro (bacterial mutation and chromosome aberration) and in vivo (micronucleus) genotoxicity studies of cloned cattle meat. The concentrations of both normal and cloned cattle meat extracts (0-10×) were tested to five strains of bacteria (Salmonella typhimurium: TA98, TA100, TA1535, and TA1537; Escherichia coli: WP2uvrA) for bacterial mutation and to Chinese hamster lung (CHL/IU) cells for chromosome aberration, respectively. For micronucleus test, ICR mice were divided into five dietary groups: commercial pellets (control), pellets containing 5% (N-5) and 10% (N-10) normal cattle meat, and pellets containing 5% (C-5) and 10% (C-10) cloned cattle meat. No test substance-related genotoxicity was noted in the five bacterial strains, CHL/IU cells, or mouse bone marrow cells, suggesting that the cloned cattle meat potentially may be safe in terms of mutagenic hazards. Thus, it can be postulated that the cloned cattle meat do not induce any harmful genotoxic effects in vitro and in vivo.

  15. Assessment of imidacloprid-induced mutagenic effects in somatic cells of Swiss albino male mice.

    PubMed

    Bagri, Preeti; Kumar, Vinod; Sikka, Anil K

    2016-10-01

    Pesticides are being used for plant protection to increase food protection and to reduce insect-borne diseases worldwide. Exposure to the pesticides may cause genotoxic effects on both the target and nontarget organisms, including man. Therefore, the mutagenicity evaluation of such pesticides has become a priority area of research. Imidacloprid (IMI), a neonicotinoid insecticide, is widely used in agriculture either alone or in combination with other insecticides. A combined approach employing micronucleus test (MNT) and chromosomal aberrations assay (CA) was utilized to assess the mutagenicity of imidacloprid in bone marrow of Swiss albino male mice. IMI suspension was prepared in 3% gum acacia and administered at doses of 5.5, 11 and 22 mg/kg body weight for 7, 14 and 28 days to mice. IMI treatment resulted in a dose and time-dependant increase in the frequencies of micronuclei per cell and chromosomal aberrations in bone marrow cells. A statistically significant increase in chromosomal aberrations and micronuclei/cell was found only after daily treatment of IMI at highest selected dose (22 mg/kg body weight) for longest selected time period (28 days) compared to the control group. Thus, daily exposure of imidacloprid at a dose level of 22 mg/kg body weight for 28 days caused mutagenic effects on the somatic cells of Swiss albino male mice.

  16. Assessment of imidacloprid-induced mutagenic effects in somatic cells of Swiss albino male mice.

    PubMed

    Bagri, Preeti; Kumar, Vinod; Sikka, Anil K

    2016-10-01

    Pesticides are being used for plant protection to increase food protection and to reduce insect-borne diseases worldwide. Exposure to the pesticides may cause genotoxic effects on both the target and nontarget organisms, including man. Therefore, the mutagenicity evaluation of such pesticides has become a priority area of research. Imidacloprid (IMI), a neonicotinoid insecticide, is widely used in agriculture either alone or in combination with other insecticides. A combined approach employing micronucleus test (MNT) and chromosomal aberrations assay (CA) was utilized to assess the mutagenicity of imidacloprid in bone marrow of Swiss albino male mice. IMI suspension was prepared in 3% gum acacia and administered at doses of 5.5, 11 and 22 mg/kg body weight for 7, 14 and 28 days to mice. IMI treatment resulted in a dose and time-dependant increase in the frequencies of micronuclei per cell and chromosomal aberrations in bone marrow cells. A statistically significant increase in chromosomal aberrations and micronuclei/cell was found only after daily treatment of IMI at highest selected dose (22 mg/kg body weight) for longest selected time period (28 days) compared to the control group. Thus, daily exposure of imidacloprid at a dose level of 22 mg/kg body weight for 28 days caused mutagenic effects on the somatic cells of Swiss albino male mice. PMID:26823062

  17. PAK4 kinase activity and somatic mutation promote carcinoma cell motility and influence inhibitor sensitivity

    PubMed Central

    Whale, Andrew D.; Dart, Anna; Holt, Mark; Jones, Gareth E.; Wells, Claire M.

    2012-01-01

    Hepatocyte growth factor (HGF) and its receptor (c-Met) are associated with cancer cell motility and invasiveness. p21-activated kinase 4 (PAK4), a potential therapeutic target, is recruited to and activated by c-Met. In response, PAK4 phosphorylates LIM kinase 1 (LIMK1) in an HGF-dependent manner in metastatic prostate carcinoma cells. PAK4 overexpression is known to induce increased cell migration speed but the requirement for kinase activity has not been established. We have used a panel of PAK4 truncations and mutations in a combination of over-expression and RNAi rescue experiments to determine the requirement for PAK4 kinase activity during carcinoma cell motility downstream of HGF. We find that neither the kinase domain alone nor a PAK4 mutant unable to bind Cdc42 is able to fully rescue cell motility in a PAK4-deficient background. Nevertheless, we find that PAK4 kinase activity and associated LIMK1 activity are essential for carcinoma cell motility, highlighting PAK4 as a potential anti-metastatic therapeutic target. We also show here that overexpression of PAK4 harboring a somatic mutation, E329K, increased the HGF-driven motility of metastatic prostate carcinoma cells. E329 lies within the G-loop region of the kinase. Our data suggest E329K mutation leads to a modest increase in kinase activity conferring resistance to competitive ATP inhibitors in addition to promoting cell migration. The existence of such a mutation may have implications for the development of PAK4-specific competitive ATP inhibitors should PAK4 be further explored for clinical inhibition. PMID:22689056

  18. The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus).

    PubMed

    Du, Fuliang; Shen, Perng-Chih; Xu, Jie; Sung, Li-Ying; Jeong, B-Seon; Lucky Nedambale, Tshimangadzo; Riesen, John; Cindy Tian, X; Cheng, Winston T K; Lee, Shan-Nan; Yang, Xiangzhong

    2006-02-01

    One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.

  19. Differences in protodermal cell wall structure in zygotic and somatic embryos of Daucus carota (L.) cultured on solid and in liquid media.

    PubMed

    Dobrowolska, Izabela; Majchrzak, Oliwia; Baldwin, Timothy C; Kurczynska, Ewa U

    2012-01-01

    The ultrastructure, cuticle, and distribution of pectic epitopes in outer periclinal walls of protodermal cells of Daucus carota zygotic and somatic embryos from solid and suspension culture were investigated. Lipid substances were present as a continuous layer in zygotic and somatic embryos cultured on solid medium. Somatic embryos from suspension cultures were devoid of cuticle. The ultrastructure of the outer walls of protodermis of embryos was similar in zygotic and somatic embryos from solid culture. Fibrillar material was observed on the surface of somatic embryos. In zygotic embryos, in cotyledons and root pectic epitopes recognised by the antibody JIM5 were observed in all cell walls. In hypocotyls of these embryos, these pectic epitopes were not present in the outer periclinal and anticlinal walls of the protodermis. In somatic embryos from solid media, distribution of pectic epitopes recognised by JIM5 was similar to that described for their zygotic counterparts. In somatic embryos from suspension culture, pectic epitopes recognised by JIM5 were detected in all cell walls. In the cotyledons and hypocotyls, a punctate signal was observed on the outside of the protodermis. Pectic epitopes recognised by JIM7 were present in all cell walls independent of embryo organs. In zygotic embryos, this signal was punctate; in somatic embryos from both cultures, this signal was uniformly distributed. In embryos from suspension cultures, a punctate signal was detected outside the surface of cotyledon and hypocotyl. These data are discussed in light of current models for embryogenesis and the influence of culture conditions on cell wall structure.

  20. Telomere elongation facilitated by trichostatin a in cloned embryos and pigs by somatic cell nuclear transfer.

    PubMed

    Kong, Qingran; Ji, Guangzhen; Xie, Bingteng; Li, Jingyu; Mao, Jian; Wang, Juan; Liu, Shichao; Liu, Lin; Liu, Zhonghua

    2014-06-01

    Telomere attrition and genomic instability are associated with organism aging. Concerns still exist regarding telomere length resetting in cloned embryos and ntES cells, and possibilities of premature aging of cloned animals achieved by somatic cell nuclear transfer (SCNT). Trichostatin A (TSA), a histone deacetylase inhibitor, effectively improves the developmental competence of cloned embryos and animals, and recently contributes to successful generation of human ntES cells by SCNT. To test the function of TSA on resetting telomere length, we analyzed telomeres in cloned blastocysts and pigs following treatment of SCNT embryos with TSA. Here, we show that telomeres of cloned pigs generated by standard SCNT methods are not effectively restored, compared with those of donor cells, however TSA significantly increases telomere lengths in cloned pigs. Telomeres elongate in cloned porcine embryos during early cleavage from one-cell to four-cell stages. Notably, TSA facilitates telomere lengthening of cloned embryos mainly at morula-blastocyst stages. Knockdown of pTert by shRNA in donor cells reduces telomerase activity in cloned blastocysts but does not abrogate telomere elongation in the TSA-treated embryos (p > 0.05). However, genes associated with recombination or telomerase-independent mechanism of alternative lengthening of telomeres (ALT) Rad50 and BLM show increased expression in TSA-treated embryos. These data suggest that TSA may promote telomere elongation of cloned porcine embryos by ALT. Together, TSA can elongate telomeres in cloned embryos and piglets, and this could be one of the mechanisms underlying improved development of cloned embryos and animals treated with TSA. PMID:24510582

  1. Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model

    PubMed Central

    Ma, Li-bing; He, Xiao-ning; Si, Wan-tong; Zheng, Yue-Mao

    2016-01-01

    Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos. PMID:26243608

  2. Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model.

    PubMed

    He, Xiao-Ying; Ma, Li-Bing; He, Xiao-Ning; Si, Wan-Tong; Zheng, Yue-Mao

    2016-06-30

    Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos. PMID:26243608

  3. Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model.

    PubMed

    He, Xiao-Ying; Ma, Li-Bing; He, Xiao-Ning; Si, Wan-Tong; Zheng, Yue-Mao

    2016-06-30

    Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos.

  4. Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid.

    PubMed

    Verma, R; Holland, M K; Temple-Smith, P; Verma, P J

    2012-01-01

    Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future.

  5. Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid.

    PubMed

    Verma, R; Holland, M K; Temple-Smith, P; Verma, P J

    2012-01-01

    Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future. PMID

  6. Effects of somatic cell count on quality and shelf-life of pasteurized fluid milk.

    PubMed

    Ma, Y; Ryan, C; Barbano, D M; Galton, D M; Rudan, M A; Boor, K J

    2000-02-01

    Milk was collected from eight Holstein cows four times before and four times after intramammary infection with Streptococcus agalactiae. Postinfection milk had significantly higher somatic cell count (SCC) (849,000 cells/ml) than preinfection milk (45,000 cells/ml). High SCC raw milk had more lipolysis and proteolysis than low SCC raw milk. Pasteurized, homogenized, 2% fat milks from pre- and postinfection periods were stored at 5 degrees C and analyzed for lipolysis, proteolysis, microbial quality, and sensory attributes at 1, 7, 14, and 21 d post processing. During refrigerated storage, the average rates of free fatty acid increase (i.e., lipolysis) and casein hydrolysis in high SCC milk were, respectively, three and two times faster than those in low SCC milk. In general, standard plate counts, coliform counts, and psychrotrophic bacterial counts of both the high and low SCC milks remained low (<100,000 cfu/ ml) during 5 degrees C storage. Low SCC milk maintained high organoleptic quality for the entire 21-d shelf-life period. However, for high SCC milk, between 14 and 21 d, sensory defects were detected, which resulted in low overall quality ratings. The sensory defects mainly included rancidity and bitterness and were consistent with higher levels of lipolysis and proteolysis. Hence, mastitis adversely affected the quality of pasteurized fluid milk. It is recommended that the fluid milk industry consider implementation of premium quality payment programs for low SCC milks.

  7. Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos.

    PubMed

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Yao, Chaogang; Zhou, Yang; Zhu, Jianguo; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

    2011-12-01

    Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.

  8. Transcriptomic Features of Bovine Blastocysts Derived by Somatic Cell Nuclear Transfer.

    PubMed

    Min, Byungkuk; Cho, Sunwha; Park, Jung Sun; Lee, Yun-Gyeong; Kim, Namshin; Kang, Yong-Kook

    2015-09-03

    Reprogramming incompletely occurs in most somatic cell nuclear transfer (SCNT) embryos, which results in misregulation of developmentally important genes and subsequent embryonic malfunction and lethality. Here we examined transcriptome profiles in single bovine blastocysts derived by in vitro fertilization (IVF) and SCNT. Different types of donor cells, cumulus cell and ear-skin fibroblast, were used to derive cSCNT and fSCNT blastocysts, respectively. SCNT blastocysts expressed 13,606 genes on average, similar to IVF (13,542). Correlation analysis found that both cSCNT and fSCNT blastocyst groups had transcriptomic features distinctive from the IVF group, with the cSCNT transcriptomes closer to the IVF ones than the fSCNT. Gene expression analysis identified 56 underrepresented and 78 overrepresented differentially expressed genes in both SCNT groups. A 400-kb locus harboring zinc-finger protein family genes in chromosome 18 were found coordinately down-regulated in fSCNT blastocysts, showing a feature of reprogramming-resistant regions. Probing into different categories of genes important for blastocyst development revealed that genes involved in trophectoderm development frequently were underrepresented, and those encoding epigenetic modifiers tended to be overrepresented in SCNT blastocysts. Our effort to identify reprogramming-resistant, differentially expressed genes can help map reprogramming error-prone loci onto the genome and elucidate how to handle the stochastic events of reprogramming to improve cloning efficiency.

  9. Transcriptomic Features of Bovine Blastocysts Derived by Somatic Cell Nuclear Transfer

    PubMed Central

    Min, Byungkuk; Cho, Sunwha; Park, Jung Sun; Lee, Yun-Gyeong; Kim, Namshin; Kang, Yong-Kook

    2015-01-01

    Reprogramming incompletely occurs in most somatic cell nuclear transfer (SCNT) embryos, which results in misregulation of developmentally important genes and subsequent embryonic malfunction and lethality. Here we examined transcriptome profiles in single bovine blastocysts derived by in vitro fertilization (IVF) and SCNT. Different types of donor cells, cumulus cell and ear-skin fibroblast, were used to derive cSCNT and fSCNT blastocysts, respectively. SCNT blastocysts expressed 13,606 genes on average, similar to IVF (13,542). Correlation analysis found that both cSCNT and fSCNT blastocyst groups had transcriptomic features distinctive from the IVF group, with the cSCNT transcriptomes closer to the IVF ones than the fSCNT. Gene expression analysis identified 56 underrepresented and 78 overrepresented differentially expressed genes in both SCNT groups. A 400-kb locus harboring zinc-finger protein family genes in chromosome 18 were found coordinately down-regulated in fSCNT blastocysts, showing a feature of reprogramming-resistant regions. Probing into different categories of genes important for blastocyst development revealed that genes involved in trophectoderm development frequently were underrepresented, and those encoding epigenetic modifiers tended to be overrepresented in SCNT blastocysts. Our effort to identify reprogramming-resistant, differentially expressed genes can help map reprogramming error-prone loci onto the genome and elucidate how to handle the stochastic events of reprogramming to improve cloning efficiency. PMID:26342001

  10. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos.

    PubMed

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue; Pang, Daxin; Ouyang, Hongsheng

    2011-07-29

    The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50μg/mL vitamin C 15h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos. PMID:21749856

  11. Exome sequencing identifies somatic mutations of DDX3X in natural killer/T-cell lymphoma.

    PubMed

    Jiang, Lu; Gu, Zhao-Hui; Yan, Zi-Xun; Zhao, Xia; Xie, Yin-Yin; Zhang, Zi-Guan; Pan, Chun-Ming; Hu, Yuan; Cai, Chang-Ping; Dong, Ying; Huang, Jin-Yan; Wang, Li; Shen, Yang; Meng, Guoyu; Zhou, Jian-Feng; Hu, Jian-Da; Wang, Jin-Fen; Liu, Yuan-Hua; Yang, Lin-Hua; Zhang, Feng; Wang, Jian-Min; Wang, Zhao; Peng, Zhi-Gang; Chen, Fang-Yuan; Sun, Zi-Min; Ding, Hao; Shi, Ju-Mei; Hou, Jian; Yan, Jin-Song; Shi, Jing-Yi; Xu, Lan; Li, Yang; Lu, Jing; Zheng, Zhong; Xue, Wen; Zhao, Wei-Li; Chen, Zhu; Chen, Sai-Juan

    2015-09-01

    Natural killer/T-cell lymphoma (NKTCL) is a malignant proliferation of CD56(+) and cytoCD3(+) lymphocytes with aggressive clinical course, which is prevalent in Asian and South American populations. The molecular pathogenesis of NKTCL has largely remained elusive. We identified somatic gene mutations in 25 people with NKTCL by whole-exome sequencing and confirmed them in an extended validation group of 80 people by targeted sequencing. Recurrent mutations were most frequently located in the RNA helicase gene DDX3X (21/105 subjects, 20.0%), tumor suppressors (TP53 and MGA), JAK-STAT-pathway molecules (STAT3 and STAT5B) and epigenetic modifiers (MLL2, ARID1A, EP300 and ASXL3). As compared to wild-type protein, DDX3X mutants exhibited decreased RNA-unwinding activity, loss of suppressive effects on cell-cycle progression in NK cells and transcriptional activation of NF-κB and MAPK pathways. Clinically, patients with DDX3X mutations presented a poor prognosis. Our work thus contributes to the understanding of the disease mechanism of NKTCL. PMID:26192917

  12. Green fluorescent protein gene-transfected peafowl somatic cells participate in the development of chicken embryos.

    PubMed

    Xi, Yongmei; Nada, Yoich; Soh, Tomoki; Fujihara, Noboru; Hattori, Masa-Aki

    2004-02-01

    This study was performed to investigate whether the embryonic somatic cells are capable of reconstituting and participating in the embryonic development of chickens to produce chimeras. In order to track the migration behavior of the donor cells, a cell line, originally isolated from an Indian peafowl embryo, was fluorescent-labeled by transfection of the cells with enhanced Green Fluorescent Protein (GFP) and Neomycin resistant (Neo) genes prior to injection into the stage X blastoderm of White Leghorn chickens. The injection was performed with a medium in the presence of 1-5% polyethylene glycol. The development of putative chimeric embryos between the stages three and 24 was examined for GFP expression under fluorescent light. To trace the peafowl cells in the developing chicken embryos, both a species-specific genetic marker originating from the mitochondrial DNA cytochrome b (cyt b) gene and a DNA fragment of GFP gene were used. Of the 185 fertile eggs manipulated, 173 developed into embryos. Fifty-five of them showed positive GFP patches in extra-embryonic tissues, and 15 expressed GFP in intra-embryonic tissues such as those of the head, heart, and gonad. PCR analysis revealed that PCR fragments for the peafowl mitochondrial DNA cyt b and GFP genes were detected in the samples of the GFP positive extra- and intra-embryonic tissues of the chimeras. The present results provide evidence that fluorescent-labeled peafowl embryonic cells carrying GFP and Neo genes are able to participate in the development of chicken embryos to generate chimeras.

  13. Green fluorescent protein gene-transfected peafowl somatic cells participate in the development of chicken embryos.

    PubMed

    Xi, Yongmei; Nada, Yoich; Soh, Tomoki; Fujihara, Noboru; Hattori, Masa-Aki

    2004-02-01

    This study was performed to investigate whether the embryonic somatic cells are capable of reconstituting and participating in the embryonic development of chickens to produce chimeras. In order to track the migration behavior of the donor cells, a cell line, originally isolated from an Indian peafowl embryo, was fluorescent-labeled by transfection of the cells with enhanced Green Fluorescent Protein (GFP) and Neomycin resistant (Neo) genes prior to injection into the stage X blastoderm of White Leghorn chickens. The injection was performed with a medium in the presence of 1-5% polyethylene glycol. The development of putative chimeric embryos between the stages three and 24 was examined for GFP expression under fluorescent light. To trace the peafowl cells in the developing chicken embryos, both a species-specific genetic marker originating from the mitochondrial DNA cytochrome b (cyt b) gene and a DNA fragment of GFP gene were used. Of the 185 fertile eggs manipulated, 173 developed into embryos. Fifty-five of them showed positive GFP patches in extra-embryonic tissues, and 15 expressed GFP in intra-embryonic tissues such as those of the head, heart, and gonad. PCR analysis revealed that PCR fragments for the peafowl mitochondrial DNA cyt b and GFP genes were detected in the samples of the GFP positive extra- and intra-embryonic tissues of the chimeras. The present results provide evidence that fluorescent-labeled peafowl embryonic cells carrying GFP and Neo genes are able to participate in the development of chicken embryos to generate chimeras. PMID:14743513

  14. In vitro somatic embryogenesis and plantlet regeneration from immature male inflorescence of adult dura and tenera palms of Elaeis guineensis (Jacq.).

