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Sample records for individual cloned mouse

  1. Cloning the laboratory mouse.

    PubMed

    Wakayama, T; Yanagimachi, R

    1999-06-01

    A brief account is given of early attempts to clone mammals (mice) by transferring cells (nuclei) of preimplantation embryos into enucleated oocytes, zygotes or blastomeres of two-cell embryos. This is followed by a brief review of recent successes using adult somatic cells: mammary gland cells for sheep, muscle cells for cattle and cumulus cells for mice. We have developed a technique for cloning the laboratory mouse by transferring cumulus cell nuclei into enucleated oocytes. With this technique, we have produced a population of over 80 cloned animals, and have carried the process over four generations. Development and fertility of these appear normal. However, the yield is very low; only approximately 1% of injected oocytes are carried to term. The challenge is now to understand the reason for this high loss. Is it a problem of technique, genomic reprogramming, somatic mutation, imprinting or incompatible cell cycle phases?

  2. Therapeutic cloning in the mouse.

    PubMed

    Mombaerts, Peter

    2003-09-30

    Nuclear transfer technology can be applied to produce autologous differentiated cells for therapeutic purposes, a concept termed therapeutic cloning. Countless articles have been published on the ethics and politics of human therapeutic cloning, reflecting the high expectations from this new opportunity for rejuvenation of the aging or diseased body. Yet the research literature on therapeutic cloning, strictly speaking, is comprised of only four articles, all in the mouse. The efficiency of derivation of embryonic stem cell lines via nuclear transfer is remarkably consistent among these reports. However, the efficiency is so low that, in its present form, the concept is unlikely to become widespread in clinical practice.

  3. Cloning and expression of mouse legumain, a lysosomal endopeptidase.

    PubMed Central

    Chen, J M; Dando, P M; Stevens, R A; Fortunato, M; Barrett, A J

    1998-01-01

    Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain. PMID:9742219

  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. Cloning, characterization and targeting of the mouse HEXA gene

    SciTech Connect

    Wakamatsu, N.; Trasler, J.M.; Gravel, R.A.

    1994-09-01

    The HEXA gene, encoding the {alpha} subunit of {beta}-hexosaminidase A, is essential for the metabolism of ganglioside G{sub M2}, and defects in this gene cause Tay-Sachs disease in humans. To elucidate the role of the gene in the nervous system of the mouse and to establish a mouse model of Tay-Sachs disease, we have cloned and characterized the HEXA gene and targeted a disruption of the gene in mouse ES cells. The mouse HEXA gene spans {approximately}26 kb and consists of 14 exons, similar to the human gene. A heterogeneous transcription initiation site was identified 21-42 bp 5{prime} of the initiator ATG, with two of the sites fitting the consensus CTCA (A = start) as seen for some weak initiator systems. Promoter analysis showed that the first 150 bp 5{prime} of the ATG contained 85% of promoter activity observed in constructs containing up to 1050 bp of 5{prime} sequence. The active region contained a sequence matching that of the adenovirus major late promoter upstream element factor. A survey of mouse tissues showed that the highest mRNA levels were in (max to min): testis (5.5 x brain cortex), adrenal, epididymis, heart, brain, lung, kidney, and liver (0.3 x brain cortex). A 12 kb BstI/SalI fragment containing nine exons was disrupted with the insertion of the bacterial neo{sup r} gene in exon 11 and was targeted into 129/Sv ES cells by homologous recombination. Nine of 153 G418 resistant clones were correctly targeted as confirmed by Southern blotting. The heterozygous ES cells were microinjected into mouse blastocysts and implanted into pseudo-pregnant mice. Nine male chimeric mice, showing that 40-95% chimerism for the 129/Sv agouti coat color marker, are being bred in an effort to generate germline transmission of the disrupted HEXA gene.

  6. Characterization of individual mouse cerebrospinal fluid proteomes

    SciTech Connect

    Smith, Jeffrey S.; Angel, Thomas E.; Chavkin, Charles; Orton, Daniel J.; Moore, Ronald J.; Smith, Richard D.

    2014-03-20

    Analysis of cerebrospinal fluid (CSF) offers key insight into the status of the central nervous system. Characterization of murine CSF proteomes can provide a valuable resource for studying central nervous system injury and disease in animal models. However, the small volume of CSF in mice has thus far limited individual mouse proteome characterization. Through non-terminal CSF extractions in C57Bl/6 mice and high-resolution liquid chromatography-mass spectrometry analysis of individual murine samples, we report the most comprehensive proteome characterization of individual murine CSF to date. Utilizing stringent protein inclusion criteria that required the identification of at least two unique peptides (1% false discovery rate at the peptide level) we identified a total of 566 unique proteins, including 128 proteins from three individual CSF samples that have been previously identified in brain tissue. Our methods and analysis provide a mechanism for individual murine CSF proteome analysis.