    PubMed

    Jayanthi, Madhavan; Susanthi, Bollarapu; Murali Mohan, Nandiganti; Mandal, Pranab Kumar

    2015-01-01

    We report here a method for plant regeneration through somatic embryogenesis from explants collected from immature male inflorescence of adult oil palm cultivated in India. Callus induction was successful from tissues of immature male inflorescence collected from both dura and tenera varieties of oil palm. A modified Y3 (Eeuwens) media supplemented with several additives and activated charcoal (3%) were used for the experiments. Out of four different auxin treatments, 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram) produced maximum callus induction (82%) and it was not significantly different from 2,4-dichlorophenoxyacetic acid (2,4-D) and a combination of 2,4-D + picloram. The callus induction obtained with auxin α-naphthalene acetic acid was only 54% and it was significantly low as compared to the other treatments. Highest embryogenesis was obtained with a combination of 2,4-D + picloram (4.9%) followed by picloram (3.4%). Genotypic variation in response to the same auxins was observed both for callus induction and embryogenesis. Callus induction and embryogenesis ranged from 42 to 72% and 6.8 to 9.35%, respectively in tenera. The formation of embryogenic calli was marked by the appearance of white to yellowish globular or nodular structures which subsequently formed clear somatic embryos. Somatic embryogenesis was asynchronous and at one time we could find different stages of embryogenesis like the globular, torpedo and the cotyledonary stages. The somatic embryos when exposed to light in the same basal media along with 6-benzyladenine (18 µM), abscisic acid (3.78 µM) and gibberellic acid (5.78 µM) regenerated into plantlets. To the best of our knowledge this is the first report o f callus induction and somatic embryogenesis from immature male inflorescence of oil palm. PMID:26085976

  15. Flow Cytometry Approach to Quantify the Viability of Milk Somatic Cell Counts after Various Physico-Chemical Treatments

    PubMed Central

    Li, Na; Richoux, Romain; Perruchot, Marie-Hélène; Boutinaud, Marion; Mayol, Jean-François; Gagnaire, Valérie

    2015-01-01

    Flow cytometry has been used as a routine method to count somatic cells in milk, and to ascertain udder health and milk quality. However, few studies investigate the viability of somatic cells and even fewer at a subpopulation level to follow up how the cells can resist to various stresses that can be encountered during technological processes. To address this issue, a flow cytometry approach was used to simultaneously identify cell types of bovine milk using cell-specific antibodies and to measure the cell viability among the identified subpopulations by using a live/dead cell viability kit. Confirmation of the cell viability was performed by using conventional microscopy. Different physico-chemical treatments were carried out on standardized cell samples, such as heat treatment, various centrifugation rates and storage in milk or in PBS pH 7.4 for three days. Cytometry gating strategy was developed by using blood cell samples stored at 4°C in PBS and milk cell samples heat-treated at 80°C for 30 min as a control for the maximum (95.9%) and minimum (0.7%) values of cell viability respectively. Cell viability in the initial samples was 39.5% for all cells and varied for each cell population from 26.7% for PMNs, to 32.6% for macrophages, and 58.3% for lymphocytes. Regarding the physico-chemical treatments applied, somatic cells did not sustain heat treatment at 60°C and 80°C in contrast to changes in centrifugation rates, for which only the higher level, i.e. 5000×g led to a cell viability decrease, down to 9.4%, but no significant changes within the cell subpopulation distribution were observed. Finally, the somatic cells were better preserved in milk after 72h storage, in particular PMNs, that maintained a viability of 34.0 ± 2.9% compared to 4.9±1.9% in PBS, while there was almost no changes for macrophages (41.7 ± 5.7% in milk vs 31.2 ± 2.4% in PBS) and lymphocytes (25.3 ± 3.0% in milk vs 11.4 ± 3.1% in PBS). This study provides a new array to better

  16. In vitro development of bison embryos using interspecies somatic cell nuclear transfer.

    PubMed

    Seaby, R P; Alexander, B; King, W A; Mastromonaco, G F

    2013-12-01

    Interspecies somatic cell nuclear transfer (interspecies SCNT) has been explored in many domestic and non-domestic animal species. However, problems arise during the development of these embryos, which may be related to species-specific differences in nuclear-cytoplasmic communication. The objectives of this study were to investigate the possibility of producing bison embryos in vitro using interspecies SCNT and assess the developmental potential of these embryos. Treatment groups consisted of cattle in vitro fertilization (IVF) and cattle SCNT as controls and wood bison SCNT, plains bison SCNT and wisent SCNT as experimental groups. Cleavage and blastocyst rates were assessed, and blastocyst quality was determined using total cell number, apoptotic incidence and relative quantification of mitochondria-related genes NRF1, MT-CYB and TFAM. These results indicate that embryos can be produced by interspecies SCNT in all bison species/subspecies (13.34-33.54% blastocyst rates). Although increased incidence of apoptosis was observed in bison SCNT blastocysts compared to cattle SCNT controls (10.45-12.69 vs 8.76, respectively) that corresponded with significantly lower cell numbers (80-87 cells vs >100 cells, respectively), no major differences were observed in the expression of NRF1, MT-CYB and TFAM. This study is the first to report the production of bison embryos by interspecies SCNT. Blastocyst development in all three bison species/subspecies was greater than the rates obtained in previous studies by IVF, which supports the potential role of SCNT for in vitro embryo production in this species. Yet, further investigation of developmental competence and the factors influencing blastocyst quality and viability is required.

  17. SMC1B is present in mammalian somatic cells and interacts with mitotic cohesin proteins

    PubMed Central

    Mannini, Linda; Cucco, Francesco; Quarantotti, Valentina; Amato, Clelia; Tinti, Mara; Tana, Luigi; Frattini, Annalisa; Delia, Domenico; Krantz, Ian D.; Jessberger, Rolf; Musio, Antonio

    2015-01-01

    Cohesin is an evolutionarily conserved protein complex that plays a role in many biological processes: it ensures faithful chromosome segregation, regulates gene expression and preserves genome stability. In mammalian cells, the mitotic cohesin complex consists of two structural maintenance of chromosome proteins, SMC1A and SMC3, the kleisin protein RAD21 and a fourth subunit either STAG1 or STAG2. Meiotic paralogs in mammals were reported for SMC1A, RAD21 and STAG1/STAG2 and are called SMC1B, REC8 and STAG3 respectively. It is believed that SMC1B is only a meiotic-specific cohesin member, required for sister chromatid pairing and for preventing telomere shortening. Here we show that SMC1B is also expressed in somatic mammalian cells and is a member of a mitotic cohesin complex. In addition, SMC1B safeguards genome stability following irradiation whereas its ablation has no effect on chromosome segregation. Finally, unexpectedly SMC1B depletion impairs gene transcription, particularly at genes mapping to clusters such as HOX and PCDHB. Genome-wide analyses show that cluster genes changing in expression are enriched for cohesin-SMC1B binding. PMID:26673124

  18. Correlation between standard plate count and somatic cell count milk quality results for Wisconsin dairy producers.

    PubMed

    Borneman, Darand L; Ingham, Steve

    2014-05-01

    The objective of this study was to determine if a correlation exists between standard plate count (SPC) and somatic cell count (SCC) monthly reported results for Wisconsin dairy producers. Such a correlation may indicate that Wisconsin producers effectively controlling sanitation and milk temperature (reflected in low SPC) also have implemented good herd health management practices (reflected in low SCC). The SPC and SCC results for all grade A and B dairy producers who submitted results to the Wisconsin Department of Agriculture, Trade, and Consumer Protection, in each month of 2012 were analyzed. Grade A producer SPC results were less dispersed than grade B producer SPC results. Regression analysis showed a highly significant correlation between SPC and SCC, but the R(2) value was very small (0.02-0.03), suggesting that many other factors, besides SCC, influence SPC. Average SCC (across 12 mo) for grade A and B producers decreased with an increase in the number of monthly SPC results (out of 12) that were ≤ 25,000 cfu/mL. A chi-squared test of independence showed that the proportion of monthly SCC results >250,000 cells/mL varied significantly depending on whether the corresponding SPC result was ≤ 25,000 or >25,000 cfu/mL. This significant difference occurred in all months of 2012 for grade A and B producers. The results suggest that a generally consistent level of skill exists across dairy production practices affecting SPC and SCC.

  19. SMC1B is present in mammalian somatic cells and interacts with mitotic cohesin proteins.

    PubMed

    Mannini, Linda; Cucco, Francesco; Quarantotti, Valentina; Amato, Clelia; Tinti, Mara; Tana, Luigi; Frattini, Annalisa; Delia, Domenico; Krantz, Ian D; Jessberger, Rolf; Musio, Antonio

    2015-01-01

    Cohesin is an evolutionarily conserved protein complex that plays a role in many biological processes: it ensures faithful chromosome segregation, regulates gene expression and preserves genome stability. In mammalian cells, the mitotic cohesin complex consists of two structural maintenance of chromosome proteins, SMC1A and SMC3, the kleisin protein RAD21 and a fourth subunit either STAG1 or STAG2. Meiotic paralogs in mammals were reported for SMC1A, RAD21 and STAG1/STAG2 and are called SMC1B, REC8 and STAG3 respectively. It is believed that SMC1B is only a meiotic-specific cohesin member, required for sister chromatid pairing and for preventing telomere shortening. Here we show that SMC1B is also expressed in somatic mammalian cells and is a member of a mitotic cohesin complex. In addition, SMC1B safeguards genome stability following irradiation whereas its ablation has no effect on chromosome segregation. Finally, unexpectedly SMC1B depletion impairs gene transcription, particularly at genes mapping to clusters such as HOX and PCDHB. Genome-wide analyses show that cluster genes changing in expression are enriched for cohesin-SMC1B binding.

  20. Cytogenetic effects of leachates from tannery solid waste on the somatic cells of Vicia faba.

    PubMed

    Chandra, Saurabh; Chauhan, L K S; Pande, P N; Gupta, S K

    2004-04-01

    The contamination of surface- and groundwater by the leaching of solid wastes generated by industrial activities as a result of water runoff and rainfall is a matter of great concern. The leachates from tannery solid waste (TSW), a major environmental pollutant, were examined for their possible genotoxic effects on the somatic cells of Vicia faba. Leachates were prepared from solid wastes procured from leather-tanning industrial sites, and V. faba seedlings were exposed to three test concentrations, 2.5%, 5%, and 10%, through soil and aqueous media for 5 days. The root tips examined for cytogenetic damage revealed that leachate of TSW significantly inhibited the mitotic index and induced significantly frequent chromosomal and mitotic aberrations (CA/MA) in a dose-dependent manner. The chemical analysis of TSW samples revealed that the chief constituents were chromium and nickel, which may cause genetic abnormalities. The frequency of aberrations was found to be higher in the root meristematic cells of Vicia faba exposed through the aqueous medium than those exposed through the soil medium. The results of the present study indicated that contamination of potable water bodies by leachates of TSW may cause genotoxicity. For the biomonitoring of complex mixtures of toxicants with the V. faba bioassay, the use of the aqueous medium seems to be a more promising method than the use of the soil medium.

  1. PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells.

    PubMed

    Chandrakanthan, Vashe; Yeola, Avani; Kwan, Jair C; Oliver, Rema A; Qiao, Qiao; Kang, Young Chan; Zarzour, Peter; Beck, Dominik; Boelen, Lies; Unnikrishnan, Ashwin; Villanueva, Jeanette E; Nunez, Andrea C; Knezevic, Kathy; Palu, Cintia; Nasrallah, Rabab; Carnell, Michael; Macmillan, Alex; Whan, Renee; Yu, Yan; Hardy, Philip; Grey, Shane T; Gladbach, Amadeus; Delerue, Fabien; Ittner, Lars; Mobbs, Ralph; Walkley, Carl R; Purton, Louise E; Ward, Robyn L; Wong, Jason W H; Hesson, Luke B; Walsh, William; Pimanda, John E

    2016-04-19

    Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

  2. Somatic mutations and affinity maturation are impaired by excessive numbers of T follicular helper cells and restored by Treg cells or memory T cells

    PubMed Central

    Preite, Silvia; Baumjohann, Dirk; Foglierini, Mathilde; Basso, Camilla; Ronchi, Francesca; Rodriguez, Blanca M. Fernandez; Corti, Davide; Lanzavecchia, Antonio

    2015-01-01

    We previously reported that Cd3e‐deficient mice adoptively transferred with CD4+ T cells generate high numbers of T follicular helper (Tfh) cells, which go on to induce a strong B‐cell and germinal center (GC) reaction. Here, we show that in this system, GC B cells display an altered distribution between the dark and light zones, and express low levels of activation‐induced cytidine deaminase. Furthermore, GC B cells from Cd3e –/– mice accumulate fewer somatic mutations as compared with GC B cells from wild‐type mice, and exhibit impaired affinity maturation and reduced differentiation into long‐lived plasma cells. Reconstitution of Cd3e –/– mice with regulatory T (Treg) cells restored Tfh‐cell numbers, GC B‐cell numbers and B‐cell distribution within dark and light zones, and the rate of antibody somatic mutations. Tfh‐cell numbers and GC B‐cell numbers and dynamics were also restored by pre‐reconstitution of Cd3e –/– mice with Cxcr5 –/– Treg cells or non‐regulatory, memory CD4+ T cells. Taken together, these findings underline the importance of a quantitatively regulated Tfh‐cell response for an efficient and long‐lasting serological response. PMID:26332258

  3. Stem Cell Basics

    MedlinePlus

    ... stem cells? What are the potential uses of human stem cells and the obstacles that must be overcome before ... two kinds of stem cells from animals and humans: embryonic stem cells and non-embryonic "somatic" or "adult" stem cells . ...

  4. Zfp281 mediates Nanog autorepression through recruitment of the NuRD complex and inhibits somatic cell reprogramming

    PubMed Central

    Fidalgo, Miguel; Faiola, Francesco; Pereira, Carlos-Filipe; Ding, Junjun; Saunders, Arven; Gingold, Julian; Schaniel, Christoph; Lemischka, Ihor R.; Silva, José C. R.; Wang, Jianlong

    2012-01-01

    The homeodomain transcription factor Nanog plays an important role in embryonic stem cell (ESC) self-renewal and is essential for acquiring ground-state pluripotency during reprogramming. Understanding how Nanog is transcriptionally regulated is important for further dissecting mechanisms of ESC pluripotency and somatic cell reprogramming. Here, we report that Nanog is subjected to a negative autoregulatory mechanism, i.e., autorepression, in ESCs, and that such autorepression requires the coordinated action of the Nanog partner and transcriptional repressor Zfp281. Mechanistically, Zfp281 recruits the NuRD repressor complex onto the Nanog locus and maintains its integrity to mediate Nanog autorepression and, functionally, Zfp281-mediated Nanog autorepression presents a roadblock to efficient somatic cell reprogramming. Our results identify a unique transcriptional regulatory mode of Nanog gene expression and shed light into the mechanistic understanding of Nanog function in pluripotency and reprogramming. PMID:22988117

  5. Extinction of expression of the translocated myc gene in somatic cell hybrids between mouse myeloma and L-cells.

    PubMed

    Greenberg, A; Hijazzi, M; Sharir, H; Cohen, L; Bergman, Y; Ber, R; Laskov, R

    1989-01-15

    Most murine plasma-cell tumors show a t(12;15) reciprocal chromosomal translocation which truncates the first exon of one of the myc gene alleles and fuses it to one of the switch regions of the immunoglobulin (Ig) heavy-chain locus. This results in constitutive activation of the translocated myc gene and the production of smaller-sized mRNA molecules, which are initiated at new sites in the first myc intron. The normal myc allele is not expressed in these myeloma cells. We have studied the expression of the translocated myc gene in somatic cell hybrids between mouse myeloma and L-cells. Our previous findings show that Ig gene expression is extinguished in such hybrids. In the present work we found that the hybrids contain the normal and translocated myc genes. In contrast to the myeloma parental cells which express the translocated myc gene, the hybrids are similar to the L-cells in expressing only the normal myc allele. Our results suggest that the L-cell, fibroblast-like phenotype, is dominant in these hybrids, and show that the translocated myc gene is expressed in a tissue-specific manner in the context of the myeloma cell, and is not expressed when subjected to a fibroblast-like cellular environment.

  6. Age-related accumulation of Ig VH gene somatic mutations in peripheral B cells from aged humans

    PubMed Central

    CHONG, Y; IKEMATSU, H; YAMAJI, K; NISHIMURA, M; KASHIWAGI, S; HAYASHI, J

    2003-01-01

    To investigate age-related alterations in human humoral immunity, we analysed Ig heavy chain variable region genes expressed by peripheral B cells from young and aged individuals. Three hundred and twenty-seven cDNA sequences, 163 µ and 164 γ transcripts with VH5 family genes, were analysed for somatic hypermutation and VHDJH recombinational features. Unmutated and mutated µ transcripts were interpreted as being from naive and memory IgM B cells, respectively. In young and aged individuals, the percentages of naive IgM among total µ transcripts were 39% and 42%, respectively. D and JH segment usage in naive IgM from aged individuals was similar to that from young individuals. The mutational frequencies of memory IgM were similar in young and aged individuals. γ transcripts, which are regarded as being from memory IgG B cells, showed a significantly higher mutational frequency (7·6%) in aged than in young individuals (5·8%) (P < 0·01). These findings suggest that VHDJH recombinational diversity was preserved, but that the accumulation of somatic mutations in the IgG VH region was increased in aged humans. The accumulation of somatic mutations in IgG B cells during ageing may imply that an age-related alteration exists in the selection and/or maintenance of peripheral memory B cells. PMID:12823279

  7. Deletion of Tuberous Sclerosis 1 in Somatic Cells of the Murine Reproductive Tract Causes Female Infertility

    PubMed Central

    Tanaka, Yoshihiro; Park, Joo Hyun; Tanwar, Pradeep S.; Kaneko-Tarui, Tomoko; Mittal, Shilpi; Lee, Ho-Joon

    2012-01-01

    Tumors develop with dysregulated activation of mammalian target of rapamycin (mTOR), the kinase activity of which is kept in an inactive state by a tumor suppressor dimer containing tuberous sclerosis 1 (TSC1) and TSC2. We examined whether conditional deletion of TSC1 by a knock-in allele of the anti-Müllerian hormone type 2 receptor (Amhr2) driving Cre expression and subsequent activation of mTOR in granulosa cells and in oviductal and uterine stromal cells affects fertility in female mice. Increased phosphorylation of ribosomal protein S6, a downstream target of activated mTOR, was observed in all AMHR2-expressing tissues examined, indicating loss of TSC1 activity. TSC1 deletion in granulosa cells led to the detection of significantly fewer primordial follicles in mutant mice at 12 wk, suggesting premature ovarian insufficiency, which might be related to the significantly increased time mutant mice spent in estrus. Although the number of good-quality ovulated oocytes was not significantly different compared with controls, there was a significantly higher number of degenerated oocytes after normal and superovulation, suggesting compromised oocyte quality, as well. Natural mating also showed severalfold higher numbers of degenerate bodies in the mutants that collected in bilateral swellings resembling hydrosalpinges that formed in all mice examined because of occlusion of the proximal oviduct. Attempts to transfer control embryos into mutant uteri also failed, indicating that implantation was compromised. Endometrial epithelial cells continued to proliferate, and quantitative RT-PCR showed that mucin 1 expression persisted during the window of implantation in mutant uteri, without any changes in progesterone receptor mRNA expression, suggesting a mechanism that does not involve disrupted estradiol-regulated progesterone receptor expression. Homozygous deletion of TSC1 in reproductive tract somatic tissues of mice rendered females completely infertile, which is

  8. A novel somatic MAPK1 mutation in primary ovarian mixed germ cell tumors.

    PubMed

    Zou, Yang; Deng, Wei; Wang, Feng; Yu, Xiao-Hong; Liu, Fa-Ying; Yang, Bi-Cheng; Huang, Mei-Zhen; Guo, Jiu-Bai; Xie, Qiu-Hua; He, Ming; Huang, Ou-Ping

    2016-02-01

    A recent exome-sequencing study revealed prevalent mitogen-activated protein kinase 1 (MAPK1) p.E322K mutation in cervical carcinoma. It remains largely unknown whether ovarian carcinomas also harbor MAPK1 mutations. As paralogous gene mutations co‑occur frequently in human malignancies, we analyzed here a total of 263 ovarian carcinomas for the presence of MAPK1 and paralogous MAPK3 mutations by DNA sequencing. A previously unreported MAPK1 p.D321N somatic mutation was identified in 2 out of 18 (11.1%) ovarian mixed germ cell tumors, while no other MAPK1 or MAPK3 mutation was detected in our samples. Of note, OCC‑115, the MAPK1‑mutated sample with bilateral cancerous ovaries affected, harbored MAPK1 mutation in the right ovary while retained the left ovary intact, implicating that the genetic alterations underlying ovarian mixed germ cell tumor may be different, even in patients with similar genetic backgrounds and tumor microenvironments. The results of evolutionary conservation and protein structure modeling analysis implicated that MAPK1 p.D321N mutation may be pathogenic. Additionally, mutations in protein phosphatase 2 regulatory subunit α (PPP2R1A), ring finger protein 43 (RNF43), DNA directed polymerase ε (POLE1), ribonuclease type III (DICER1), CCCTC‑binding factor (CTCF), ribosomal protein L22 (RPL22), DNA methyltransferase 3α (DNMT3A), transformation/transcription domain‑associated protein (TRRAP), isocitrate dehydrogenase (IDH)1 and IDH2 were not detected in ovarian mixed germ cell tumors, implicating these genetic alterations may be not associated with MAPK1 mutation in the development of this malignancy. The present study identified a previously unreported MAPK1 mutation in ovarian mixed germ cell tumors for the first time, and this mutation may be actively involved in the tumorigenesis of this disease.

  9. Dynamics of somatic cell counts and intramammary infections across the dry period.

    PubMed

    Pantoja, J C F; Hulland, C; Ruegg, P L

    2009-07-01

    The objectives of this research were to study the relationship between somatic cell count (SCC) and intramammary infection (IMI) across the dry period and the risk of subclinical mastitis at the first dairy herd improvement (DHI) test of the subsequent lactation. A secondary objective was to determine SCC test characteristics for diagnosis of IMI at both the cow and quarter levels. A total of 218 cows from a university herd were enrolled at dry-off. Duplicate quarter milk samples were collected from all quarters at dry-off, calving and on the day of the first DHI test. Somatic cell count status across the dry period was defined based on the comparison of quarter SCC from dry-off and the post-calving sampling periods and comparison of composite SCC from DHI samples from the last test and first test of the following lactation. Of new IMI detected from post-calving milk samples (n=45), 46.7, 26.7 and 11% were caused by CNS, Streptococci and Gram-negative bacteria, respectively. Of cured IMI at post-calving (n=91), 61.5, 23.1 and 9.9% had CNS, Streptococci and Coryneforms isolated from dry-off milk samples. The most frequent microorganisms related to cured IMI were CNS (33%). Of chronically infected quarters across the dry period (n=10), only one had the same species of pathogen isolated from dry-off and post-calving samples. The sensitivity of a SCC threshold of 200,000 cells/mL for detection of subclinical IMI was 0.64, 0.69 and 0.65 for milk samples obtained at dry-off, post-calving and first DHI test, respectively. The specificity was 0.66, 0.84 and 0.93 for milk samples obtained at dry-off, post-calving and first DHI test, respectively. Quarters with SCC> or =200,000 cells/mL at both dry-off and post-calving sampling periods were 20.4 times more likely to be subclinically infected by a major pathogen (rather than being uninfected) and 5.6 times more likely to be subclinically infected by a minor pathogen (rather than being uninfected) at the first DHI test than

  10. Cell-Specific mRNA Profiling of the Caenorhabditis elegans Somatic Gonadal Precursor Cells Identifies Suites of Sex-Biased and Gonad-Enriched Transcripts

    PubMed Central

    Kroetz, Mary B.; Zarkower, David

    2015-01-01

    The Caenorhabditis elegans somatic gonad differs greatly between the two sexes in its pattern of cell divisions, migration, and differentiation. Despite decades of study, the genetic pathways directing early gonadal development and establishing sexual dimorphism in the gonad remain largely unknown. To help define the genetic networks that regulate gonadal development, we employed cell-specific RNA-seq. We identified transcripts present in the somatic gonadal precursor cells and their daughter cells of each sex at the onset of sexual differentiation. We identified several hundred gonad-enriched transcripts, including the majority of known regulators of early gonadal development, and transgenic reporter analysis confirmed the effectiveness of this approach. Before the division of the somatic gonad precursors, few sex-biased gonadal transcripts were detectable; less than 6 hr later, after their division, we identified more than 250 sex-biased transcripts, of which about a third were enriched in the somatic gonad compared to the whole animal. This indicates that a robust sex-biased developmental program, some of it gonad-specific, initiates in the somatic gonadal precursor cells around the time of their first division. About 10% of male-biased transcripts had orthologs with male-biased expression in the early mouse gonad, suggesting possible conservation of gonad sex differentiation. Cell-specific analysis also identified approximately 70 previously unannotated mRNA isoforms that are enriched in the somatic gonad. Our data illustrate the power of cell-specific transcriptome analysis and suggest that early sex differentiation in the gonad is controlled by a relatively small suite of differentially expressed genes, even after dimorphism has become apparent. PMID:26497144

  11. Meiotic behavior of pollen mother cells in relation to ploidy level of somatic hybrids between Solanum tuberosum and S. chacoense.

    PubMed

    Guo, Xianpu; Xie, Conghua; Cai, Xingkui; Song, Botao; He, Li; Liu, Jun

    2010-11-01

    Potato somatic hybrids obtained by protoplast fusion between Solanum tuberosum (4x) and Solanum chacoense (2x) were investigated for genome stability and meiotic behavior associated with the pollen viability in order to elucidate the mechanism influencing the fertility of the somatic hybrids. The ploidy level detections conducted in 2004 and 2007 demonstrated that 68 out of 108 somatic hybrids had their ploidy level changed to be uniform and euploidy after successive in vitro subcultures, which mainly occurred in octaploids, aneuploids, and mixoploids, while 74% hexaploids were still stable in their genome dosage in 2007. Different types of abnormal meiotic behavior were observed during the development of pollen mother cells (PMCs) including the formation of univalents, multivalents, laggard chromosomes, and chromosomal bridges, as well as triads and polyads. A higher proportion of abnormal meiosis seemed to be accompanied with a genome dosage higher than the hexaploids expected in this study. A significant positive correlation between defective PMCs and the number of small pollen grains and negative correlation between number of small pollen grains and pollen viability strongly suggested that abnormal meiosis could be a causal factor influencing the fertility of the somatic hybrids. The hexaploids with stable genome dosage and a certain level of fertility will have great potential in a potato breeding program.

  12. Potential of adipose-derived mesenchymal stem cells and skeletal muscle-derived satellite cells for somatic cell nuclear transfer mediated transgenesis in Arbas Cashmere goats.

    PubMed

    Ren, Yu; Wu, Haiqing; Ma, Yuzhen; Yuan, Jianlong; Liang, Hao; Liu, Dongjun

    2014-01-01

    Somatic cell nuclear transfer is used to generate genetic models for research and new, genetically modified livestock varieties. Goat fetal fibroblast cells (gFFCs) are the predominant nuclear donors in Cashmere goat transgenic cloning, but have disadvantages. We evaluated the potential of goat adipose-derived mesenchymal stem cells (gADSCs) and goat skeletal muscle-derived satellite cells (gMDSCs) for somatic cell nuclear transfer, evaluating their proliferation, pluripotency, transfection efficiency and capacity to support full term development of embryos after additive gene transfer or homologous recombination. gADSCs and gMDSCs were isolated by enzyme digestion and differentiated into neurocytes, myotube cells and insulin-producing cells. Neuron-specific enolase, fast muscle myosin and insulin expression were determined by immunohistochemistry. Following somatic cell nuclear transfer with donor cells derived from gADSCs, gMDSCs and gFFCs, transfection and cloning efficiencies were compared. Red fluorescent protein levels were determined by quantitative PCR and western blotting. 5-Methylcytosine, H4K5, H4K12 and H3K18 were determined immunohistochemically. gADSCs and gMDSCs were maintained in culture for up to 65 passages, whereas gFFCs could be passaged barely more than 15 times. gADSCs and gMDSCs had higher fluorescent colony forming efficiency and greater convergence (20%) and cleavage (10%) rates than gFFCs, and exhibited differing H4K5 histone modification patterns after somatic cell nuclear transfer and in vitro cultivation. After transfection with a pDsRed2-1 expression plasmid, the integrated exogenous genes did not influence the pluripotency of gADSCs-pDsRed2-1 or gMDSCs-pDsRed2-1. DsRed2 mRNA expression by cloned embryos derived from gADSCs-pDsRed2-1 or gMDSCs-pDsRed2-1 was more than twice that of gFFCs-pDsRed2-1 embryos (P<0.01). Pregnancy rates of gADSCs-pDsRed2-1 and gMDSCs-pDsRed2-1 recipients were higher than those of gFFCs-pDsRed2-1 recipients (P

  13. Potential of adipose-derived mesenchymal stem cells and skeletal muscle-derived satellite cells for somatic cell nuclear transfer mediated transgenesis in Arbas Cashmere goats.

    PubMed

    Ren, Yu; Wu, Haiqing; Ma, Yuzhen; Yuan, Jianlong; Liang, Hao; Liu, Dongjun

    2014-01-01

    Somatic cell nuclear transfer is used to generate genetic models for research and new, genetically modified livestock varieties. Goat fetal fibroblast cells (gFFCs) are the predominant nuclear donors in Cashmere goat transgenic cloning, but have disadvantages. We evaluated the potential of goat adipose-derived mesenchymal stem cells (gADSCs) and goat skeletal muscle-derived satellite cells (gMDSCs) for somatic cell nuclear transfer, evaluating their proliferation, pluripotency, transfection efficiency and capacity to support full term development of embryos after additive gene transfer or homologous recombination. gADSCs and gMDSCs were isolated by enzyme digestion and differentiated into neurocytes, myotube cells and insulin-producing cells. Neuron-specific enolase, fast muscle myosin and insulin expression were determined by immunohistochemistry. Following somatic cell nuclear transfer with donor cells derived from gADSCs, gMDSCs and gFFCs, transfection and cloning efficiencies were compared. Red fluorescent protein levels were determined by quantitative PCR and western blotting. 5-Methylcytosine, H4K5, H4K12 and H3K18 were determined immunohistochemically. gADSCs and gMDSCs were maintained in culture for up to 65 passages, whereas gFFCs could be passaged barely more than 15 times. gADSCs and gMDSCs had higher fluorescent colony forming efficiency and greater convergence (20%) and cleavage (10%) rates than gFFCs, and exhibited differing H4K5 histone modification patterns after somatic cell nuclear transfer and in vitro cultivation. After transfection with a pDsRed2-1 expression plasmid, the integrated exogenous genes did not influence the pluripotency of gADSCs-pDsRed2-1 or gMDSCs-pDsRed2-1. DsRed2 mRNA expression by cloned embryos derived from gADSCs-pDsRed2-1 or gMDSCs-pDsRed2-1 was more than twice that of gFFCs-pDsRed2-1 embryos (P<0.01). Pregnancy rates of gADSCs-pDsRed2-1 and gMDSCs-pDsRed2-1 recipients were higher than those of gFFCs-pDsRed2-1 recipients (P

  14. Potential of Adipose-Derived Mesenchymal Stem Cells and Skeletal Muscle-Derived Satellite Cells for Somatic Cell Nuclear Transfer Mediated Transgenesis in Arbas Cashmere Goats

    PubMed Central

    Yuan, Jianlong; Liang, Hao; Liu, Dongjun

    2014-01-01

    Somatic cell nuclear transfer is used to generate genetic models for research and new, genetically modified livestock varieties. Goat fetal fibroblast cells (gFFCs) are the predominant nuclear donors in Cashmere goat transgenic cloning, but have disadvantages. We evaluated the potential of goat adipose-derived mesenchymal stem cells (gADSCs) and goat skeletal muscle-derived satellite cells (gMDSCs) for somatic cell nuclear transfer, evaluating their proliferation, pluripotency, transfection efficiency and capacity to support full term development of embryos after additive gene transfer or homologous recombination. gADSCs and gMDSCs were isolated by enzyme digestion and differentiated into neurocytes, myotube cells and insulin-producing cells. Neuron-specific enolase, fast muscle myosin and insulin expression were determined by immunohistochemistry. Following somatic cell nuclear transfer with donor cells derived from gADSCs, gMDSCs and gFFCs, transfection and cloning efficiencies were compared. Red fluorescent protein levels were determined by quantitative PCR and western blotting. 5-Methylcytosine, H4K5, H4K12 and H3K18 were determined immunohistochemically. gADSCs and gMDSCs were maintained in culture for up to 65 passages, whereas gFFCs could be passaged barely more than 15 times. gADSCs and gMDSCs had higher fluorescent colony forming efficiency and greater convergence (20%) and cleavage (10%) rates than gFFCs, and exhibited differing H4K5 histone modification patterns after somatic cell nuclear transfer and in vitro cultivation. After transfection with a pDsRed2-1 expression plasmid, the integrated exogenous genes did not influence the pluripotency of gADSCs–pDsRed2-1 or gMDSCs–pDsRed2-1. DsRed2 mRNA expression by cloned embryos derived from gADSCs–pDsRed2-1 or gMDSCs–pDsRed2-1 was more than twice that of gFFCs–pDsRed2-1 embryos (P<0.01). Pregnancy rates of gADSCs–pDsRed2-1 and gMDSCs–pDsRed2-1 recipients were higher than those of gFFCs–pDsRed2

  15. Isolation, culture and characterisation of somatic cells derived from semen and milk of endangered sheep and eland antelope.

    PubMed

    Nel-Themaat, L; Gómez, M C; Damiani, P; Wirtu, G; Dresser, B L; Bondioli, K R; Lyons, L A; Pope, C E; Godke, R A

    2007-01-01

    Semen and milk are potential sources of somatic cells for genome banks. In the present study, we cultured and characterised cells from: (1) cooled sheep milk; (2) fresh, cooled and frozen-thawed semen from Gulf Coast native (GCN) sheep (Ovis aries); and (3) fresh eland (Taurotragus oryx) semen. Cells attached to the culture surface from fresh (29%), cooled (43%) and slow-frozen (1 degrees C/min; 14%) ram semen, whereas no attachment occurred in the fast-frozen (10 degrees C/min) group. Proliferation occurred in fresh (50%) and cooled (100%) groups, but no cells proliferated after passage 1 (P1). Eland semen yielded cell lines (100%) that were cryopreserved at P1. In samples from GCN and cross-bred milk, cell attachment (83% and 95%, respectively) and proliferation (60% and 37%, respectively) were observed. Immunocytochemical detection of cytokeratin indicated an epithelial origin of semen-derived cells, whereas milk yielded either fibroblasts, epithelial or a mixture of cell types. Deoxyribonucleic acid microsatellite analysis using cattle-derived markers confirmed that eland cells were from the semen donor. Eland epithelial cells were transferred into eland oocytes and 12 (71%), six (35%) and two (12%) embryos cleaved and developed to morulae or blastocyst stages, respectively. In conclusion, we have developed a technique for obtaining somatic cells from semen. We have also demonstrated that semen-derived cells can serve as karyoplast donors for nuclear transfer. PMID:17524303

  16. Transcript levels of several epigenome regulatory genes in bovine somatic donor cells are not correlated with their cloning efficiency.

    PubMed

    Zhou, Wenli; Sadeghieh, Sanaz; Abruzzese, Ronald; Uppada, Subhadra; Meredith, Justin; Ohlrichs, Charletta; Broek, Diane; Polejaeva, Irina

    2009-09-01

    Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

  17. The Drosophila BCL6 homolog ken and barbie promotes somatic stem cell self-renewal in the testis niche

    PubMed Central

    Issigonis, Melanie; Matunis, Erika

    2012-01-01

    Stem cells sustain tissue regeneration by their remarkable ability to replenish the stem cell pool and to generate differentiating progeny. Signals from local microenvironments, or niches, control stem cell behavior. In the Drosophila testis, a group of somatic support cells called the hub creates a stem cell niche by locally activating the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway in two adjacent types of stem cells: germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Here, we find that ken and barbie (ken) is autonomously required for the self-renewal of CySCs but not GSCs. Furthermore, Ken misexpression in the CySC lineage induces the cell-autonomous self-renewal of somatic cells as well as the nonautonomous self-renewal of germ cells outside the niche. Thus, Ken, like Stat92E and its targets ZFH1 (Leatherman and Dinardo, 2008) and Chinmo (Flaherty et al., 2010), is necessary and sufficient for CySC renewal. However, ken is not a JAK-STAT target in the testis, but instead acts in parallel to Stat92E to ensure CySC self-renewal. Ken represses a subset of Stat92E targets in the embryo (Arbouzova et al., 2006) suggesting that Ken maintains CySCs by repressing differentiation factors. In support of this hypothesis, we find that the global JAKSTAT inhibitor Protein tyrosine phosphatase 61F (Ptp61F) is a JAK-STAT target in the testis that is repressed by Ken. Together, our work demonstrates that Ken has an important role in the in the inhibition of CySC differentiation. Studies of ken may inform our understanding of its vertebrate orthologue B-Cell Lymphoma 6 (BCL6) and how misregulation of this oncogene leads to human lymphomas. PMID:22580161

  18. Single-Cell RNA-Seq with Waterfall Reveals Molecular Cascades underlying Adult Neurogenesis.

    PubMed

    Shin, Jaehoon; Berg, Daniel A; Zhu, Yunhua; Shin, Joseph Y; Song, Juan; Bonaguidi, Michael A; Enikolopov, Grigori; Nauen, David W; Christian, Kimberly M; Ming, Guo-li; Song, Hongjun

    2015-09-01

    Somatic stem cells contribute to tissue ontogenesis, homeostasis, and regeneration through sequential processes. Systematic molecular analysis of stem cell behavior is challenging because classic approaches cannot resolve cellular heterogeneity or capture developmental dynamics. Here we provide a comprehensive resource of single-cell transcriptomes of adult hippocampal quiescent neural stem cells (qNSCs) and their immediate progeny. We further developed Waterfall, a bioinformatic pipeline, to statistically quantify singe-cell gene expression along a de novo reconstructed continuous developmental trajectory. Our study reveals molecular signatures of adult qNSCs, characterized by active niche signaling integration and low protein translation capacity. Our analyses further delineate molecular cascades underlying qNSC activation and neurogenesis initiation, exemplified by decreased extrinsic signaling capacity, primed translational machinery, and regulatory switches in transcription factors, metabolism, and energy sources. Our study reveals the molecular continuum underlying adult neurogenesis and illustrates how Waterfall can be used for single-cell omics analyses of various continuous biological processes.

  19. Meiotic Recombination in Somatic Cell Nuclear Transfer Bulls and Their Offspring

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In mammals, homologous chromosome pairing and recombination are essential events for meiosis. The generation of reciprocal exchanges of genetic material ensure both genetic diversity and the proper segregation of homologous chromosomes. With the advent of reproductive biotechnologies such as somat...

  20. Gamete derivation from embryonic stem cells, induced pluripotent stem cells or somatic cell nuclear transfer-derived embryonic stem cells: state of the art

    PubMed Central

    Easley, Charles A.; Simerly, Calvin R.; Schatten, Gerald

    2015-01-01

    Generating gametes from pluripotent stem cells (PSCs) has many scientific justifications and several biomedical rationales. Here, we consider several strategies for deriving gametes from PSCs from mice and primates (human and non-human) and their anticipated strengths, challenges and limitations. Although the ‘Weismann barrier’, which separates the mortal somatic cell lineages from the potentially immortal germline, has long existed, breakthroughs first in mice and now in humans are artificially creating germ cells from somatic cells. Spermatozoa with full reproductive viability establishing multiple generations of seemingly normal offspring have been reported in mice and, in humans, haploid spermatids with correct parent-of-origin imprints have been obtained. Similar progress with making oocytes has been published using mouse PSCs differentiated in vitro into primordial germ cells, which are then cultured after xenografting reconstructed artificial ovaries. Progress in making human oocytes artificially is proving challenging. The usefulness of these artificial gametes, from assessing environmental exposure toxicity to optimising medical treatments to prevent negative off-target effects on fertility, may prove invaluable, as may basic discoveries on the fundamental mechanisms of gametogenesis. PMID:25472048

  1. Serial cloning of pigs by somatic cell nuclear transfer: restoration of phenotypic normality during serial cloning.

    PubMed

    Cho, Seong-Keun; Kim, Jae-Hwan; Park, Jong-Yi; Choi, Yun-Jung; Bang, Jae-Il; Hwang, Kyu-Chan; Cho, Eun-Jeong; Sohn, Sea-Hwan; Uhm, Sang Jun; Koo, Deog-Bon; Lee, Kyung-Kwang; Kim, Teoan; Kim, Jin-Hoi

    2007-12-01

    Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce first-generation (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age- and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the

  2. Telomere-to-centromere ratio of bovine clones, embryos, gametes, fetal cells, and adult cells.

    PubMed

    Meerdo, Lora N; Reed, William A; White, Kenneth L

    2005-01-01

    In 1997, Dolly, the first animal cloned from an adult cell, was born. It was announced in 1999 that Dolly might be aging faster than normal because her telomeres were shorter than age-matched control sheep. Telomeres, a repeated DNA sequence located at the ends of linear chromosomes, allow for base pair loss during DNA replication. Telomere shortening acts as a "mitotic clock," leading to replicative senescence. By using whole cell lysate and slot-blot analysis, we determined the telomere-to-centromere ratio (T/C) for bovine gametes, embryos, fetal tissues (brain, heart, lung, kidney, uterus, ovary, and skin), adult donor cells, and cloned embryos. Our data indicates a consistency in T/C among the various fetal tissues. The T/C of sperm is significantly lower than in oocytes. The T/C decreases from the oocyte to the 2-8-cell stage embryo, increases dramatically at the morula stage, and decreases at the blastocyst stage. Our data shows no significant difference in T/C between cloned embryos and in vitro fertilized (IVF) embryos, but there is a significant difference between cloned embryos and adult donor cells. In conclusion, the enucleated bovine oocyte has the ability to reestablish the telomere length of adult somatic cell donor nuclei. PMID:15996118

  3. Single-Cell, Genome-wide Sequencing Identifies Clonal Somatic Copy-Number Variation in the Human Brain

    PubMed Central

    Cai, Xuyu; Evrony, Gilad D.; Lehmann, Hillel S.; Elhosary, Princess C.; Mehta, Bhaven K.; Poduri, Annapurna; Walsh, Christopher A.