  7. Genetic heterogeneity of induced pluripotent stem cells: results from 24 clones derived from a single C57BL/6 mouse.

    PubMed

    Li, Cheng; Klco, Jeffery M; Helton, Nichole M; George, Daniel R; Mudd, Jacqueline L; Miller, Christopher A; Lu, Charles; Fulton, Robert; O'Laughlin, Michelle; Fronick, Catrina; Wilson, Richard K; Ley, Timothy J

    2015-01-01

    Induced pluripotent stem cells (iPSCs) have tremendous potential as a tool for disease modeling, drug testing, and other applications. Since the generation of iPSCs "captures" the genetic history of the individual cell that was reprogrammed, iPSC clones (even those derived from the same individual) would be expected to demonstrate genetic heterogeneity. To assess the degree of genetic heterogeneity, and to determine whether some cells are more genetically "fit" for reprogramming, we performed exome sequencing on 24 mouse iPSC clones derived from skin fibroblasts obtained from two different sites of the same 8-week-old C57BL/6J male mouse. While no differences in the coding regions were detected in the two parental fibroblast pools, each clone had a unique genetic signature with a wide range of heterogeneity observed among the individual clones: a total of 383 iPSC variants were validated for the 24 clones (mean 16.0/clone, range 0-45). Since these variants were all present in the vast majority of the cells in each clone (variant allele frequencies of 40-60% for heterozygous variants), they most likely preexisted in the individual cells that were reprogrammed, rather than being acquired during reprogramming or cell passaging. We then tested whether this genetic heterogeneity had functional consequences for hematopoietic development by generating hematopoietic progenitors in vitro and enumerating colony forming units (CFUs). While there was a range of hematopoietic potentials among the 24 clones, only one clone failed to differentiate into hematopoietic cells; however, it was able to form a teratoma, proving its pluripotent nature. Further, no specific association was found between the mutational spectrum and the hematopoietic potential of each iPSC clone. These data clearly highlight the genetic heterogeneity present within individual fibroblasts that is captured by iPSC generation, and suggest that most of the changes are random, and functionally benign.

  8. Genetic Heterogeneity of Induced Pluripotent Stem Cells: Results from 24 Clones Derived from a Single C57BL/6 Mouse

    PubMed Central

    Li, Cheng; Klco, Jeffery M.; Helton, Nichole M.; George, Daniel R.; Mudd, Jacqueline L.; Miller, Christopher A.; Lu, Charles; Fulton, Robert; O'Laughlin, Michelle; Fronick, Catrina; Wilson, Richard K.; Ley, Timothy J.

    2015-01-01

    Induced pluripotent stem cells (iPSCs) have tremendous potential as a tool for disease modeling, drug testing, and other applications. Since the generation of iPSCs “captures” the genetic history of the individual cell that was reprogrammed, iPSC clones (even those derived from the same individual) would be expected to demonstrate genetic heterogeneity. To assess the degree of genetic heterogeneity, and to determine whether some cells are more genetically “fit” for reprogramming, we performed exome sequencing on 24 mouse iPSC clones derived from skin fibroblasts obtained from two different sites of the same 8-week-old C57BL/6J male mouse. While no differences in the coding regions were detected in the two parental fibroblast pools, each clone had a unique genetic signature with a wide range of heterogeneity observed among the individual clones: a total of 383 iPSC variants were validated for the 24 clones (mean 16.0/clone, range 0–45). Since these variants were all present in the vast majority of the cells in each clone (variant allele frequencies of 40–60% for heterozygous variants), they most likely preexisted in the individual cells that were reprogrammed, rather than being acquired during reprogramming or cell passaging. We then tested whether this genetic heterogeneity had functional consequences for hematopoietic development by generating hematopoietic progenitors in vitro and enumerating colony forming units (CFUs). While there was a range of hematopoietic potentials among the 24 clones, only one clone failed to differentiate into hematopoietic cells; however, it was able to form a teratoma, proving its pluripotent nature. Further, no specific association was found between the mutational spectrum and the hematopoietic potential of each iPSC clone. These data clearly highlight the genetic heterogeneity present within individual fibroblasts that is captured by iPSC generation, and suggest that most of the changes are random, and functionally benign

  9. Rapid cloning of any rearranged mouse immunoglobulin variable genes

    SciTech Connect

    Dattamajumdar, A.K.; Jacobson, D.P.; Hood, L.E.; Osman, G.E.

    1996-12-31

    Immunoglobulins (Ig) have been the focus of extensive study for several decades and have become an important research area for immunologists and molecular biologists. The use of polymerase chain reaction (PCR) technology has accelerated the cloning, sequencing, and characterization of genes of the immune system. However, cloning and sequencing the Ig variable (V) genes using the PCR technology has been a challenging task, primarily due to the very diverse nature of Ig V region genes. We have developed a simple, rapid, and reproducible PCR-based technique to clone any rearranged mouse Ig heavy or light chain genes. A close examination of all Ig heavy and light chain V gene families has resulted in the design of 5{prime} and 3{prime} universal primers from regions that are highly conserved across all heavy or light chain V gene families, and the joining or constant regions, respectively. We present our strategy for designing universal primers for Ig V gene families. These primers were able to rapidly amplify the rearranged Ig V genes, belonging to diverse Ig V gene families from very different cell lines, i.e., J558, MOPC-21, 36-60, and a chicken ovalbumin specific B-cell hybridoma. In addition, the present study provides the complete alignment of nucleotide sequences of all heavy and light chain variable gene families. This powerful method of cloning Ig V genes, therefore, allows rapid and precise analysis of B-cell hybridomas, B-cell repertoire, and B-cell ontogeny. 55 refs., 5 figs., 2 tabs.

  10. Measurement of background translocation frequencies in individuals with clones

    SciTech Connect

    Wade, M.J.

    1996-08-01

    In the leukemia case the unseparated B and T lymphocytes had a high translocation frequency even after 0.0014, respectively. After purging all clones from the data, the translocation frequencies for Bio 8 and Bio 23 were 0.00750.0014 and 0.0073 metaphases were scored for chromosomal aberrations,, specifically reciprocal translocations, using fluorescence in situ hybridization (FISH). Metaphase spreads were used from two healthy, unexposed individuals (not exposed to radiation, chemotherapy or radiotherapy) and one early B- precursor acute lymphocytic leukemia (ALL) patient (metaphase spreads from both separated T lymphocytes and unseparated B and T lymphocytes were scored). All three individuals had an abnormally high translocation frequency. The high translocation frequencies resulted from clonal expansion of specific translocated chromosomes. I show in this thesis that by purging (discounting or removing) clones from the data of unexposed individuals, one can obtain true background translocation frequencies. In two cases, Bio 8 and Bio 23, the measured translocation frequency for chromosomes 1, 2 and 4 was 0.0124 purging all of the clones from the data. This high translocation frequency may be due to a low frequency of some clones and may not be recognized. The separated T lymphocytes had a higher translocation frequency than expected.

  11. Successful mouse cloning of an outbred strain by trichostatin A treatment after somatic nuclear transfer.

    PubMed

    Kishigami, Satoshi; Bui, Hong-Thuy; Wakayama, Sayaka; Tokunaga, Kenzo; Van Thuan, Nguyen; Hikichi, Takafusa; Mizutani, Eiji; Ohta, Hiroshi; Suetsugu, Rinako; Sata, Tetsutaro; Wakayama, Teruhiko

    2007-02-01

    Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, extremely low success rates have plagued this technique for a decade. Further in mice, the "clonable" strains have been limited to mainly hybrid F1 strains such as B6D2F1. Recently, we established a new efficient cloning technique using trichostatin A (TSA) which leads to a 2-5 fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. To further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4-5% success rates, which is comparable to 5-7% of B6D2F1. Thus, the TSA treatment is the first cloning technique to allow us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from hybrid strains, but also enables mouse cloning from normally "unclonable" strains.