    2014-01-01

    SUMMARY De novo copy-number variants (CNVs) can cause neuropsychiatric disease, but the degree to which they occur somatically, and during development, is unknown. Single-cell whole-genome sequencing (WGS) in >200 single cells, including >160 neurons from three normal and two pathological human brains, sensitively identified germline trisomy of chromosome 18 but found most (≥95%) neurons in normal brain tissue to be euploid. Analysis of a patient with hemimegalencephaly (HMG) due to a somatic CNV of chromosome 1q found unexpected tetrasomy 1q in ~20% of neurons, suggesting that CNVs in a minority of cells can cause widespread brain dysfunction. Single-cell analysis identified large (>1 Mb) clonal CNVs in lymphoblasts and in single neurons from normal human brain tissue, suggesting that some CNVs occur during neurogenesis. Many neurons contained one or more large candidate private CNVs, including one at chromosome 15q13.2-13.3, a site of duplication in neuropsychiatric conditions. Large private and clonal somatic CNVs occur in normal and diseased human brains. PMID:25159146

  4. Somatic hypermutation of immunoglobulin genes: lessons from proliferating cell nuclear antigenK164R mutant mice.

    PubMed

    Langerak, Petra; Krijger, Peter H L; Heideman, Marinus R; van den Berk, Paul C M; Jacobs, Heinz

    2009-03-12

    Proliferating cell nuclear antigen (PCNA) encircles DNA as a ring-shaped homotrimer and, by tethering DNA polymerases to their template, PCNA serves as a critical replication factor. In contrast to high-fidelity DNA polymerases, the activation of low-fidelity translesion synthesis (TLS) DNA polymerases seems to require damage-inducible monoubiquitylation (Ub) of PCNA at lysine residue 164 (PCNA-Ub). TLS polymerases can tolerate DNA damage, i.e. they can replicate across DNA lesions. The lack of proofreading activity, however, renders TLS highly mutagenic. The advantage is that B cells use mutagenic TLS to introduce somatic mutations in immunoglobulin (Ig) genes to generate high-affinity antibodies. Given the critical role of PCNA-Ub in activating TLS and the role of TLS in establishing somatic mutations in immunoglobulin genes, we analysed the mutation spectrum of somatically mutated immunoglobulin genes in B cells from PCNAK164R knock-in mice. A 10-fold reduction in A/T mutations is associated with a compensatory increase in G/C mutations-a phenotype similar to Poleta and mismatch repair-deficient B cells. Mismatch recognition, PCNA-Ub and Poleta probably act within one pathway to establish the majority of mutations at template A/T. Equally relevant, the G/C mutator(s) seems largely independent of PCNAK(164) modification.

  5. Recombinogenic activity of Pantoprazole(®) in somatic cells of Drosophila melanogaster.

    PubMed

    Lopes, Jeyson Césary; Machado, Nayane Moreira; Saturnino, Rosiane Soares; Nepomuceno, Júlio César

    2015-03-01

    Pantoprazole(®) is one of the leading proton pump inhibitors (PPIs) used in the treatment of a variety of diseases related to the upper gastrointestinal tract. However, studies have shown an increased risk of developing gastric cancer, intestinal metaplasia and hyperplasia of endocrine cells with prolonged use. In the present study, the somatic mutation and recombination test (SMART) was employed to determine the mutagenic effects of Pantoprazole on Drosophila melanogaster. Repeated treatments with Pantoprazole were performed on 72-hour larvae of the standard (ST) and high bioactivation (HB) crosses at concentrations of 2.5, 5.0, and 10.0 μM. In addition, doxorubicin (DXR) was administered at 0.4 mM, as a positive control. When administered to ST descendants, total number of spots were statistically significant at 2.5 and 5.0 μM concentrations. For HB descendants, a significant increase in the total number of spots was observed among the marked transheterozygous (MH) flies. Through analysis of balancer heterozygous (BH) descendants, recombinogenic effects were observed at all concentrations in descendants of the HB cross. In view of these experimental conditions and results, it was concluded that Pantoprazole is associated with recombinogenic effects in Drosophila melanogaster. PMID:25983631

  6. A somatic cell hybrid panel for pig regional gene mapping characterized by molecular cytogenetics.

    PubMed

    Yerle, M; Echard, G; Robic, A; Mairal, A; Dubut-Fontana, C; Riquet, J; Pinton, P; Milan, D; Lahbib-Mansais, Y; Gellin, J

    1996-01-01

    A panel of 27 pig x rodent somatic cell hybrids was produced and characterized cytogenetically. The first step of this study consisted of hybridizing a SINE probe to GTG-banded metaphases of each hybrid clone in order to count and identify the normal pig chromosomes and to detect rearranged ones. The second step consisted of using the DNA of each clone as a probe after pIRS-PCR (porcine interspersed repetitive sequence-polymerase chain reaction) amplification to highly enrich it in pig sequences. These probes, hybridized to normal pig metaphase chromosomes, enabled the identification of the complete porcine complement in the hybrid lines. Whole chromosomes and fragments were characterized quickly and precisely, and results were compared. In addition to this cytogenetic characterization, molecular verification was also carried out by using primers specific to six microsatellites and to one gene previously mapped to pig chromosomes. The results obtained allow us to conclude that we have produced a panel that is informative for all porcine chromosomes. This panel constitutes a highly efficient tool to establish not only assignments of genes and markers but also regional localizations on pig chromosomes. PMID:8697807

  7. The IgH 3′ regulatory region controls somatic hypermutation in germinal center B cells

    PubMed Central

    Rouaud, Pauline; Vincent-Fabert, Christelle; Saintamand, Alexis; Fiancette, Rémi; Marquet, Marie; Robert, Isabelle; Reina-San-Martin, Bernardo; Pinaud, Eric

    2013-01-01

    Interactions with cognate antigens recruit activated B cells into germinal centers where they undergo somatic hypermutation (SHM) in V(D)J exons for the generation of high-affinity antibodies. The contribution of IgH transcriptional enhancers in SHM is unclear. The Eμ enhancer upstream of Cμ has a marginal role, whereas the influence of the IgH 3′ regulatory region (3′RR) enhancers (hs3a, hs1,2, hs3b, and hs4) is controversial. To clarify the latter issue, we analyzed mice lacking the whole 30-kb extent of the IgH 3′RR. We show that SHM in VH rearranged regions is almost totally abrogated in 3′RR-deficient mice, whereas the simultaneous Ig heavy chain transcription rate is only partially reduced. In contrast, SHM in κ light chain genes remains unaltered, acquitting for any global SHM defect in our model. Beyond class switch recombination, the IgH 3′RR is a central element that controls heavy chain accessibility to activation-induced deaminase modifications including SHM. PMID:23825188

  8. Somatic Copy Number Alterations Associated with Japanese or Endometriosis in Ovarian Clear Cell Adenocarcinoma

    PubMed Central

    Okamoto, Aikou; Sehouli, Jalid; Yanaihara, Nozomu; Hirata, Yukihiro; Braicu, Ioana; Kim, Byoung-Gie; Takakura, Satoshi; Saito, Misato; Yanagida, Satoshi; Takenaka, Masataka; Yamaguchi, Noriko; Morikawa, Asuka; Tanabe, Hiroshi; Yamada, Kyosuke; Yoshihara, Kosuke; Enomoto, Takayuki; Itamochi, Hiroaki; Kigawa, Junzo; Matsumura, Noriomi; Konishi, Ikuo; Aida, Satoshi; Aoki, Yuko; Ishii, Nobuya; Ochiai, Kazunori; Akiyama, Tetsu; Urashima, Mitsuyoshi

    2015-01-01

    When compared with other epithelial ovarian cancers, the clinical characteristics of ovarian clear cell adenocarcinoma (CCC) include 1) a higher incidence among Japanese, 2) an association with endometriosis, 3) poor prognosis in advanced stages, and 4) a higher incidence of thrombosis as a complication. We used high resolution comparative genomic hybridization (CGH) to identify somatic copy number alterations (SCNAs) associated with each of these clinical characteristics of CCC. The Human Genome CGH 244A Oligo Microarray was used to examine 144 samples obtained from 120 Japanese, 15 Korean, and nine German patients with CCC. The entire 8q chromosome (minimum corrected p-value: q = 0.0001) and chromosome 20q13.2 including the ZNF217 locus (q = 0.0078) were amplified significantly more in Japanese than in Korean or German samples. This copy number amplification of the ZNF217 gene was confirmed by quantitative real-time polymerase chain reaction (Q-PCR). ZNF217 RNA levels were also higher in Japanese tumor samples than in non-Japanese samples (P = 0.027). Moreover, endometriosis was associated with amplification of EGFR gene (q = 0.047), which was again confirmed by Q-PCR and correlated with EGFR RNA expression. However, no SCNAs were significantly associated with prognosis or thrombosis. These results indicated that there may be an association between CCC and ZNF217 amplification among Japanese patients as well as between endometriosis and EGFR gene amplifications. PMID:25658832

  9. Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT)

    SciTech Connect

    Do, Minhwa; Jang, Won-Gu; Hwang, Jeong Hee; Jang, Hoon; Kim, Eun-Jung; Jeong, Eun-Jeong; Shim, Hosup; Hwang, Sung Soo; Oh, Keon Bong; Byun, Sung June; Kim, Jin-Hoi; Lee, Jeong Woong

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer We success serial SCNT through the third generation using pig fibroblasts. Black-Right-Pointing-Pointer Donor-specific mtDNA in the recloned pigs was detected. Black-Right-Pointing-Pointer SCNT affect mtDNA mounts. -- Abstract: Somatic cell nuclear transfer (SCNT) has been established for the transmission of specific nuclear DNA. However, the fate of donor mitochondrial DNA (mtDNA) remains unclear. Here, we examined the fate of donor mtDNA in recloned pigs through third generations. Fibroblasts of recloned pigs were obtained from offspring of each generation produced by fusion of cultured fibroblasts from a Minnesota miniature pig (MMP) into enucleated oocytes of a Landrace pig. The D-loop regions from the mtDNA of donor and recipient differ at nucleotide sequence positions 16050 (A{yields}T), 16062 (T{yields}C), and 16135 (G{yields}A). In order to determine the fate of donor mtDNA in recloned pigs, we analyzed the D-loop region of the donor's mtDNA by allele-specific PCR (AS-PCR) and real-time PCR. Donor mtDNA was successfully detected in all recloned offspring (F1, F2, and F3). These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction.

  10. Recombinogenic activity of Pantoprazole® in somatic cells of Drosophila melanogaster

    PubMed Central

    Lopes, Jeyson Césary; Machado, Nayane Moreira; Saturnino, Rosiane Soares; Nepomuceno, Júlio César

    2015-01-01

    Pantoprazole® is one of the leading proton pump inhibitors (PPIs) used in the treatment of a variety of diseases related to the upper gastrointestinal tract. However, studies have shown an increased risk of developing gastric cancer, intestinal metaplasia and hyperplasia of endocrine cells with prolonged use. In the present study, the somatic mutation and recombination test (SMART) was employed to determine the mutagenic effects of Pantoprazole on Drosophila melanogaster. Repeated treatments with Pantoprazole were performed on 72-hour larvae of the standard (ST) and high bioactivation (HB) crosses at concentrations of 2.5, 5.0, and 10.0 μM. In addition, doxorubicin (DXR) was administered at 0.4 mM, as a positive control. When administered to ST descendants, total number of spots were statistically significant at 2.5 and 5.0 μM concentrations. For HB descendants, a significant increase in the total number of spots was observed among the marked transheterozygous (MH) flies. Through analysis of balancer heterozygous (BH) descendants, recombinogenic effects were observed at all concentrations in descendants of the HB cross. In view of these experimental conditions and results, it was concluded that Pantoprazole is associated with recombinogenic effects in Drosophila melanogaster. PMID:25983631

  11. Generalized Potential of Adult Neural Stem Cells

    NASA Astrophysics Data System (ADS)

    Clarke, Diana L.; Johansson, Clas B.; Wilbertz, Johannes; Veress, Biborka; Nilsson, Erik; Karlström, Helena; Lendahl, Urban; Frisén, Jonas

    2000-06-01

    The differentiation potential of stem cells in tissues of the adult has been thought to be limited to cell lineages present in the organ from which they were derived, but there is evidence that some stem cells may have a broader differentiation repertoire. We show here that neural stem cells from the adult mouse brain can contribute to the formation of chimeric chick and mouse embryos and give rise to cells of all germ layers. This demonstrates that an adult neural stem cell has a very broad developmental capacity and may potentially be used to generate a variety of cell types for transplantation in different diseases.

  12. Lactoperoxidase activity in milk is correlated with somatic cell count in dairy cows.

    PubMed

    Isobe, N; Kubota, H; Yamasaki, A; Yoshimura, Y

    2011-08-01

    Lactoperoxidase (LPO) is a milk protein with antimicrobial function. The present study was undertaken to examine the correlation between LPO activity and somatic cell count (SCC) in milk to use LPO activity as an indicator of mastitis. Composite milk of 36 cows and quarter milk of 3 cows were collected once per week from 0 to 300 d postpartum and twice per day for 1 wk, respectively. For the measurement of LPO activity, milk was mixed with tetramethylbenzidine solution and incubated at 37°C for 30 min, followed by the measurement of optical density. When only milk with low SCC (132±12×10(3) cells/mL) was used, a significant decrease in LPO activity was detected in primiparous cows from 0 to 4 mo postpartum. Lactoperoxidase activities of primiparous cows in mo 1, 2, and 3 postpartum were significantly higher than those in multiparous cows. When composite milk was divided based on LPO activity, the SCC was significantly higher in the groups with LPO activity >5 and from 3 to 3.9 U/mL in the second- and fourth-parity cows, respectively, compared with the group with LPO activity <2U/mL. Extremely high SCC were found in the ≥fifth-parity cows, even in low-LPO activity groups. In the case of quarter milk, higher LPO activity was associated with increased SCC in all 3 cows. The percentage of quarter milk samples with high SCC (4,062±415×10(3) cells/mL) increased with an increase in the LPO activity. The percentage of quarter milk samples with high SCC was 50.0 to 100% in the milk with LPO activity ≥5 U/mL. These results indicate that the correlation of LPO activity to the SCC in bovine milk may point to the potential use of the former as an indicator of SCC.

  13. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    SciTech Connect

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue; Pang, Daxin; Ouyang, Hongsheng

    2011-07-29

    Highlights: {yields} Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. {yields} The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. {yields} A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 {mu}g/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  14. [Reduced performance and high somatic cell counts in a dairy herd fed high amounts of brewers' grain].

    PubMed

    Wenzinger, B

    2013-09-01

    The present case report describes a herd problem on a Holstein Friesian dairy farm in Switzerland, which could be attributed to the feeding of high amounts of wet brewers' grain over several months. Apathy and reduced general appearance, reduced feed intake as well as a decline in milk yield could be observed. A strong increase in milk somatic cell counts as well as an increase in the incidence of mastitis could be found. The milk fat content was highly elevated in all cows, whereas the milk protein content was reduced. The exclusion of wet brewers' grain from the partial mixed ration resulted in a considerable improvement of the general appearance of the cows and a decrease of the milk somatic cell counts. Feed that is easily spoiled could be a health risk for animals, particularly under hot and humid weather conditions and if fed in high amounts.

  15. Reproductive semi-cloning respecting biparental embryo origin: embryos from syngamy between a gamete and a haploidized somatic cell.

    PubMed

    Tesarik, J

    2002-08-01

    Embryos formed by somatic cell nuclear transfer to enucleated oocytes (cloning) have given rise to viable offspring in several mammalian species. The possibility of future application of this technique to human assisted reproduction (reproductive cloning) has been widely debated. On this background there is current discussion of the potential for a cloning-derived technique, which aims at syngamy between a gamete nucleus from one parent and a somatic cell nucleus from the other. Critical analysis of the clinical indications, the current state of the art, biological concerns and ethical considerations relative to this technique, called here reproductive semi-cloning, are presented. Such a technique requires validation by further research before it can be considered as a treatment option. This debate explores issues raised by the technique.

  16. Effects of season, milking routine and cow cleanliness on bacterial and somatic cell counts of bulk tank milk.

    PubMed

    Zucali, Maddalena; Bava, Luciana; Tamburini, Alberto; Brasca, Milena; Vanoni, Laura; Sandrucci, Anna

    2011-11-01

    The aim of the study was to investigate the effects of season, cow cleanliness and milking routine on bacterial and somatic cell counts of bulk tank milk. A total of 22 dairy farms in Lombardy (Italy) were visited three times in a year in different seasons. During each visit, samples of bulk tank milk were taken for bacterial and somatic cell counts; swabs from the teat surface of a group of cows were collected after teat cleaning and before milking. Cow cleanliness was assessed by scoring udder, flanks and legs of all milking cows using a 4-point scale system. Season affected cow cleanliness with a significantly higher percentage of non-clean (NC) cows during Cold compared with Mild season. Standard plate count (SPC), laboratory pasteurization count (LPC), coliform count (CC) and somatic cell count, expressed as linear score (LS), in milk significantly increased in Hot compared with Cold season. Coagulase-positive staphylococci on teat swabs showed higher counts in Cold season in comparison with the other ones. The effect of cow cleanliness was significant for SPC, psychrotrophic bacterial count (PBC), CC and Escherichia coli in bulk tank milk. Somatic cell count showed a relationship with udder hygiene score. Milking operation routine strongly affected bacterial counts and LS of bulk tank milk: farms that accomplished a comprehensive milking scheme including two or more operations among forestripping, pre-dipping and post-dipping had lower teat contamination and lower milk SPC, PBC, LPC, CC and LS than farms that did not carry out any operation.

  17. [Isolation and nuclear transfer of ES-like cells colonies derived from embryos being cloning of bovine somatic].

    PubMed

    Dong, Ya-Juan; Bai, Xue-Jin; Li, Jian-Dong; Suzuki, Tatsuyuki

    2003-02-01

    In this experiment, it was designed to carry out proliferous culture of bovine blastocysts(day 7) derived from embryos cloned through bovine somatic cell nuclear transfer, isolating and passaging of ES cells. The cells of blastocysts, which were planted on feeder layer, formed small colonies within 24 h. The nest-shape colonies occurred after culturing for 2-3 days. After the colonies in the same shape were isolated and passaged 4-5 times, many different size colonies with monolayer of multi-cells appeared. The colonies that had been passaged 4-5 times were planted into 4-wells multi-dishes without feeder layer. The colonies with monolayer of multi-cells appeared after 24 h, spread all over the bottom of the dishes, emerged epidermis-like cells that appeared reticulate after 4-7 days. These cells were used as donor cells to carry out nuclear transfer. The results showed that 80% (40/50) of the reconstructed embryos cleaved, 5% (2/40) and 2.5% (1/40) of them developed to the morulaes and blastocyst stage, respectively. It revealed that ES-like cells derived embryos constructed through somatic cell nuclear transfer have the developmental potentials.

  18. Reproductive and growth performance in Jin Hua pigs cloned from somatic cell nuclei and the meat quality of their offspring.

    PubMed

    Shibata, Masatoshi; Otake, Masayoshi; Tsuchiya, Seiko; Chikyu, Mikio; Horiuchi, Atsushi; Kawarasaki, Tatsuo

    2006-10-01

    Somatic cell cloning is expected to be a valuable method for conserving genetic resources in pigs. In this study, we compared the reproductive and growth performance of Jin Hua cloned pigs with that of naturally bred Jin Hua pigs. In addition, we generated offspring from the cloned sows and examined the productivity and quality of meat in the progeny. The birth weights and growth rates of somatic cell-cloned pigs were similar to those of Jin Hua pigs. The cloned pigs reached puberty very early, and this is typical of the Jin Hua breed. Furthermore, reproductive performance, in terms of traits such as gestation period, litter size, and raising rate in the cloned pigs were similar to Jin Hua pigs. Although the offspring of the cloned (OC) pigs had lower birth weights than the Jin Hua breed, the daily weight gain of the OC pigs was significantly higher, especially at the finishing stage. The carcass quality of the OC pigs had similar characteristics to the Jin Hua breed, namely thick back fat and a small loin area. Furthermore, the meat qualities of the OC pigs were similar to those of Jin Hua pigs in terms of intramuscular fat content and tenderness. These results demonstrate that cloned pigs and their offspring were similar to the Jin Hua breed in most of the growth, reproductive, and meat productive performances. This strongly suggests that pigs cloned from somatic cell nuclei have the potential to be a valuable genetic resource for breeding.

  19. Expression Profiling of Innate Immune Genes in Milk Somatic Cells During Subclinical Mastitis in Crossbred Dairy Cows.

    PubMed

    Karthikeyan, A; Radhika, G; Aravindhakshan, T V; Anilkumar, K

    2016-10-01

    Innate immune mechanism plays a key role in mammary defense, from recognition of pathogens to activation of nonspecific and specific immunity involved in elimination of pathogens. Expression profiles of innate immune response genes namely Toll like receptor 2 (TLR-2), Peptidoglycan recognition protein 1 (PGLYRP-1), Interleukin 8 receptor (IL-8 R), L-Selectin (SELL), and Osteopontin (OPN) in milk somatic cells of subclinical mastitis (SCM) affected crossbred cows were investigated under this study at transcript level using quantitative real time polymerase chain reaction (qRT-PCR). Dairy cows in mid lactation were screened for SCM using California Mastitis Test (CMT), Somatic Cell Count (SCC) and Electrical Conductivity test (EC). Based on results of SCM screening tests, crossbred cows were clustered into two groups with four Staphylococcus aureus infected SCM cows and four apparently healthy cows. The expressions levels of TLR-2, PGLYRP-1, IL-8 R, SELL, and OPN in milk somatic cells of SCM affected cows were significantly higher (p < 0.05) than healthy cows. These genes could be considered as candidate genes for innate immune response against S. aureus SCM infection.

  20. A comparison of reproductive characteristics of boars generated by somatic cell nuclear transfer to highly related conventionally produced boars.

    PubMed

    Williams, N E; Walker, S C; Reeves, D E; Sherrer, E; Galvin, J M; Polejaeva, I; Rampacek, G; Benyshek, L; Christenson, R K; Graves, W M; Pratt, S L

    2006-01-01

    This study compares the reproductive performance of boars produced by somatic cell nuclear transfer versus conventional breeding. Two different genotypes were selected for comparison: terminal cross line 1 (TX1) and terminal cross line 2 (TX2). The boars selected for comparison from TX1 were three cloned boars, produced by somatic cell nuclear transfer and the conventionally produced progenitor of the clones. The boars selected for comparison from TX2 were a cloned boar produced by somatic cell nuclear transfer and two conventionally produced half sibling boars that were offspring of the progenitor of the clone. Semen from each boar was collected, extended, evaluated and shipped offsite. Upon arrival, the semen was reevaluated and utilized for artificial insemination of 89 commercial gilts, at least 12 gilts per boar, producing 625 piglets. Pregnancy rates were determined at day 30 and 110 of gestation; and farrowing rate and gestation length were recorded. Differences were observed in some of the semen characteristics analyzed with the clones usually possessing superior semen quality to the control, this likely being a result of age differences amongst the clones and controls. Additionally no differences were noted between the clones and controls (progenitor) or between individual boars within genetic line for pregnancy rates, gestation length or any of the litter parameters examined between the clones and controls. These data further support previous reports with limited numbers that the reproductive capabilities of cloned boars are equal to that of conventionally produced boars.