  12. Placentomegaly in cloned mouse concepti caused by expansion of the spongiotrophoblast layer.

    PubMed

    Tanaka, S; Oda, M; Toyoshima, Y; Wakayama, T; Tanaka, M; Yoshida, N; Hattori, N; Ohgane, J; Yanagimachi, R; Shiota, K

    2001-12-01

    Hypertrophic placenta, or placentomegaly, has been reported in cloned cattle and mouse concepti, although their placentation processes are quite different from each other. It is therefore tempting to assume that common mechanisms underlie the impact of somatic cell cloning on development of the trophoblast cell lineage that gives rise to the greater part of fetal placenta. To characterize the nature of placentomegaly in cloned mouse concepti, we histologically examined term cloned mouse placentas and assessed expression of a number of genes. A prominent morphological abnormality commonly found among all cloned mouse placentas examined was expansion of the spongiotrophoblast layer, with an increased number of glycogen cells and enlarged spongiotrophoblast cells. Enlargement of trophoblast giant cells and disorganization of the labyrinth layer were also seen. Despite the morphological abnormalities, in situ hybridization analysis of spatiotemporally regulated placenta-specific genes did not reveal any drastic disturbances. Although repression of some imprinted genes was found in Northern hybridization analysis, it was concluded that this was mostly due to the reduced proportion of the labyrinth layer in the entire placenta, not to impaired transcriptional activity. Interestingly, however, cloned mouse fetuses appeared to be smaller than those of litter size-matched controls, suggesting that cloned mouse fetuses were under a latent negative effect on their growth, probably because the placentas are not fully functional. Thus, a major cause of placentomegaly is expansion of the spongiotrophoblast layer, which consequently disturbs the architecture of the layers in the placenta and partially damages its function.

  13. Cloning and expression of hepatic synaptotagmin 1 in mouse.

    PubMed

    Sancho-Knapik, Sara; Guillén, Natalia; Osada, Jesús

    2015-05-15

    Mouse hepatic synaptotagmin 1 (SYT1) cDNA was cloned, characterized and compared to the brain one. The hepatic transcript was 1807 bp in length, smaller than the brain, and only encoded by 9 of 11 gene exons. In this regard, 5'-and 3'-untranslated regions were 66 and 476 bp, respectively; the open reading frame of 1266 bp codified for a protein of 421 amino acids, identical to the brain, with a predicted molecular mass of 47.4 kDa and highly conserved across different species. Immunoblotting of protein showed two isoforms of higher molecular masses than the theoretical prediction based on amino acid sequence suggesting posttranslational modifications. Subcellular distribution of protein isoforms corresponded to plasma membrane, lysosomes and microsomes and was identical between the brain and liver. Nonetheless, the highest molecular weight isoform was smaller in the liver, irrespective of subcellular location. Quantitative mRNA tissue distribution showed that it was widely expressed and that the highest values corresponded to the brain, followed by the liver, spleen, abdominal fat, intestine and skeletal muscle. These findings indicate tissue-specific splicing of the gene and posttranslational modification and the variation in expression in the different tissues might suggest a different requirement of SYT1 for the specific function in each organ. PMID:25735570

  14. "Mouse Clone Model" for evaluating the immunogenicity and tumorigenicity of pluripotent stem cells.

    PubMed

    Zhang, Gang; Zhang, Yi

    2015-01-01

    To investigate the immune-rejection and tumor-formation potentials of induced pluripotent stem cells and other stem cells, we devised a model-designated the "Mouse Clone Model"-which combined the theory of somatic animal cloning, tetraploid complementation, and induced pluripotent stem cells to demonstrate the applicability of stem cells for transplantation therapy. PMID:26687081

  15. "Mouse Clone Model" for evaluating the immunogenicity and tumorigenicity of pluripotent stem cells.

    PubMed

    Zhang, Gang; Zhang, Yi

    2015-12-18

    To investigate the immune-rejection and tumor-formation potentials of induced pluripotent stem cells and other stem cells, we devised a model-designated the "Mouse Clone Model"-which combined the theory of somatic animal cloning, tetraploid complementation, and induced pluripotent stem cells to demonstrate the applicability of stem cells for transplantation therapy.

  16. Cloning the mouse homologue of the human lysosomal acid {alpha}-glucosidase gene

    SciTech Connect

    Ding, J.H.; Yang, B.Z.; Liu, H.M.

    1994-09-01

    Pompe disease (GSD II) is an autosomal recessive disorder caused by a deficiency of lysosomal acid {alpha}-glucosidase (GAA). In an attempt to create a mouse model for Pompe disease, we isolated and characterized the gene encoding the mouse homologue of the human GAA. Twenty clones that extend from exon 2 to the poly(A) tail were isolated from a mouse liver cDNA library, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-2 were cloned by RACE from mouse liver RNA. The full-length liver GAA cDNA contains 3365 nucleotides with a coding region of 2859 nucleotides and a 394 base pair 3{prime}-nontranslated region. The deduced amino acid sequence of the mouse GAA shows 84% identity to the human GAA. Southern blot analysis demonstrated that the mouse GAA was encoded by a single copy gene. Then six bacteriophages containing DNA from the GAA gene were isolated by screening 10{sup 6} phage plaques of a mouse 129 genomic library using a mouse GAA cDNA as a probe. From one of these bacteriophages, an 11-kilobase EcoRI fragment containing exons 3 to 15 was subcloned and sequenced. Work is in progress using this genomic clone to disrupt the GAA gene in murine embryonic stem cells in order to create GSD II mice.