  1. Genetic parameters for lactation traits of milking ewes: protein content and composition, fat, somatic cells and individual laboratory cheese yield

    PubMed Central

    Othmane, Med Houcine; Carriedo, Juan Antonio; San Primitivo, Fermin; De la Fuente, Luis Fernando

    2002-01-01

    The effects of some environmental variation factors and the genetic parameters for total milk traits (fat content, protein content, casein content, serum protein content, lactation mean of individual laboratory cheese yield (LILCY), lactation mean of somatic cell count (LSCC), and milk yield) were estimated from the records of 1 111 Churra ewes. Genetic parameters were estimated by multivariate REML. Heritability for fat content was low (0.10) as is usually found in the Churra breed. Heritabilities for protein content, casein content, serum protein content, LILCY, milk yield and somatic cell count were 0.31, 0.30, 0.22, 0.09, 0.26 and 0.11, respectively. The highest heritability estimates were for protein and casein contents. Casein content is not advisable as an alternative to protein content as a selection criterion for cheese yield improvement; it does not have any compelling advantages and its measurement is costly. Our results for LSCC indicated that efforts should focus on improving the level of management rather than selecting for somatic cells, in the actual conditions of the Churra breed. PMID:12427387

  2. Are we Genomic Mosaics? Variations of the Genome of Somatic Cells can Contribute to Diversify our Phenotypes.

    PubMed

    Astolfi, P A; Salamini, F; Sgaramella, V

    2010-09-01

    Theoretical and experimental evidences support the hypothesis that the genomes and the epigenomes may be different in the somatic cells of complex organisms. In the genome, the differences range from single base substitutions to chromosome number; in the epigenome, they entail multiple postsynthetic modifications of the chromatin. Somatic genome variations (SGV) may accumulate during development in response both to genetic programs, which may differ from tissue to tissue, and to environmental stimuli, which are often undetected and generally irreproducible. SGV may jeopardize physiological cellular functions, but also create novel coding and regulatory sequences, to be exposed to intraorganismal Darwinian selection. Genomes acknowledged as comparatively poor in genes, such as humans', could thus increase their pristine informational endowment. A better understanding of SGV will contribute to basic issues such as the "nature vs nurture" dualism and the inheritance of acquired characters. On the applied side, they may explain the low yield of cloning via somatic cell nuclear transfer, provide clues to some of the problems associated with transdifferentiation, and interfere with individual DNA analysis. SGV may be unique in the different cells types and in the different developmental stages, and thus explain the several hundred gaps persisting in the human genomes "completed" so far. They may compound the variations associated to our epigenomes and make of each of us an "(epi)genomic" mosaic. An ensuing paradigm is the possibility that a single genome (the ephemeral one assembled at fertilization) has the capacity to generate several different brains in response to different environments.

  3. Are we Genomic Mosaics? Variations of the Genome of Somatic Cells can Contribute to Diversify our Phenotypes.

    PubMed

    Astolfi, P A; Salamini, F; Sgaramella, V

    2010-09-01

    Theoretical and experimental evidences support the hypothesis that the genomes and the epigenomes may be different in the somatic cells of complex organisms. In the genome, the differences range from single base substitutions to chromosome number; in the epigenome, they entail multiple postsynthetic modifications of the chromatin. Somatic genome variations (SGV) may accumulate during development in response both to genetic programs, which may differ from tissue to tissue, and to environmental stimuli, which are often undetected and generally irreproducible. SGV may jeopardize physiological cellular functions, but also create novel coding and regulatory sequences, to be exposed to intraorganismal Darwinian selection. Genomes acknowledged as comparatively poor in genes, such as humans', could thus increase their pristine informational endowment. A better understanding of SGV will contribute to basic issues such as the "nature vs nurture" dualism and the inheritance of acquired characters. On the applied side, they may explain the low yield of cloning via somatic cell nuclear transfer, provide clues to some of the problems associated with transdifferentiation, and interfere with individual DNA analysis. SGV may be unique in the different cells types and in the different developmental stages, and thus explain the several hundred gaps persisting in the human genomes "completed" so far. They may compound the variations associated to our epigenomes and make of each of us an "(epi)genomic" mosaic. An ensuing paradigm is the possibility that a single genome (the ephemeral one assembled at fertilization) has the capacity to generate several different brains in response to different environments. PMID:21358981

  4. Expression Profiling of Innate Immune Genes in Milk Somatic Cells During Subclinical Mastitis in Crossbred Dairy Cows.

    PubMed

    Karthikeyan, A; Radhika, G; Aravindhakshan, T V; Anilkumar, K

    2016-10-01

    Innate immune mechanism plays a key role in mammary defense, from recognition of pathogens to activation of nonspecific and specific immunity involved in elimination of pathogens. Expression profiles of innate immune response genes namely Toll like receptor 2 (TLR-2), Peptidoglycan recognition protein 1 (PGLYRP-1), Interleukin 8 receptor (IL-8 R), L-Selectin (SELL), and Osteopontin (OPN) in milk somatic cells of subclinical mastitis (SCM) affected crossbred cows were investigated under this study at transcript level using quantitative real time polymerase chain reaction (qRT-PCR). Dairy cows in mid lactation were screened for SCM using California Mastitis Test (CMT), Somatic Cell Count (SCC) and Electrical Conductivity test (EC). Based on results of SCM screening tests, crossbred cows were clustered into two groups with four Staphylococcus aureus infected SCM cows and four apparently healthy cows. The expressions levels of TLR-2, PGLYRP-1, IL-8 R, SELL, and OPN in milk somatic cells of SCM affected cows were significantly higher (p < 0.05) than healthy cows. These genes could be considered as candidate genes for innate immune response against S. aureus SCM infection. PMID:27565875

  5. In Vitro Ectopic Behavior of Porcine Spermatogonial Germ Cells and Testicular Somatic Cells.

    PubMed

    Lee, Kyung Hoon; Lee, Won Young; Do, Jung Tae; Park, Chan Kyu; Kim, Nam Hyung; Kim, Jin Hoi; Chung, Hak Jae; Kim, Dong Woon; Song, Hyuk

    2016-08-01

    Embryonic body-like colony formation is a unique pattern in male germ cell cultures, including spermatogonial stem cells. However, detailed information of the colony formation has not yet been sufficiently reported in male germ cell culture. To elucidate the formation of germ cell-derived colony (GDC), glial cell-derived neurotrophic factor receptor alpha-1 (GFRα-1)-positive pig germ cells were isolated using an immunomagnetic cell isolation method and labeled with red- or green-fluorescent dye. In GDC culture, red-fluorescent-labeled germ cells were evenly distributed in the wells from day 1 to 4, and they clustered together at the time of GDC formation on day 6. Interestingly, feeder cells migrated to the site of colony formation as spermatogonia carriers. Furthermore, when freshly prepared green-labeled GFRα-1-positive germ cells were added, mixed-fluorescent dye (red and green) colonies were observed. On bromodeoxyuridine (BrdU) treatment, 58% ± 3.13% of germ cells were positive to protein gene product 9.5 but negative to BrdU cells. Immunocytochemistry and reverse transcription-polymerase chain reaction results showed that cultured GDC cells were positive to stem cell- and pig germ cell-specific marker genes. In conclusion, in vitro formation of GDCs is mainly dependent on the aggregation of single germ cells as well as on the slow proliferation of germ cells.

  6. In Vitro Ectopic Behavior of Porcine Spermatogonial Germ Cells and Testicular Somatic Cells.

    PubMed

    Lee, Kyung Hoon; Lee, Won Young; Do, Jung Tae; Park, Chan Kyu; Kim, Nam Hyung; Kim, Jin Hoi; Chung, Hak Jae; Kim, Dong Woon; Song, Hyuk

    2016-08-01

    Embryonic body-like colony formation is a unique pattern in male germ cell cultures, including spermatogonial stem cells. However, detailed information of the colony formation has not yet been sufficiently reported in male germ cell culture. To elucidate the formation of germ cell-derived colony (GDC), glial cell-derived neurotrophic factor receptor alpha-1 (GFRα-1)-positive pig germ cells were isolated using an immunomagnetic cell isolation method and labeled with red- or green-fluorescent dye. In GDC culture, red-fluorescent-labeled germ cells were evenly distributed in the wells from day 1 to 4, and they clustered together at the time of GDC formation on day 6. Interestingly, feeder cells migrated to the site of colony formation as spermatogonia carriers. Furthermore, when freshly prepared green-labeled GFRα-1-positive germ cells were added, mixed-fluorescent dye (red and green) colonies were observed. On bromodeoxyuridine (BrdU) treatment, 58% ± 3.13% of germ cells were positive to protein gene product 9.5 but negative to BrdU cells. Immunocytochemistry and reverse transcription-polymerase chain reaction results showed that cultured GDC cells were positive to stem cell- and pig germ cell-specific marker genes. In conclusion, in vitro formation of GDCs is mainly dependent on the aggregation of single germ cells as well as on the slow proliferation of germ cells. PMID:27328332

  7. Persistent chromosome aberrations in the somatic cells of A-bomb survivors, Hiroshima and Nagasaki.

    PubMed

    Awa, A A

    1991-03-01

    Current status of knowledge on the radiation-induced chromosome aberrations persisting since their induction in 1945 to date in the somatic cells of A-bomb survivors in Hiroshima and Nagasaki is reviewed. Dose-response relationship for chromosome aberration frequencies observed with the use of the old A-bomb dosimetry system (T65D) is also demonstrable based on the new dosimetry system (DS86). Despite the fact that the remarkable decrease in the amount of neutron component relative to the total dose in Hiroshima, there still exist inter-city differences in aberration frequency per unit dose both for kerma and bone marrow dose; the dose-square term is smaller in Hiroshima than in Nagasaki. The differential contribution of neutron radiation may be responsible in some part for the observed difference between Hiroshima and Nagasaki, although proof still remains to be obtained. There is a wide variability of the frequency of cells with chromosome aberrations between survivors within a given dose range. Random errors in the dose estimates assigned to individual survivors seem responsible, to a large extent, for the observed overdispersions in aberration frequencies in both cities. New molecular biology-oriented techniques to differentially stain specific chromosomes using fluorescence in situ hybridization with chromosome-specific composite DNA probes seem extremely promising for future rapid, accurate and extensive screening of reciprocal translocations observed predominantly in A-bomb survivors. Such data may be utilized to establish a better biological dosimetry system, especially for those persons who are irradiated in vivo many years before cytogenetic examinations.

  8. Short communication: Bulk milk somatic cell penalties in herds enrolled in Dairy Herd Improvement programs.

    PubMed

    Hand, K J; Godkin, M A; Kelton, D F

    2012-01-01

    The objective of this study was to determine the effect of somatic cell count (SCC) monitoring at the cow level through Dairy Herd Improvement (DHI) programs on the risk of bulk tank SCC (BTSCC) penalties. For the year 2009, BTSCC for all producers in Ontario were examined, for a total of 2,898 DHI herds, 1,186 non-DHI herds, and 48,250 BTSCC records. Two penalty levels were examined, where BTSCC exceeded 499,000 (P500) and 399,000 (P400) cells/mL. Data were modeled first to determine the odds of a BTSCC exceeding a set penalty threshold and second to determine the odds of incurring a penalty under the Ontario Milk Act. All data were modeled as a generalized mixed model with a binary link function. Random effects included herd, fixed effects included season of BTSCC (summer, May to September, and winter, October to April), total milk shipped per month (L), fat paid per month (kg), protein paid per month (kg), and participation or not in the DHI program. The likelihood of a BTSCC exceeding a penalty threshold in a non-DHI herd compared with a DHI herd was significantly greater than 1 at both penalty levels, where the odds ratios were estimated to be 1.42 [95% confidence interval (CI): 1.19 to 1.69] and 1.38 (95% CI: 1.25 to 1.54) for P500 and P400, respectively. The likelihood of incurring a BTSCC penalty (where 3 out of 4 consecutive BTSCC exceeded penalty thresholds) was not significantly different at P500; however, it was significantly different for P400, where the odds ratio was estimated to be 1.42 (95% CI: 1.12 to 1.81).

  9. Protein profiles of bovine placenta derived from somatic cell nuclear transfer.

    PubMed

    Kim, Hong Rye; Kang, Jae Ku; Yoon, Jong Taek; Seong, Hwan Hoo; Jung, Jin Kwan; Lee, Hong Mie; Sik Park, Chang; Jin, Dong Il

    2005-11-01

    Practical application of animal cloning by somatic cell nuclear transfer (SCNT) has been hampered by an extremely low success rate. To address whether placental dysfunction in SCNT causes fetal loss during pregnancy, we have used a global proteomics approach using 2-DE and MS to analyze the differential protein patterns of three placentae from the afterbirth of cases of postnatal death, derived from SCNT of Korean Native cattle, and three normal placentae obtained from the afterbirth of fetuses derived from artificial insemination. Proteins within a pI range of 4.0-7.0 and 6.0-9.0 were analyzed separately by 2-DE in triplicate. A total of approximately 2000 spots were detected in placental 2-DE gels stained with CBB. In the comparison of normal and SCNT samples, 60 spots were identified as differentially expressed proteins, of which 33 spots were up-regulated proteins in SCNT placentae, while 27 spots were down-regulated proteins. Most of the proteins identified in this analysis appeared to be related with protein repair or protection, cytoskeleton, signal transduction, immune system, metabolism, extracellular matrix and remodeling, transcription regulation, cell structure or differentiation and ion transport. One of up-regulated proteins in SCNT was TIMP-2 protein known to be related to extracellular matrix and remodeling during pregnancy. Western blot analysis showed an increased level of TIMP-2 in SCNT placenta compared to normal. Our results revealed composite profiles of key proteins involved in abnormal placenta derived from SCNT, and suggested expression abnormality of these genes in SCNT placenta, resulting in fetal losses following SCNT.

  10. Trichostatin A affects histone acetylation and gene expression in porcine somatic cell nucleus transfer embryos.

    PubMed

    Cervera, R P; Martí-Gutiérrez, N; Escorihuela, E; Moreno, R; Stojkovic, M

    2009-11-01

    Epigenetic aberrancies likely preclude correct and complete nuclear reprogramming after somatic cell nucleus transfer (SCNT) and may underlie the observed reduced viability of cloned embryos. In the current study, we tested the effects of the histone deacetylase-inhibitor trichostatin A (TSA) on preimplantation development and on histone acetylation and the gene expression of nucleus transfer (NT) porcine (Sus scrofa) embryos. Our results showed that 5 nM TSA for 26 h after reconstitution resulted in embryos (NTTSA) that reached the blastocyst stage at a higher level (48.1% vs. 20.2%) and increased number of cells (105.0 vs. 75.3) than that of the control (NTC) embryos. In addition, and unlike the NTC embryos, the treated embryos displayed a global acetylated histone H4 at lysine 8 profile similar to the in vitro-fertilized (IVF) and cultured embryos during the preimplantation development. Finally, we determined that several transcription factors exert a dramatic amount of genetic control over pluripotency, including Oct4, Nanog, Cdx2, and Rex01, the imprinting genes Igf2 and Igf2r, and the histone deacetyltransferase gene Hdac2. The NT blastocysts showed similar levels of Oct4, Cdx2, and Hdac2 but lower levels of Nanog than those of the IVF blastocyst. However, the NTTSA blastocysts showed similar levels of Rex01, Igf2, and Igf2r as those of IVF blastocysts, whereas the NTC blastocysts showed significantly lower levels for those genes. Our results suggest that TSA improves porcine SCNT preimplantation development and affects the acetylated status of the H4K8, rendering acetylation levels similar to those of the IVF counterparts.

  11. Effect of an automated dipping and backflushing system on somatic cell counts.

    PubMed

    Olde Riekerink, R G M; Ohnstad, I; van Santen, B; Barkema, H W

    2012-09-01

    Postmilking teat disinfection is an effective management practice to prevent transmission of contagious mastitis pathogens from cow to cow. With farms increasing in size and an increase in the number of rotary milking parlors, the need for automation of postmilking teat disinfection is mounting. Automated teat dipping and backflushing (ADB) systems have existed for some years, but their effect on udder health was never examined in a field study on commercial dairy farms. The objectives of this study were, therefore, to evaluate the effect of introducing an ADB system in a herd on (1) bulk milk somatic cell count (SCC), (2) individual cow SCC, and (3) the proportion of newly elevated SCC. Dairy herd improvement data were collected over a 30-mo period on 25 sets of 3 farms. Each set of 3 farms contained a farm that installed an ADB system, one that disinfected teats using dipping after milking, and one that sprayed teats after milking. Data were analyzed using linear mixed models. Bulk milk SCC on farms that sprayed or dipped before installing an ADB system were 16,000 and 30,000 cells/mL lower in the period 6 to 18 mo after installation, respectively, than on farms that continued spraying or dipping the teats after milking. In the same period after installing an ADB system, proportions of cows with elevated SCC were 4.3 and 1.2% lower, respectively, compared with spraying and with dipping. Similarly, proportions of cows that had newly elevated SCC were 1.5% lower and 0.3% higher, respectively, compared with farms that sprayed or dipped. Installing an ADB system had a beneficial effect on bulk milk SCC, individual cow SCC, and the proportion of newly elevated SCC. The effect was most prominent in the period 6 to 18 mo after installation of an ADB system. PMID:22916897

  12. Somatic cell nuclear transfer alters peri-implantation trophoblast differentiation in bovine embryos.

    PubMed

    Arnold, Daniel R; Bordignon, Vilceu; Lefebvre, Réjean; Murphy, Bruce D; Smith, Lawrence C

    2006-08-01

    Abnormal placental development limits success in ruminant pregnancies derived from somatic cell nuclear transfer (SCNT), due to reduction in placentome number and consequently, maternal/fetal exchange. In the primary stages of an epithelial-chorial association, the maternal/fetal interface is characterized by progressive endometrial invasion by specialized trophoblast binucleate/giant cells (TGC). We hypothesized that dysfunctional placentation in SCNT pregnancies results from aberration in expression of genes known to be necessary for trophoblast proliferation (Mash2), differentiation (Hand1), and function (IFN-tau and PAG-9). We, therefore, compared the expression of these factors in trophoblast from bovine embryos derived from artificial insemination (AI), in vitro fertilization (IVF), and SCNT prior to (day 17) and following (day 40 of gestation) implantation, as well as TGC densities and function. In preimplantation embryos, Mash2 mRNA was more abundant in SCNT embryos compared to AI, while Hand1 was highest in AI and IVF relative to SCNT embryos. IFN-tau mRNA abundance did not differ among groups. PAG-9 mRNA was undetectable in SCNT embryos, present in IVF embryos and highest in AI embryos. In postimplantation pregnancies, SCNT fetal cotyledons displayed higher Mash2 and Hand1 than AI and IVF tissues. Allelic expression of Mash2 was not different among the groups, which suggests that elevated mRNA expression was not due to altered imprinting status of Mash2. The day 40 SCNT cotyledons had the fewest number of TGC compared to IVF and AI controls. Thus, expression of genes critical to normal placental development is altered in SCNT bovine embryos, and this is expected to cause abnormal trophoblast differentiation and contribute to pregnancy loss.

  13. Hard ewe's milk cheese manufactured from milk of three different groups of somatic cell counts.

    PubMed

    Jaeggi, J J; Govindasamy-Lucey, S; Berger, Y M; Johnson, M E; McKusick, B C; Thomas, D L; Wendorff, W L

    2003-10-01

    As ovine milk production increases in the United States, somatic cell count (SCC) is increasingly used in routine ovine milk testing procedures as an indicator of flock health. Ovine milk was collected from 72 East Friesian-crossbred ewes that were machine milked twice daily. The milk was segregated and categorized into three different SCC groups: < 100,000 (group I); 100,000 to 1,000,000 (group II); and > 1,000,000 cells/ ml (group III). Milk was stored frozen at -19 degrees C for 4 mo. Milk was then thawed at 7 degrees C over a 3-d period before pasteurization and cheese making. Casein (CN) content and CN-to-true protein ratio decreased with increasing SCC group 3.99, 3.97, to 3.72% CN, and 81.43, 79.72, and 79.32% CN to true protein ratio, respectively. Milk fat varied from 5.49, 5.67, and 4.86% in groups I, II, and III, respectively. Hard ewe's milk cheese was made from each of the three different SCC groups using a Manchego cheese manufacturing protocol. As the level of SCC increased, the time required for visual flocculation increased, and it took longer to reach the desired firmness for cutting the coagulum. The fat and moisture contents were lower in the highest SCC cheeses. After 3 mo, total free fatty acids (FFA) contents were significantly higher in the highest SCC cheeses. Butyric and caprylic acids levels were significantly higher in group III cheeses at all stages of ripening. Cheese graders noted rancid or lipase flavor in the highest SCC level cheeses at each of the sampling points, and they also deducted points for more body and textural defects in these cheeses at 6 and 9 mo.

  14. Hard ewe's milk cheese manufactured from milk of three different groups of somatic cell counts.

    PubMed

    Jaeggi, J J; Govindasamy-Lucey, S; Berger, Y M; Johnson, M E; McKusick, B C; Thomas, D L; Wendorff, W L

    2003-10-01

    As ovine milk production increases in the United States, somatic cell count (SCC) is increasingly used in routine ovine milk testing procedures as an indicator of flock health. Ovine milk was collected from 72 East Friesian-crossbred ewes that were machine milked twice daily. The milk was segregated and categorized into three different SCC groups: < 100,000 (group I); 100,000 to 1,000,000 (group II); and > 1,000,000 cells/ ml (group III). Milk was stored frozen at -19 degrees C for 4 mo. Milk was then thawed at 7 degrees C over a 3-d period before pasteurization and cheese making. Casein (CN) content and CN-to-true protein ratio decreased with increasing SCC group 3.99, 3.97, to 3.72% CN, and 81.43, 79.72, and 79.32% CN to true protein ratio, respectively. Milk fat varied from 5.49, 5.67, and 4.86% in groups I, II, and III, respectively. Hard ewe's milk cheese was made from each of the three different SCC groups using a Manchego cheese manufacturing protocol. As the level of SCC increased, the time required for visual flocculation increased, and it took longer to reach the desired firmness for cutting the coagulum. The fat and moisture contents were lower in the highest SCC cheeses. After 3 mo, total free fatty acids (FFA) contents were significantly higher in the highest SCC cheeses. Butyric and caprylic acids levels were significantly higher in group III cheeses at all stages of ripening. Cheese graders noted rancid or lipase flavor in the highest SCC level cheeses at each of the sampling points, and they also deducted points for more body and textural defects in these cheeses at 6 and 9 mo. PMID:14594225

  15. A proteomic perspective on the changes in milk proteins due to high somatic cell count.

    PubMed

    Zhang, L; Boeren, S; van Hooijdonk, A C M; Vervoort, J M; Hettinga, K A

    2015-08-01

    Although cows with subclinical mastitis have no difference in the appearance of their milk, milk composition and milk quality are altered because of the inflammation. To know the changes in milk quality with different somatic cell count (SCC) levels, 5 pooled bovine milk samples with SCC from 10(5) to 10(6) cells/mL were analyzed qualitatively and quantitatively using both one-dimension sodium dodecyl sulfate PAGE and filter-aided sample preparation coupled with dimethyl labeling, both followed by liquid chromatography tandem mass spectrometry. Minor differences were found on the qualitative level in the proteome from milk with different SCC levels, whereas the concentration of milk proteins showed remarkable changes. Not only immune-related proteins (cathelicidins, IGK protein, CD59 molecule, complement regulatory protein, lactadherin), but also proteins with other biological functions (e.g., lipid metabolism: platelet glycoprotein 4, butyrophilin subfamily 1 member A1, perilipin-2) were significantly different in milk from cows with high SCC level compared with low SCC level. The increased concentration of protease inhibitors in the milk with higher SCC levels may suggest a protective role in the mammary gland against protease activity. Prostaglandin-H2 D-isomerase showed a linear relation with SCC, which was confirmed with an ELISA. However, the correlation coefficient was lower in individual cows compared with bulk milk. These results indicate that prostaglandin-H2 D-isomerase may be used as an indicator to evaluate bulk milk quality and thereby reduce the economic loss in the dairy industry. The results from this study reflect the biological phenomena occurring during subclinical mastitis and in addition provide a potential indicator for the detection of bulk milk with high SCC. PMID:26094216

  16. Adult stem cells and tissue repair.

    PubMed

    Körbling, M; Estrov, Z; Champlin, R

    2003-08-01

    Recently, adult stem cells originating from bone marrow or peripheral blood have been suggested to contribute to repair and genesis of cells specific for liver, cardiac and skeletal muscle, gut, and brain tissue. The mechanism involved has been termed transdifferentiation, although other explanations including cell fusion have been postulated. Using adult stem cells to generate or repair solid organ tissue obviates the immunologic, ethical, and teratogenic issues that accompany embryonic stem cells.

  17. The Jak-STAT target Chinmo prevents sex transformation of adult stem cells in the Drosophila testis niche

    PubMed Central

    Ma, Qing; Wawersik, Matthew; Matunis, Erika L.

    2014-01-01

    Local signals maintain adult stem cells in many tissues. Whether the sexual identity of adult stem cells must also be maintained was not known. In the adult Drosophila testis niche, local Jak-STAT signaling promotes somatic cyst stem cell (CySC) renewal through several effectors, including the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo). Here, we find that Chinmo also prevents feminization of CySCs. Chinmo promotes expression of the canonical male sex determination factor DoublesexM (DsxM) within CySCs and their progeny, and ectopic expression of DsxM in the CySC lineage partially rescues the chinmo sex transformation phenotype, placing Chinmo upstream of DsxM. The Dsx homologue DMRT1 prevents the male-to female conversion of differentiated somatic cells in the adult mammalian testis, but its regulation is not well understood. Our work indicates that sex maintenance occurs in adult somatic stem cells, and that this highly conserved process is governed by effectors of niche signals. PMID:25453558

  18. Cloning mammary cell cDNAs from 17q12-q23 using interspecific somatic cell hybrids and subtractive hybridization

    SciTech Connect

    Cerosaletti, K.M.; Shapero, M.H.; Fournier, R.E.K.

    1995-01-01

    We have cloned human genes that are encoded in the region 17q12-q23 and expressed in breast tissue using interspecific somatic cell hybrids and subtractive hybridization. Two mouse microcell hybrids containing fragments of human chromosome 17 with a nonoverlap region at 17q12-q23 were generated by microcell transfer. Radiolabeled cDNA was synthesized from the hybrid cell containing the 17q12-q23 interval and was subtracted with an excess of RNA from the hybrid cell lacking the interval. Resulting cDNA probes enriched for sequences from 17q12-q23 were used to screen a human premenopausal breast cDNA library, and 60 cDNAs were identified. Three of these cDNAs mapped to the hybrid cell nonoverlap region. These cDNAs were expressed in mammary epithelial cell hybrids, although none appeared to be breast-specific. Sequence analysis of the cDNAs revealed that clone 93A represents a previously unidentified gene, clone 98C has homology to an expressed sequence tag from goat mammary tissue, and clone 200A is identical to the human homologue of the Drosophila melanogaster flightless-I gene. These genes map outside a 1-cM region linked to early onset familial breast cancer but may be useful genetic markers in the 17q12-q23 region. 47 refs., 6 figs.

  19. G protein-coupled receptors in stem cell maintenance and somatic reprogramming to pluripotent or cancer stem cells.

    PubMed

    Choi, Hye Yeon; Saha, Subbroto Kumar; Kim, Kyeongseok; Kim, Sangsu; Yang, Gwang-Mo; Kim, BongWoo; Kim, Jin-hoi; Cho, Ssang-Goo

    2015-02-01

    G protein-coupled receptors (GPCRs) are a large class of transmembrane receptors categorized into five distinct families: rhodopsin, secretin, adhesion, glutamate, and frizzled. They bind and regulate 80% of all hormones and account for 20-50% of the pharmaceuticals currently on the market. Hundreds of GPCRs integrate and coordinate the functions of individual cells, mediating signaling between various organs. GPCRs are crucial players in tumor progression, adipogenesis, and inflammation. Several studies have also confirmed their central roles in embryonic development and stem cell maintenance. Recently, GPCRs have emerged as key players in the regulation of cell survival, proliferation, migration, and self-renewal in pluripotent (PSCs) and cancer stem cells (CSCs). Our study and other reports have revealed that the expression of many GPCRs is modulated during the generation of induced PSCs (iPSCs) or CSCs as well as during CSC sphere formation. These GPCRs may have crucial roles in the regulation of selfrenewal and other biological properties of iPSCs and CSCs. This review addresses the current understanding of the role of GPCRs in stem cell maintenance and somatic reprogramming to PSCs or CSCs.

  20. G protein-coupled receptors in stem cell maintenance and somatic reprogramming to pluripotent or cancer stem cells

    PubMed Central

    Choi, Hye Yeon; Saha, Subbroto Kumar; Kim, Kyeongseok; Kim, Sangsu; Yang, Gwang-Mo; Kim, BongWoo; Kim, Jin-hoi; Cho, Ssang-Goo

    2015-01-01

    G protein-coupled receptors (GPCRs) are a large class of transmembrane receptors categorized into five distinct families: rhodopsin, secretin, adhesion, glutamate, and frizzled. They bind and regulate 80% of all hormones and account for 20-50% of the pharmaceuticals currently on the market. Hundreds of GPCRs integrate and coordinate the functions of individual cells, mediating signaling between various organs. GPCRs are crucial players in tumor progression, adipogenesis, and inflammation. Several studies have also confirmed their central roles in embryonic development and stem cell maintenance. Recently, GPCRs have emerged as key players in the regulation of cell survival, proliferation, migration, and self-renewal in pluripotent (PSCs) and cancer stem cells (CSCs). Our study and other reports have revealed that the expression of many GPCRs is modulated during the generation of induced PSCs (iPSCs) or CSCs as well as during CSC sphere formation. These GPCRs may have crucial roles in the regulation of selfrenewal and other biological properties of iPSCs and CSCs. This review addresses the current understanding of the role of GPCRs in stem cell maintenance and somatic reprogramming to PSCs or CSCs. [BMB Reports 2015; 48(2): 68-80] PMID:25413305

  1. Unequal chromosome division and inter-genomic translocation occurred in somatic cells of wheat-rye allopolyploid.

    PubMed

    Tang, Zongxiang; Fu, Shulan; Yan, Benju; Zhang, Huaiqiong; Ren, Zhenglong

    2012-03-01

    Newly synthesized wheat-rye allopolyploids were investigated by genomic in situ hybridization, over the first, second, third and fourth allopolyploid generations. Inter and intra chromosome connections were observed in 12 root-tip cells of CA4.4.7 (S(2) generation), and translocations between wheat and rye chromosomes were also detected in five root-tip cells. In root-tip cells of CA4.4.7.5 and CA4.4.7.2.2 (S(3) and S(4) generation), the chromosome connections occurred again, a dissociative small rye segment was detected in seven cells of CA4.4.7.5. In plants MSV6.1 and MSV6.5 (S(1) generation), almost half of the root-tip cells contained 13 rye chromosomes and the rest held 12 rye chromosomes, and all the cells of the two plants contained 42 wheat chromosomes. Five pairing configurations of rye chromosomes, including 5 II + 3 I, 6 II + 1 I, 6 II, 5 II + 2 I and 4 II + 4 I, were observed in pollen mother cells of the two plants. The two plants' progeny, including S(2), S(3), and S(4) generation plants, contained 42 wheat chromosomes and 12 rye chromosomes. Therefore, the inter chromosome translocation and unequal chromosome division could occur in somatic cells of wide hybrids. The unequal chromosome division in somatic cell could induce chromosome elimination at the early stages of allopolyploidization.

  2. Rex Rabbit Somatic Cell Nuclear Transfer with In Vitro-Matured Oocytes.

    PubMed

    Liu, Yong; Wang, Huili; Lu, Jinhua; Miao, Yiliang; Cao, Xinyan; Zhang, Ling; Wu, Xiaoqing; Wu, Fengrui; Ding, Biao; Wang, Rong; Luo, Mingjiu; Li, Wenyong; Tan, Jinghe

    2016-06-01

    Somatic cell nuclear transfer (SCNT) requires large numbers of matured oocytes. In vitro-matured (IVM) oocytes have been used in SCNT in many animals. We investigated the use of IVM oocytes in Rex rabbit SCNT using Rex rabbit ovaries obtained from a local abattoir. The meiotic ability of oocytes isolated from follicles of different diameters was studied. Rex rabbit SCNT was optimized for denucleation, activation, and donor cell synchronization. Rex rabbit oocytes grew to the largest diameter (110 μm) when the follicle diameter was 1.0 mm. Oocytes isolated from <0.5-mm follicles lacked the ability to resume meiosis. More than 90% of these oocytes remained in the germinal vesicle (GV) stage after in vitro culture (IVC) for 18 h. Oocytes isolated from >0.7-mm follicles acquired maturation ability. More than 90% of these oocytes matured after IVC for 18 h. The developmental potential of oocytes isolated from >1-mm follicles was greater than that of oocytes isolated from 0.7- to 1.0-mm follicles. The highest activation rates for IVM Rex rabbit oocytes were seen after treatment with 2.5 μM ionomycin for 5 min followed by 2 mM 6-dimethylaminopurine (6-DMAP) and 5 μg/mL cycloheximide (CHX) for 1 h. Ionomycin induced the chromatin of IVM oocytes to protrude from the oocyte surface, promoting denucleation. Fetal fibroblast cells (FFCs) and cumulus cells (CCs) were more suitable for Rex rabbit SCNT than skin fibroblast cells (SFCs) (blastocyst rate was 35.6 ± 2.2% and 38.0 ± 6.0% vs. 19.7 ± 3.1%). The best fusion condition was a 2DC interval for 1 sec, 1.6 kV/cm voltages, and 40 μsec duration in 0.28 M mannitol. In conclusion, the in vitro maturation of Rex rabbit oocytes and SCNT procedures were studied systematically and optimized in this study. PMID:27159389

  3. Control of intramammary infections in goats: impact on somatic cell counts.

    PubMed

    Poutrel, B; de Crémoux, R; Ducelliez, M; Verneau, D

    1997-02-01

    Udder-half infections were recorded throughout a lactation for 1,060 goats belonging to eight commercial herds. Bacteriological examination from aseptic milk samples and somatic cell counts (SCC) determined by Fossomatic cell counting were performed at the beginning, the middle, and the end of lactation. Coagulase-negative staphylococci (CNS) were the prevalent microorganisms isolated. Geometric means of SCC for uninfected halves or halves infected by CNS or major pathogens were 272 x 10(3) cells/mL, 932,000 x 10(3) cells/mL and 2,443,000 x 10(3) cells/mL, respectively. Two field trials were carried out for evaluation of effectiveness of systematic treatment at drying-off (1 syringe by half) by a combination of penicillin, nafcillin, and dihydrostreptomycin labeled for bovines. In the first trial, all goats (n = 217) of two herds were treated immediately after the last milking, and two herds (n = 196) were used as untreated controls. In the second trial, 215 goats were treated at drying-off. There were no untreated controls. Dry period cures were determined by bacteriological examination of udder-half milk samples collected aseptically at drying-off and 2 wk after parturition. Impact of treatment on SCC was determined from composite milk samples collected monthly after kidding. At parturition, in the first trial, 40 of 202 (19.8%) udder halves were spontaneously cured in the control group vs 169 of 217 (77.9%) in the treatment group. In the second trial, 141 out of 215 treated halves were cured. During the first 75 d in lactation, geometric mean SCC was significantly lower for treated goats than for control goats. After 75 d, SCC for treated and control goats were similar. These data suggest that other methods are required to prevent new intramammary infections throughout the lactation in order to keep a low SCC in goat milk. To determine whether this could be accomplished through teat dipping, half of the goats in five commercial herds were dipped (n = 294) after

  4. The recent history of somatic cloning in mammals.

    PubMed

    Brem, Gottfried; Kühholzer, Birgit

    2002-01-01

    The history of somatic cell nuclear transfer (NT) in mammals is full of exciting experiments and findings regarding the technique and outcome of NT, despite only covering a period of 6 years. The production of Dolly, for the first time demonstrating cloning from an adult somatic cell, had a great impact on subsequent studies. However, the more progress we make, the more obvious it becomes how little we know about the processes during NT, specifically how reprogramming events occur. Therefore, it is certainly challenging to continue investigating every step of somatic cell NT more intensively, starting from the donor cell, (type, cell cycle, synchronization, population doublings) and continuing until the cloned offspring are born and even further, to see how and if NT has an influence on health, viability, quantitative traits, and reproduction of cloned individuals.

  5. The recent history of somatic cloning in mammals.

    PubMed

    Brem, Gottfried; Kühholzer, Birgit

    2002-01-01

    The history of somatic cell nuclear transfer (NT) in mammals is full of exciting experiments and findings regarding the technique and outcome of NT, despite only covering a period of 6 years. The production of Dolly, for the first time demonstrating cloning from an adult somatic cell, had a great impact on subsequent studies. However, the more progress we make, the more obvious it becomes how little we know about the processes during NT, specifically how reprogramming events occur. Therefore, it is certainly challenging to continue investigating every step of somatic cell NT more intensively, starting from the donor cell, (type, cell cycle, synchronization, population doublings) and continuing until the cloned offspring are born and even further, to see how and if NT has an influence on health, viability, quantitative traits, and reproduction of cloned individuals. PMID:12006157

  6. Using a nano-flare probe to detect RNA in live donor cells prior to somatic cell nuclear transfer.

    PubMed

    Fu, Bo; Ren, Liang; Liu, Di; Ma, Jian-Zhang; An, Tie-Zhu; Yang, Xiu-Qin; Ma, Hong; Guo, Zhen-Hua; Zhu, Meng; Bai, Jing

    2016-01-01

    Many transgenes are silenced in mammalian cells (donor cells used for somatic cell nuclear transfer [SCNT]). Silencing correlated with a repressed chromatin structure or suppressed promoter, and it impeded the production of transgenic animals. Gene transcription studies in live cells are challenging because of the drawbacks of reverse-transcription polymerase chain reaction and fluorescence in situ hybridization. Nano-flare probes provide an effective approach to detect RNA in living cells. We used 18S RNA, a housekeeping gene, as a reference gene. This study aimed to establish a platform to detect RNA in single living donor cells using a Nano-flare probe prior to SCNT and to verify the safety and validity of the Nano-flare probe in order to provide a technical foundation for rescuing silenced transgenes in transgenic cloned embryos. We investigated cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts, characterized the distribution of the 18S RNA-Nano-flare probe in living cells and investigated the effect of the 18S RNA-Nano-flare probe on the development of cloned embryos after SCNT. The cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts was dose-dependent, and 18S RNA was detected using the 18S RNA-Nano-flare probe. In addition, treating donor cells with 500 pM 18S RNA-Nano-flare probe did not have adverse effects on the development of SCNT embryos at the pre-implantation stage. In conclusion, we established a preliminary platform to detect RNA in live donor cells using a Nano-flare probe prior to SCNT.

  7. Diethylnitrosamine-induced expression of germline-specific genes and pluripotency factors, including vasa and oct4, in medaka somatic cells.

    PubMed

    Shen, Jialing; Yokota, Shinpei; Yokoi, Hayato; Suzuki, Tohru

    2016-09-16

    Various methods have been developed to reprogram mammalian somatic cells into pluripotent cells as well as to directly reprogram somatic cells into other cell lineages. We are interested in applying these methods to fish, and here, we examined whether mRNA expression of germline-specific genes (vasa, nanos2, -3) and pluripotency factors (oct4, sox2, c-myc, nanog) is inducible in somatic cells of Japanese medaka (Oryzias latipes). We found that the expression of vasa is induced in the gut and regenerating fin by exposure to a carcinogen, diethylnitrosamine (DEN). Induction of vasa in the gut started on the 5th day of treatment with >50 ppm DEN. In addition, nanos2, -3, oct4, sox2, klf4, c-myc, and nanog were also expressed simultaneously in some vasa-positive gut and regenerating fin samples. Vasa-positive cells were detected by immunohistochemistry (IHC) in the muscle surrounding the gut and in the wound epidermis, blastema, and fibroblast-like cells in regenerating fin. In vasa:GFP transgenic medaka, green fluorescent protein (GFP) fluorescence appeared in the wound epidermis and fibroblast-like cells in the regenerating fin following DEN exposure, in agreement with the IHC data. Our data show that mRNA expression of genes relevant to germ cell specification and pluripotency can be induced in fish somatic cells by exposure to DEN, suggesting the possibility of efficient and rapid cell reprogramming of fish somatic cells. PMID:27514449

  8. Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer.

    PubMed

    Bao, Lei; Chen, HaiDe; Jong, UiMyong; Rim, CholHo; Li, WenLing; Lin, XiJuan; Zhang, Dan; Luo, Qiong; Cui, Chun; Huang, HeFeng; Zhang, Yan; Xiao, Lei; Fu, ZhiXin

    2014-02-01

    Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs. Conventional gene targeting in pig somatic cells is extremely inefficient. Zinc-finger nuclease (ZFN) technology has been shown to be a powerful tool for efficiently inducing mutations in the genome. However, ZFN-mediated targeting in pigs has rarely been achieved. Here, we used ZFNs to knock out the porcine α-1, 3-galactosyl-transferase (GGTA1) gene, which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation. Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1. Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4% and 5.2%, respectively. The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer (SCNT). Three GGTA1 null piglets were born, and one knockout primary fibroblast cell line was established from a cloned fetus. Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane. Functionally, GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum. This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs. GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation. PMID:24430555

  9. Somatic cell nuclear transfer: origins, the present position and future opportunities.

    PubMed

    Wilmut, Ian; Bai, Yu; Taylor, Jane

    2015-10-19

    Nuclear transfer that involves the transfer of the nucleus from a donor cell into an oocyte or early embryo from which the chromosomes have been removed was considered first as a means of assessing changes during development in the ability of the nucleus to control development. In mammals, development of embryos produced by nuclear transfer depends upon coordination of the cell cycles of donor and recipient cells. Our analysis of nuclear potential was completed in 1996 when a nucleus from an adult ewe mammary gland cell controlled development to term of Dolly the sheep. The new procedure has been used to target the first precise genetic modification into livestock; however, the greatest inheritance of the Dolly experiment was to make biologists think differently. If unknown factors in the recipient oocyte could reprogramme the nucleus to a stage very early in development then there must be other ways of making that change. Within 10 years, two laboratories working independently established protocols by which the introduction of selected transcription factors changes a small proportion of the treated cells to pluripotent stem cells. This ability to produce 'induced pluripotent stem cells' is providing revolutionary new opportunities in research and cell therapy.

  10. Somatic cell nuclear transfer: origins, the present position and future opportunities.

    PubMed

    Wilmut, Ian; Bai, Yu; Taylor, Jane

    2015-10-19

    Nuclear transfer that involves the transfer of the nucleus from a donor cell into an oocyte or early embryo from which the chromosomes have been removed was considered first as a means of assessing changes during development in the ability of the nucleus to control development. In mammals, development of embryos produced by nuclear transfer depends upon coordination of the cell cycles of donor and recipient cells. Our analysis of nuclear potential was completed in 1996 when a nucleus from an adult ewe mammary gland cell controlled development to term of Dolly the sheep. The new procedure has been used to target the first precise genetic modification into livestock; however, the greatest inheritance of the Dolly experiment was to make biologists think differently. If unknown factors in the recipient oocyte could reprogramme the nucleus to a stage very early in development then there must be other ways of making that change. Within 10 years, two laboratories working independently established protocols by which the introduction of selected transcription factors changes a small proportion of the treated cells to pluripotent stem cells. This ability to produce 'induced pluripotent stem cells' is providing revolutionary new opportunities in research and cell therapy. PMID:26416677

  11. Cells with Stem Cell Characteristics in Somatic Compartments of the Ovary

    PubMed Central

    Kossowska-Tomaszczuk, Katarzyna; De Geyter, Christian

    2013-01-01

    Antral follicular growth in the ovary is characterized by rapid expansion of granulosa cells accompanied by a rising complexity of their functionality. Within two weeks the number of human granulosa cells increases from less than 500,000 to more than 50 millions cells per follicle and differentiates into groups of cells with a variety of specialized functions involved in steroidogenesis, nursing the oocyte, and forming a functional syncitium. Both the rapid proliferation and different specialized functions of the granulosa cells can only be explained through the involvement of stem cells. However, luteinizing granulosa cells were believed to be terminally differentiated cells. Only recently, stem and progenitor cells with FSH-receptor activity were identified in populations of luteinizing granulosa cells obtained during oocyte collected for assisted reproduction. In the presence of the leukaemia-inhibiting factor (LIF), it was possible to culture a subpopulation of the luteinizing granulosa cells over prolonged time periods. Furthermore, when embedded in a matrix consisting of collagen type I, these cells continued to express the FSH receptor over prolonged time periods, developed globular formations that surrogated as follicle-like structures, providing a promising tool for reproductive biology. PMID:23484108

  12. Progenitor cells in the adult pancreas.

    PubMed

    Holland, Andrew M; Góñez, L Jorge; Harrison, Leonard C

    2004-01-01

    The beta-cell mass in the adult pancreas possesses the ability to undergo limited regeneration following injury. Identifying the progenitor cells involved in this process and understanding the mechanisms leading to their maturation will open new avenues for the treatment of type 1 diabetes. However, despite steady advances in determining the molecular basis of early pancreatic development, the identification of pancreatic stem cells or beta-cell progenitors and the molecular mechanisms underlying beta-cell regeneration remain unclear. Recent advances in the directed differentiation of embryonic and adult stem cells has heightened interest in the possible application of stem cell therapy in the treatment of type 1 diabetes. Drawing on the expanding knowledge of pancreas development, beta-cell regeneration and stem cell research, this review focuses on progenitor cells in the adult pancreas as a potential source of beta-cells. PMID:14737742

  13. Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer.

    PubMed

    Jin, Jun-Xue; Li, Suo; Gao, Qing-Shan; Hong, Yu; Jin, Long; Zhu, Hai-Ying; Yan, Chang-Guo; Kang, Jin-Dan; Yin, Xi-Jun

    2013-10-01

    The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering the epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. In the current study, we examined the effects of LBH589 (panobinostat), a novel broad-spectrum HDACi, on the nuclear reprogramming and development of pig SCNT embryos in vitro. In experiment 1, we compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with 50 nM LBH589 for 24 hours showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with 5 or 500 nM LBH589 (32.4% vs. 11.8%, 12.1%, and 10.0%, respectively, P < 0.05). In experiment 2, we examined the in vitro developmental competence of nuclear transfer embryos treated with 50 nM LBH589 for various intervals after activation and 6-dimethylaminopurine. Embryos treated for 24 hours had higher rates of blastocyst formation than the other groups. In experiment 3, when the acetylation of H4K12 was examined in SCNT embryos treated for 6 hours with 50 nM LBH589 by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control. In experiment 4, LBH589-treated nuclear transfer and control embryos were transferred into surrogate mothers, resulting in three (100%) and two (66.7%) pregnancies, respectively. In conclusion, LBH589 enhances the nuclear reprogramming and developmental potential of SCNT embryos by altering the epigenetic status and expression, and increasing blastocyst quality.

  14. Longitudinal study of reproductive performance of female cattle produced by somatic cell nuclear transfer.

    PubMed

    Polejaeva, Irina A; Broek, Diane M; Walker, Shawn C; Zhou, Wenli; Walton, Mark; Benninghoff, Abby D; Faber, David C

    2013-01-01

    The objective of this study was to determine whether or not reproductive performance in cattle produced by somatic cell nuclear transfer (SCNT) is significantly different from that of their genetic donors. To address this question, we directed two longitudinal studies using different embryo production procedures: (1) superovulation followed by artificial insemination (AI) and embryo collection and (2) ultrasound-guided ovum pick-up followed by in vitro fertilization (OPU-IVF). Collectively, these two studies represent the largest data set available for any species on the reproductive performance of female clones and their genetic donors as measured by their embryo production outcomes in commercial embryo production program. The large-scale study described herein was conducted over a six-year period of time and provides a unique comparison of 96 clones to the 40 corresponding genetic donors. To our knowledge, this is the first longitudinal study on the reproductive performance of cattle clones using OPU-IVF. With nearly 2,000 reproductive procedures performed and more than 9,200 transferable embryos produced, our observations show that the reproductive performance of cattle produced by SCNT is not different compared to their genetic donors for the production of transferable embryos after either AI followed by embryo collection (P = 0.77) or OPU-IVF (P = 0.97). These data are in agreement with previous reports showing that the reproductive capabilities of cloned cattle are equal to that of conventionally produced cattle. In conclusion, results of this longitudinal study once again demonstrate that cloning technology, in combination with superovulation, AI and embryo collection or OPU-IVF, provides a valuable tool for faster dissemination of superior maternal genetics.

  15. Monitoring nonlactating cow intramammary infection dynamics using DHI somatic cell count data.

    PubMed

    Cook, N B; Bennett, T B; Emery, K M; Nordlund, K V

    2002-05-01

    Although the nonlactating period presents a risk for intramammary infection, efficient systems to monitor infection status of recently calved cows have not been developed, and benchmarks for interpretation have not been established. Individual cow somatic cell count (SCC) data for the current and previous six monthly Dairy Herd Improvement milk tests and the last SCC of the previous lactation and first SCC of the current lactation were summarized for all milking cows in a selection of Wisconsin dairy herds. Prevalence of infection, herd new infection rate, fresh cow contribution to herd new infection rate, dry cow new infection rate, heifer new infection rate, and dry cow cure rate were estimated using a threshold of 200,000/ml. In 145 herds, mean (range) heifer new infection rate was 21.3% (0 to 58%). The cut-point for the 10th percentile of herds was 8%. Mean (range) dry cow new infection rate in cows that were uninfected at the last test before dry off was 22.4% (0 to 71%), and the cut-point for the 10th percentile of herds was 9%. Although nonlactating cow and heifer new infection rates increased with weighted 6-mo mean herd SCC, the between-herd variation was large, suggesting that on-farm factors are important in determining the rates of infection. In a subset of 51 Wisconsin dairy herds, significant monthly variation in weighted SCC, prevalence, herd new infection rate, and fresh cow contribution to herd new infection rate were detected. Elevations in SCC and prevalence of infection during the summer (July through September) were associated with significant increases in fresh cow and herd new infection rates.

  16. Longitudinal Study of Reproductive Performance of Female Cattle Produced by Somatic Cell Nuclear Transfer

    PubMed Central

    Polejaeva, Irina A.; Broek, Diane M.; Walker, Shawn C.; Zhou, Wenli; Walton, Mark; Benninghoff, Abby D.; Faber, David C.

    2013-01-01

    The objective of this study was to determine whether or not reproductive performance in cattle produced by somatic cell nuclear transfer (SCNT) is significantly different from that of their genetic donors. To address this question, we directed two longitudinal studies using different embryo production procedures: (1) superovulation followed by artificial insemination (AI) and embryo collection and (2) ultrasound-guided ovum pick-up followed by in vitro fertilization (OPU-IVF). Collectively, these two studies represent the largest data set available for any species on the reproductive performance of female clones and their genetic donors as measured by their embryo production outcomes in commercial embryo production program. The large-scale study described herein was conducted over a six-year period of time and provides a unique comparison of 96 clones to the 40 corresponding genetic donors. To our knowledge, this is the first longitudinal study on the reproductive performance of cattle clones using OPU-IVF. With nearly 2,000 reproductive procedures performed and more than 9,200 transferable embryos produced, our observations show that the reproductive performance of cattle produced by SCNT is not different compared to their genetic donors for the production of transferable embryos after either AI followed by embryo collection (P = 0.77) or OPU-IVF (P = 0.97). These data are in agreement with previous reports showing that the reproductive capabilities of cloned cattle are equal to that of conventionally produced cattle. In conclusion, results of this longitudinal study once again demonstrate that cloning technology, in combination with superovulation, AI and embryo collection or OPU-IVF, provides a valuable tool for faster dissemination of superior maternal genetics. PMID:24391930

  17. Trichostatin A Rescues the Disrupted Imprinting Induced by Somatic Cell Nuclear Transfer in Pigs

    PubMed Central

    Huan, Yanjun; Zhu, Jiang; Huang, Bo; Mu, Yanshuang; Kong, Qingran; Liu, Zhonghua

    2015-01-01

    Imprinting disorders induced by somatic cell nuclear transfer (SCNT) usually lead to the abnormalities of cloned animals and low cloning efficiency. Histone deacetylase inhibitors have been shown to improve gene expression, genomic methylation reprogramming and the development of cloned embryos, however, the imprinting statuses in these treated embryos and during their subsequent development remain poorly studied. In this study, we investigated the dynamics of H19/Igf2 methylation and transcription in porcine cloned embryos treated with trichostatin A (TSA), and examined H19/Igf2 imprinting patterns in cloned fetuses and piglets. Our results showed that compared with the maintenance of H19/Igf2 methylation in fertilized embryos, cloned embryos displayed aberrant H19/Igf2 methylation and lower H19/Igf2 transcripts. When TSA enhanced the development of cloned embryos, the disrupted H19/Igf2 imprinting was largely rescued in these treated embryos, more similar to those detected in fertilized counterparts. Further studies displayed that TSA effectively rescued the disrupted imprinting of H19/Igf2 in cloned fetuses and piglets, prevented the occurrence of cloned fetus and piglet abnormalities, and enhanced the full-term development of cloned embryos. In conclusion, our results demonstrated that aberrant imprinting induced by SCNT led to the abnormalities of cloned fetuses and piglets and low cloning efficiency, and TSA rescued the disrupted imprinting in cloned embryos, fetuses and piglets, and prevented the occurrence of cloned fetus and piglet abnormalities, thereby improving the development of cloned embryos. This study would have important implications in improving cloning efficiency and the health of cloned animals. PMID:25962071

  18. Somatic cell nuclear transfer: origins, the present position and future opportunities

    PubMed Central

    Wilmut, Ian; Bai, Yu; Taylor, Jane

    2015-01-01

    Nuclear transfer that involves the transfer of the nucleus from a donor cell into an oocyte or early embryo from which the chromosomes have been removed was considered first as a means of assessing changes during development in the ability of the nucleus to control development. In mammals, development of embryos produced by nuclear transfer depends upon coordination of the cell cycles of donor and recipient cells. Our analysis of nuclear potential was completed in 1996 when a nucleus from an adult ewe mammary gland cell controlled development to term of Dolly the sheep. The new procedure has been used to target the first precise genetic modification into livestock; however, the greatest inheritance of the Dolly experiment was to make biologists think differently. If unknown factors in the recipient oocyte could reprogramme the nucleus to a stage very early in development then there must be other ways of making that change. Within 10 years, two laboratories working independently established protocols by which the introduction of selected transcription factors changes a small proportion of the treated cells to pluripotent stem cells. This ability to produce ‘induced pluripotent stem cells’ is providing revolutionary new opportunities in research and cell therapy. PMID:26416677

  19. Nuclear transfer of synchronized African wild cat somatic cells into enucleated domestic cat oocytes

    USGS Publications Warehouse

    Gomez, M.C.; Jenkins, J.A.; Giraldo, A.; Harris, R.F.; King, A.; Dresser, B.L.; Pope, C.E.

    2003-01-01

    The African wild cat is one of the smallest wild cats and its future is threatened by hybridization with domestic cats. Nuclear transfer, a valuable tool for retaining genetic variability, offers the possibility of species continuation rather than extinction. The aim of this study was to investigate the ability of somatic cell nuclei of the African wild cat (AWC) to dedifferentiate within domestic cat (DSH) cytoplasts and to support early development after nuclear transplantation. In experiment 1, distributions of AWC and DSH fibroblasts in each cell-cycle phase were assessed by flow cytometry using cells cultured to confluency and disaggregated with pronase, trypsin, or mechanical separation. Trypsin (89.0%) and pronase (93.0%) yielded higher proportions of AWC nuclei in the G0/G1 phase than mechanical separation (82.0%). In contrast, mechanical separation yielded higher percentages of DSH nuclei in the G0/G1 phase (86.6%) than pronase (79.7%) or trypsin (74.2%) treatments. In both species, pronase induced less DNA damage than trypsin. In experiment 2, the effects of serum starvation, culture to confluency, and exposure to roscovitine on the distribution of AWC and DSH fibroblasts in various phases of the cell cycle were determined. Flow cytometry analyses revealed that the dynamics of the cell cycle varied as culture conditions were modified. Specifically, a higher percentage of AWC and DSH nuclei were in the G0/G1 phase after cells were serum starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition (61% vs. 88%), or after roscovitine (56% vs. 84%) treatment, respectively. In experiment 3, we evaluated the effects of cell synchronization and oocyte maturation (in vivo vs. in vitro) on the reconstruction and development of AWC-DSH- and DSH-DSH-cloned embryos. The method of cell synchronization did not affect the fusion and cleavage rate because only a slightly higher percentage of fused couplets cleaved when donor nuclei

  20. Somatic-cell mutation induced by short exposures to cigarette smoke in urate-null, oxidative stress-sensitive Drosophila.

    PubMed

    Uchiyama, Tomoyo; Koike, Ryota; Yuma, Yoko; Okamoto, Keinosuke; Arimoto-Kobayashi, Sakae; Suzuki, Toshinori; Negishi, Tomoe

    2016-01-01

    We previously reported that a urate-null strain of Drosophila is hypersensitive to cigarette smoke (CS), and we suggested that CS induces oxidative stress in Drosophila because uric acid is a potent antioxidant. Although the carcinogenic risk of CS exposure is widely recognized; documentation of in vivo genotoxic activity of environmental CS, especially gaseous-phase CS, remains inconclusive. To date, somatic-cell mutations in Drosophila resulting from exposure to CS have not been detected via the somatic mutation and recombination test (wing spot test) with wild-type flies, a widely used Drosophila assay for the detection of somatic-cell mutation; moreover, genotoxicity has not been documented via a DNA repair test that involves DNA repair-deficient Drosophila. In this study, we used a new Drosophila strain (y v ma-l; mwh) to examine the mutagenicity induced by gaseous-phase CS; these flies are urate-null due to a mutation in ma-l, and they are heterozygous for multiple wing hair (mwh), a mutation that functions as a marker for somatic-cell mutation. In an assay with this newly developed strain, a superoxide anion-producing weed-killer, paraquat, exhibited significant mutagenicity; in contrast, paraquat was hardly mutagenic with a wild-type strain. Drosophila larvae were exposed to CS for 2, 4 or 6h, and then kept at 25°C on instant medium until adulthood. After eclosion, mutant spots, which consisted of mutant hairs on wings, were scored. The number of mutant spots increased significantly in an exposure time-dependent manner in the urate-null females (ma-l (-/-)), but not in the urate-positive females (ma-l (+/-)). In this study, we showed that short-term exposure to CS was mutagenic in this in vivo system. In addition, we obtained suggestive data regarding reactive oxygen species production in larva after CS exposure using the fluorescence probe H2DCFDA. These results suggest that oxidative damage, which might be countered by uric acid, was partly responsible

  1. Human somatic cells subjected to genetic induction with six germ line-related factors display meiotic germ cell-like features

    PubMed Central

    Medrano, Jose V.; Martínez-Arroyo, Ana M.; Míguez, Jose M.; Moreno, Inmaculada; Martínez, Sebastián; Quiñonero, Alicia; Díaz-Gimeno, Patricia; Marqués-Marí, Ana I.; Pellicer, Antonio; Remohí, Jose; Simón, Carlos

    2016-01-01

    The in vitro derivation of human germ cells has attracted interest in the last years, but their direct conversion from human somatic cells has not yet been reported. Here we tested the ability of human male somatic cells to directly convert into a meiotic germ cell-like phenotype by inducing them with a combination of selected key germ cell developmental factors. We started with a pool of 12 candidates that were reduced to 6, demonstrating that ectopic expression of the germ line-related genes PRDM1, PRDM14, LIN28A, DAZL, VASA and SYCP3 induced direct conversion of somatic cells (hFSK (46, XY), and hMSC (46, XY)) into a germ cell-like phenotype in vitro. Induced germ cell-like cells showed a marked switch in their transcriptomic profile and expressed several post-meiotic germ line related markers, showed meiotic progression, evidence of epigenetic reprogramming, and approximately 1% were able to complete meiosis as demonstrated by their haploid status and the expression of several post-meiotic markers. Furthermore, xenotransplantation assays demonstrated that a subset of induced cells properly colonize the spermatogonial niche. Knowledge obtained from this work can be used to create in vitro models to study gamete-related diseases in humans. PMID:27112843

  2. Effective Oocyte Vitrification and Survival Techniques for Bovine Somatic Cell Nuclear Transfer.

    PubMed

    Park, Min Jee; Lee, Seung Eun; Kim, Eun Young; Lee, Jun Beom; Jeong, Chang Jin; Park, Se Pill

    2015-06-01

    Bovine somatic cell nuclear transfer (SCNT) using vitrified-thawed (VT) oocytes has been studied; however, the cloning efficiency of these oocytes is not comparable with that of nonvitrified (non-V) fresh oocytes. This study sought to optimize the survival and cryopreservation of VT oocytes for SCNT. Co-culture with feeder cells that had been preincubated for 15 h significantly improved the survival of VT oocytes and their in vitro developmental potential following SCNT in comparison to co-culture with feeder cells that had been preincubated for 2, 5, or 24 h (p<0.05). Spindle assessment via the Oosight Microscopy Imaging System and microtubule staining revealed that vitrified metaphase II oocytes (VT group) were not suitable for SCNT. However, enucleating and/or activating oocytes prior to freezing enhanced their developmental potential and suitability for SCNT. The cloning efficiency of the enucleated-activated-vitrified-thawed (EAVT) group (21.6%) was better than that of the other vitrification groups [enucleated-vitrified-thawed (EVT) group, 13.7%; VT group, 15.0%; p<0.05] and was comparable with that of the non-V group (25.9%). The reactive oxygen species level was significantly lower in the EAVT group than in the other vitrification groups (p<0.05). mRNA levels of maternal genes (ZAR1, BMP15, and NLRP5) and a stress gene (HSF1) were lower in the vitrification groups than in the non-V group (p<0.05), whereas the level of phospho-p44/42 mitogen-activated protein kinase did not differ among the groups. Among the vitrification groups, blastocysts in the EAVT group had the best developmental potential, as judged by their high mRNA expression of developmental potential-related genes (POU5f1, Interferon-tau, and SLC2A5) and their low expression of proapoptotic (CASP3) and stress (Hsp70) genes. This study demonstrates that SCNT using bovine frozen-thawed oocytes can be successfully achieved using optimized vitrification and co-culture techniques.

  3. Effective Oocyte Vitrification and Survival Techniques for Bovine Somatic Cell Nuclear Transfer.

    PubMed

    Park, Min Jee; Lee, Seung Eun; Kim, Eun Young; Lee, Jun Beom; Jeong, Chang Jin; Park, Se Pill

    2015-06-01

    Bovine somatic cell nuclear transfer (SCNT) using vitrified-thawed (VT) oocytes has been studied; however, the cloning efficiency of these oocytes is not comparable with that of nonvitrified (non-V) fresh oocytes. This study sought to optimize the survival and cryopreservation of VT oocytes for SCNT. Co-culture with feeder cells that had been preincubated for 15 h significantly improved the survival of VT oocytes and their in vitro developmental potential following SCNT in comparison to co-culture with feeder cells that had been preincubated for 2, 5, or 24 h (p<0.05). Spindle assessment via the Oosight Microscopy Imaging System and microtubule staining revealed that vitrified metaphase II oocytes (VT group) were not suitable for SCNT. However, enucleating and/or activating oocytes prior to freezing enhanced their developmental potential and suitability for SCNT. The cloning efficiency of the enucleated-activated-vitrified-thawed (EAVT) group (21.6%) was better than that of the other vitrification groups [enucleated-vitrified-thawed (EVT) group, 13.7%; VT group, 15.0%; p<0.05] and was comparable with that of the non-V group (25.9%). The reactive oxygen species level was significantly lower in the EAVT group than in the other vitrification groups (p<0.05). mRNA levels of maternal genes (ZAR1, BMP15, and NLRP5) and a stress gene (HSF1) were lower in the vitrification groups than in the non-V group (p<0.05), whereas the level of phospho-p44/42 mitogen-activated protein kinase did not differ among the groups. Among the vitrification groups, blastocysts in the EAVT group had the best developmental potential, as judged by their high mRNA expression of developmental potential-related genes (POU5f1, Interferon-tau, and SLC2A5) and their low expression of proapoptotic (CASP3) and stress (Hsp70) genes. This study demonstrates that SCNT using bovine frozen-thawed oocytes can be successfully achieved using optimized vitrification and co-culture techniques. PMID:25984830

  4. Effect of sensor systems for cow management on milk production, somatic cell count, and reproduction.

    PubMed

    Steeneveld, W; Vernooij, J C M; Hogeveen, H

    2015-06-01

    To improve management on dairy herds, sensor systems have been developed that can measure physiological, behavioral, and production indicators on individual cows. It is not known whether using sensor systems also improves measures of health and production in dairy herds. The objective of this study was to investigate the effect of using sensor systems on measures of health and production in dairy herds. Data of 414 Dutch dairy farms with (n=152) and without (n=262) sensor systems were available. For these herds, information on milk production per cow, days to first service, first calving age, and somatic cell count (SCC) was provided for the years 2003 to 2013. Moreover, year of investment in sensor systems was available. For every farm year, we determined whether that year was before or after the year of investment in sensor systems on farms with an automatic milking system (AMS) or a conventional milking system (CMS), or whether it was a year on a farm that never invested in sensor systems. Separate statistical analyses were performed to determine the effect of sensor systems for mastitis detection (color, SCC, electrical conductivity, and lactate dehydrogenase sensors), estrus detection for dairy cows, estrus detection for young stock, and other sensor systems (weighing platform, rumination time sensor, fat and protein sensor, temperature sensor, milk temperature sensor, urea sensor, β-hydroxybutyrate sensor, and other sensor systems). The AMS farms had a higher average SCC (by 12,000 cells/mL) after sensor investment, and CMS farms with a mastitis detection system had a lower average SCC (by 10,000 cells/mL) in the years after sensor investment. Having sensor systems was associated with a higher average production per cow on AMS farms, and with a lower average production per cow on CMS farms in the years after investment. The most likely reason for this lower milk production after investment was that on 96% of CMS farms, the sensor system investment occurred

  5. Ultrastructural comparison of porcine putative embryonic stem cells derived by in vitro fertilization and somatic cell nuclear transfer

    PubMed Central

    YOO, Hyunju; KIM, Eunhye; HWANG, Seon-Ung; YOON, Junchul David; JEON, Yubyeol; PARK, Kyu-Mi; KIM, Kyu-Jun; JIN, Minghui; LEE, Chang-Kyu; LEE, Eunsong; KIM, Hyunggee; KIM, Gonhyung; HYUN, Sang-Hwan

    2016-01-01

    The ultrastructure of porcine putative embryonic stem cells and porcine fetal fibroblasts (PFFs) was analyzed by transmission electron microscopy. The aim of this study was to compare the features of organelles in in vitro fertilization (IVF) derived porcine embryonic stem cells (IVF-pESCs) and somatic cell nuclear transfer (SCNT) derived pESCs (SCNT-pESCs). Also, the features of organelles in high-passage IVF-pESCs were compared with those in low-passage cells. The ultrastructure of PFFs showed rare microvilli on the cell surfaces, polygonal or irregular nuclei with one to two reticular-shaped nucleoli and euchromatin, low cytoplasm-to-nucleus ratios, rare ribosomes, rare rough endoplasmic reticulum, elongated mitochondria, rich lysosomes and rich phagocytic vacuoles. IVF-pESCs showed rare microvilli on the cell surfaces, round or irregular nuclei with one to two reticular-shaped nucleoli and euchromatin, low cytoplasm-to-nucleus ratios, rich ribosomes, long stacks of rough endoplasmic reticulum, elongated mitochondria, rare lysosomes and rare autophagic vacuoles. By contrast, SCNT-pESCs showed rich microvilli with various lengths and frequencies on the cell surfaces, polygonal nuclei with one reticular shaped nucleoli and heterochromatin, high cytoplasm-to-nucleus ratios, rare ribosomes, rare rough endoplasmic reticulum, round mitochondria, rich lysosomes and rich phagocytic vacuoles with clear intercellular junctions. Furthermore, high-passage IVF-pESCs showed irregularly shaped colonies, pyknosis and numerous lysosomes associated with autophagic vacuoles showing signs of apoptosis. In conclusion, this study confirms that the ultrastructural characteristics of pESCs differ depending on their origin. These ultrastructural characteristics might be useful in biomedical research using pESCs, leading to new insights regarding regenerative medicine and tissue repair. PMID:26821870

  6. Recents patents for isolating, delivering and tracking adult stem cells in regenerative medicine.

    PubMed

    Fierabracci, Alessandra

    2010-06-01

    The field of regenerative medicine offers nowadays the potential to significantly impact a wide spectrum of healthcare issues, from insulin-dependent diabetes mellitus (Type 1 diabetes, T1D) to cardiovascular disease. In tissue engineering biomaterials, biological factors, regeneration competent cells are used in the process of creating functional tissue. Regarding the type of stem or progenitor cells which represents the best candidate for therapy, embryonic stem cells have been considered the master cells capable of differentiating into every type of cells either in vitro or in vivo, in spite of serious ethical concerns. Nevertheless experimental evidence suggests that adult stem cells and even terminally differentiated somatic cells under appropriate microenvironmental treatments can be reprogrammed and contribute to a much wider spectrum of differentiated progeny than previously anticipated. One of the main goals is to exploit novel technologies aiming to isolate, expand and enrich sources of regeneration competent cells, especially adult somatic stem cells. Researchers are also trying to develop innovative strategies for effectively delivering regenerative cell populations and to implement 'tracking' tools to verify their engraftment and destiny in vivo. Here we review recent patents on the field issued over the past five years.

  7. Parkinson's Disease in a Dish: What Patient Specific-Reprogrammed Somatic Cells Can Tell Us about Parkinson's Disease, If Anything?

    PubMed

    Drouin-Ouellet, J; Barker, R A

    2012-01-01

    Technologies allowing for the derivation of patient-specific neurons from somatic cells are emerging as powerful in vitro tools to investigate the intrinsic cellular pathological behaviours of the diseases that affect these patients. While the use of patient-derived neurons to model Parkinson's disease (PD) has only just begun, these approaches have allowed us to begin investigating disease pathogenesis in a unique way. In this paper, we discuss the advances made in the field of cellular reprogramming to model PD and discuss the pros and cons associated with the use of such cells.

  8. Disruption of exogenous eGFP gene using RNA-guided endonuclease in bovine transgenic somatic cells.

    PubMed

    Choi, WooJae; Yum, SooYoung; Lee, SongJeon; Lee, WonWu; Lee, JiHyun; Kim, SeokJoong; Koo, OkJae; Lee, ByeongChun; Jang, Goo

    2015-12-01

    Genome-editing technologies are considered to be an important tool for generating gene knockout cattle models. Here, we report highly efficient disruption of a chromosomally integrated eGFP gene in bovine somatic cells using RNA-guided endonucleases, a new class of programmable nucleases developed from a bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system. In the present study, we obtained homogenously eGFP-expressing primary fibroblasts from cloned bovine transgenic embryonic tissues and employed them for further analysis. CRISPR/Cas9 plasmids specifically targeting the eGFP gene were transfected into the eGFP fibroblasts by electroporation. After 10 days of culture, more than 40% of the cells had lost eGFP expression in fluorescence activated cell sorting (FACS) analysis. Targeted sequences of the transfected cells were analyzed, and various small indel mutations (6-203 bp deletions) in the target sequence were found. The fibroblasts mutated with the CRISPR/Cas9 system were applied for somatic cell nuclear transfer, and the reconstructed embryos were successfully developed into the blastocyst stage. In conclusion, the CRISPR/Cas9 system was successfully utilized in bovine cells and cloned embryos. This will be a useful technique to develop livestock transgenesis for agricultural science. PMID:25424059

  9. An ideal oocyte activation protocol and embryo culture conditions for somatic cell nuclear transfer using sheep oocytes.

    PubMed

    Patel, Hiren; Chougule, Shruti; Chohan, Parul; Shah, Naval; Bhartiya, Deepa

    2014-10-01

    Pluripotent stem cells are possibly the best candidates for regenerative medicine, and somatic cell nuclear transfer (SCNT) is one of the viable options to make patient-specific embryonic stem cells. Till date efficacy of SCNT embryos is very low and requires further improvement like ideal oocyte activation and in vitro culture system. The aim of the present study was to evaluate ideal oocyte activation using different stimulation protocols and to study the effect of cumulus co-culture conditions on embryo development. Results demonstrate that between electric stimulation and chemical stimulation using calcium ionomycin and ionophore, best oocyte activation was obtained using calcium ionomycin (5 microM for 5 min) which resulted in 83% cleavage followed by 7% of early blastocyst which further increased to 15% when a cumulus bed was also introduced during embryo culture. Sequential modified Charles Rosenkrans 2 (mCR2) medium was used for embryo culture in which glucose levels were increased from 1 mM to 5 mM from Day 3 onwards. SCNT using cumulus cells as donor somatic cell, calcium ionomycin to activate the reconstructed oocyte and embryo culture on a cumulus bed in sequential mCR2 medium, resulted in the development of 6% embryos to early blastocyst stage. Such technological advances will make SCNT a viable option to make patient-specific pluripotent stem cell lines in near future.

  10. Somatically Hypermutated Plasmodium-Specific IgM(+) Memory B Cells Are Rapid, Plastic, Early Responders upon Malaria Rechallenge.

    PubMed

    Krishnamurty, Akshay T; Thouvenel, Christopher D; Portugal, Silvia; Keitany, Gladys J; Kim, Karen S; Holder, Anthony; Crompton, Peter D; Rawlings, David J; Pepper, Marion

    2016-08-16

    Humoral immunity consists of pre-existing antibodies expressed by long-lived plasma cells and rapidly reactive memory B cells (MBC). Recent studies of MBC development and function after protein immunization have uncovered significant MBC heterogeneity. To clarify functional roles for distinct MBC subsets during malaria infection, we generated tetramers that identify Plasmodium-specific MBCs in both humans and mice. Long-lived murine Plasmodium-specific MBCs consisted of three populations: somatically hypermutated immunoglobulin M(+) (IgM(+)) and IgG(+) MBC subsets and an unmutated IgD(+) MBC population. Rechallenge experiments revealed that high affinity, somatically hypermutated Plasmodium-specific IgM(+) MBCs proliferated and gave rise to antibody-secreting cells that dominated the early secondary response to parasite rechallenge. IgM(+) MBCs also gave rise to T cell-dependent IgM(+) and IgG(+)B220(+)CD138(+) plasmablasts or T cell-independent B220(-)CD138(+) IgM(+) plasma cells. Thus, even in competition with IgG(+) MBCs, IgM(+) MBCs are rapid, plastic, early responders to a secondary Plasmodium rechallenge and should be targeted by vaccine strategies. PMID:27473412

  11. Adult stem cells in neural repair: Current options, limitations and perspectives.

    PubMed

    Mariano, Eric Domingos; Teixeira, Manoel Jacobsen; Marie, Suely Kazue Nagahashi; Lepski, Guilherme

    2015-03-26

    Stem cells represent a promising step for the future of regenerative medicine. As they are able to differentiate into any cell type, tissue or organ, these cells are great candidates for treatments against the worst diseases that defy doctors and researchers around the world. Stem cells can be divided into three main groups: (1) embryonic stem cells; (2) fetal stem cells; and (3) adult stem cells. In terms of their capacity for proliferation, stem cells are also classified as totipotent, pluripotent or multipotent. Adult stem cells, also known as somatic cells, are found in various regions of the adult organism, such as bone marrow, skin, eyes, viscera and brain. They can differentiate into unipotent cells of the residing tissue, generally for the purpose of repair. These cells represent an excellent choice in regenerative medicine, every patient can be a donor of adult stem cells to provide a more customized and efficient therapy against various diseases, in other words, they allow the opportunity of autologous transplantation. But in order to start clinical trials and achieve great results, we need to understand how these cells interact with the host tissue, how they can manipulate or be manipulated by the microenvironment where they will be transplanted and for how long they can maintain their multipotent state to provide a full regeneration.

  12. Enhanced homologous recombination is induced by alpha-particle radiation in somatic cells of Arabidopsis thaliana

    NASA Astrophysics Data System (ADS)

    Bian, Po; Liu, Ping; Wu, Yuejin

    Almost 9 percent of cosmic rays which strike the earth's atmosphere are alpha particles. As one of the ionizing radiations (IR), its biological effects have been widely studied. However, the plant genomic instability induced by alpha-particle radiation was not largely known. In this research, the Arabidopsis thaliana transgenic for GUS recombination substrate was used to evaluate the genomic instability induced by alpha-particle radiation (3.3MeV). The pronounced effects of systemic exposure to alpha-particle radiation on the somatic homologous recombination frequency (HRF) were found at different doses. The 10Gy dose of radiation induced the maximal HRF which was 1.9-fold higher than the control. The local radiation of alpha-particle (10Gy) on root also resulted in a 2.5-fold increase of somatic HRF in non-radiated aerial plant, indicating that the signal(s) of genomic instability was transferred to non-radiated parts and initiated their genomic instability. Concurrent treatment of seedlings of Arabidopsis thaliana with alpha-particle and DMSO(ROS scavenger) both in systemic and local radiation signifi- cantly suppressed the somatic HR, indicating that the free radicals produced by alpha-particle radiation took part in the production of signal of genomic instability rather than the signal transfer. Key words: alpha-particle radiation, somatic homologous recombination, genomic instability

  13. Invited review: effect of udder health management practices on herd somatic cell count.

    PubMed

    Dufour, S; Fréchette, A; Barkema, H W; Mussell, A; Scholl, D T

    2011-02-01

    A systematic review of the scientific literature on relationships between management practices used on dairy farms and herd somatic cell count (SCC) was undertaken to distinguish those management practices that have been consistently shown to be associated with herd SCC from those lacking evidence of association. Relevant literature was identified using a combination of database searches (PubMed, Medline, CAB, Agricola, and Web of Science) and iterative screening of references. To be included in the review, a manuscript had to be published after 1979 in French, English, or Dutch; study design had to be other than case report or case series; herds studied had to be composed of ≥ 40 milking cows producing on average ≥ 7,000kg of milk in 305 d; interventions studied had to be management practices applied at the herd level and used as udder health control strategies; and SCC had to be measured using electronic cell counting methods. The 36 manuscripts selected were mainly observational cross-sectional studies; 8 manuscripts dealt exclusively with automatic milking systems and 4 with management of calves and heifers and its effect on SCC in early lactation heifers. Most practices having consistent associations with SCC were related to milking procedures: wearing gloves during milking, using automatic take-offs, using postmilking teat dipping, milking problem cows last, yearly inspection of the milking system, and use of a technique to keep cows standing following milking; all were consistently associated with lower herd SCC. Other practices associated with lower SCC were the use of a freestall system, sand bedding, cleaning the calving pen after each calving, surveillance of dry-cow udders for mastitis, use of blanket dry-cow therapy, parenteral selenium supplementation, udder hair management, and frequent use of the California Mastitis Test. Regarding SCC of heifers, most of the consistent associations reported were related to interventions made during the

  14. Effects of injectable trace mineral supplementation in lactating dairy cows with elevated somatic cell counts.

    PubMed

    Ganda, E K; Bisinotto, R S; Vasquez, A K; Teixeira, A G V; Machado, V S; Foditsch, C; Bicalho, M; Lima, F S; Stephens, L; Gomes, M S; Dias, J M; Bicalho, R C

    2016-09-01

    Objectives of this clinical trial were to evaluate the effects of injectable trace mineral supplementation (ITMS) on somatic cell count (SCC), linear score (LS), milk yield, milk fat and protein contents, subclinical mastitis cure, and incidence of clinical mastitis in cows with elevated SCC. Holstein cows from a commercial dairy farm in New York were evaluated for subclinical mastitis, defined as SCC ≥200×10(3) cells/mL on the test day preceding enrollment. Cows with a history of treatment for clinical mastitis in the current lactation and those pregnant for more than 150d were not eligible for enrollment. Cows fitting inclusion criteria were randomly allocated to 1 of 2 treatment groups. Cows assigned to ITMS (n=306) received 1 subcutaneous injection containing zinc (300mg), manganese (50mg), selenium (25mg), and copper (75mg) at enrollment (d 0). Control cows (CTRL; n=314) received 1 subcutaneous injection of sterile saline solution. Following treatment, visual assessment of milk was performed daily, and cows with abnormal milk (i.e., presence of flakes, clots, or serous milk) were diagnosed with clinical mastitis (CM). Chronic clinical mastitis was defined as cows with 3 or more cases of CM. Milk yield, milk fat and protein contents, SCC, and LS were evaluated once monthly. Additionally, randomly selected animals were sampled to test serum concentrations of selected minerals on d0 and 30 (n=30 cows/treatment). Treatment did not affect serum concentrations of calcium, magnesium, phosphorus, potassium, copper, iron, manganese, selenium, and zinc on d30. Injectable supplementation with trace minerals did not improve overall cure of subclinical mastitis (CTRL=42.8 vs. ITMS=46.5%), although a tendency was observed in cows with 3 or more lactations (CTRL=27.1 vs. ITMS=40.0%). Supplementation did not reduce treatment incidence of CM (CTRL=48.2 vs. ITMS=41.7%); however, it tended to reduce the proportion of cows diagnosed with chronic CM (CTRL=16.9 vs. ITMS=12

  15. Primed Pluripotent Cell Lines Derived from Various Embryonic Origins and Somatic Cells in Pig

    PubMed Central

    Park, Jin-Kyu; Kim, Hye-Sun; Uh, Kyung-Jun; Choi, Kwang-Hwan; Kim, Hyeong-Min; Lee, Taeheon; Yang, Byung-Chul; Kim, Hyun-Jong; Ka, Hak-Hyun; Kim, Heebal; Lee, Chang-Kyu

    2013-01-01

    Since pluripotent embryonic stem cell (ESC) lines were first derived from the mouse, tremendous efforts have been made to establish ESC lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to maintain an ESC-like state in pluripotent porcine cell lines due to the frequent occurrence of spontaneous differentiation into an epiblast stem cell (EpiSC)-like state during culture. We have been able to derive EpiSC-like porcine ESC (pESC) lines from blastocyst stage porcine embryos of various origins, including in vitro fertilized (IVF), in vivo derived, IVF aggregated, and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells (piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) into porcine fibroblast cells. In this study, we analyzed characteristics such as marker expression, pluripotency and the X chromosome inactivation status in female of our EpiSC-like pESC lines along with our piPSC line. Our results show that these cell lines demonstrate the expression of genes associated with the Activin/Nodal and FGF2 pathways along with the expression of pluripotent markers Oct4, Sox2, Nanog, SSEA4, TRA 1–60 and TRA 1–81. Furthermore all of these cell lines showed in vitro differentiation potential, the X chromosome inactivation in female and a normal karyotype. Here we suggest that the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines. PMID:23326334

  16. Temporal trends in bulk tank somatic cell count and total bacterial count in Irish dairy herds during the past decade.

    PubMed

    Berry, D P; O'Brien, B; O'Callaghan, E J; Sullivan, K O; Meaney, W J

    2006-10-01

    The objective of this study was to document temporal trends in bulk tank somatic cell count (SCC) and total bacterial counts (TBC) in Irish dairy herds during the years 1994 to 2004. Three milk processors participated in the study, providing data on 2,754,270 individual bulk tank SCC and 2,056,992 individual bulk tank TBC records from 9,113 herds. Somatic cell counts decreased during the years 1994 to 2000, followed by an annual increase thereafter of more than 2,000 cells/mL. A tendency existed for TBC to decrease over time. Across all years, bulk tank SCC were the lowest in April and highest in November; TBC were the lowest in May and highest in December. The significant seasonal pattern observed in herd SCC and TBC was an artifact of seasonal calving in Ireland. In general, herds selling more milk had lower bulk tank SCC and TBC. Herds having the highest SCC (i.e., > 450,000 cells/mL) and the lowest SCC (i.e., < or = 150,000 cells/mL) both contributed substantially to the mean SCC of the milk pool collected by the milk processors. Derived transition matrices showed that between adjacent years, herds had the greatest probability of remaining in the same annual mean SCC or TBC category.

  17. Case Study: Somatic Sprouts and Halo-Like Amorphous Materials of the Purkinje Cells in Huntington's Disease.

    PubMed

    Sakai, Kenji; Ishida, Chiho; Morinaga, Akiyoshi; Takahashi, Kazuya; Yamada, Masahito

    2015-12-01

    We described a 63-year-old Japanese female with genetically confirmed Huntington's disease who showed unusual pathological findings in the cerebellum. This case exhibited typical neuropathological features as Huntington's disease, including severe degeneration of the neostriatum and widespread occurrence of ubiquitin and expanded polyglutamine-positive neuronal intranuclear and intracytoplasmic inclusions. The cerebellum was macroscopically unremarkable; however, somatic sprouts and halo-like amorphous materials of Purkinje cell with a large amount of torpedoes were noteworthy. Furthermore, the Purkinje cells were found to have granular cytoplasmic inclusions. Somatic sprouting is a form of degenerated Purkinje cell exhibited in