  17. Cloning and sequence analysis of a cDNA clone coding for the mouse GM2 activator protein.

    PubMed Central

    Bellachioma, G; Stirling, J L; Orlacchio, A; Beccari, T

    1993-01-01

    A cDNA (1.1 kb) containing the complete coding sequence for the mouse GM2 activator protein was isolated from a mouse macrophage library using a cDNA for the human protein as a probe. There was a single ATG located 12 bp from the 5' end of the cDNA clone followed by an open reading frame of 579 bp. Northern blot analysis of mouse macrophage RNA showed that there was a single band with a mobility corresponding to a size of 2.3 kb. We deduce from this that the mouse mRNA, in common with the mRNA for the human GM2 activator protein, has a long 3' untranslated sequence of approx. 1.7 kb. Alignment of the mouse and human deduced amino acid sequences showed 68% identity overall and 75% identity for the sequence on the C-terminal side of the first 31 residues, which in the human GM2 activator protein contains the signal peptide. Hydropathicity plots showed great similarity between the mouse and human sequences even in regions of low sequence similarity. There is a single N-glycosylation site in the mouse GM2 activator protein sequence (Asn151-Phe-Thr) which differs in its location from the single site reported in the human GM2 activator protein sequence (Asn63-Val-Thr). Images Figure 1 PMID:7689829

  18. How to improve the success rate of mouse cloning technology.

    PubMed

    Thuan, Nguyen Van; Kishigami, Satoshi; Wakayama, Teruhiko

    2010-02-01

    It has now been 13 years since the first cloned mammal Dolly the sheep was generated from somatic cells using nuclear transfer (SCNT). Since then, this technique has been considered an important tool not only for animal reproduction but also for regenerative medicine. However, the success rate is still very low and the mechanisms involved in genomic reprogramming are not yet clear. Moreover, the NT technique requires donated fresh oocyte, which raises ethical problems for production of human cloned embryo. For this reason, the use of induced pluripotent stem cells for genomic reprogramming and for regenerative medicine is currently a hot topic in this field. However, we believe that the NT approach remains the only valid way for the study of reproduction and basic biology. For example, only the NT approach can reveal dynamic and global modifications in the epigenome without using genetic modification, and it can generate offspring from a single cell or even a frozen dead body. Thanks to much hard work by many groups, cloning success rates are increasing slightly year by year, and NT cloning is now becoming a more applicable method. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.

  19. How to improve the success rate of mouse cloning technology.

    PubMed

    Thuan, Nguyen Van; Kishigami, Satoshi; Wakayama, Teruhiko

    2010-02-01

    It has now been 13 years since the first cloned mammal Dolly the sheep was generated from somatic cells using nuclear transfer (SCNT). Since then, this technique has been considered an important tool not only for animal reproduction but also for regenerative medicine. However, the success rate is still very low and the mechanisms involved in genomic reprogramming are not yet clear. Moreover, the NT technique requires donated fresh oocyte, which raises ethical problems for production of human cloned embryo. For this reason, the use of induced pluripotent stem cells for genomic reprogramming and for regenerative medicine is currently a hot topic in this field. However, we believe that the NT approach remains the only valid way for the study of reproduction and basic biology. For example, only the NT approach can reveal dynamic and global modifications in the epigenome without using genetic modification, and it can generate offspring from a single cell or even a frozen dead body. Thanks to much hard work by many groups, cloning success rates are increasing slightly year by year, and NT cloning is now becoming a more applicable method. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications. PMID:20203432

  20. Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer

    SciTech Connect

    Kishigami, Satoshi . E-mail: kishigami@cdb.riken.jp; Mizutani, Eiji; Ohta, Hiroshi; Hikichi, Takafusa; Thuan, Nguyen Van; Wakayama, Sayaka; Bui, Hong-Thuy; Wakayama, Teruhiko

    2006-02-03

    The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently, we elucidated by using round spermatids that, after nuclear transfer, treatment of zygotes with trichostatin A (TSA), an inhibitor of histone deacetylase, can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions [S. Kishigami, N. Van Thuan, T. Hikichi, H. Ohta, S. Wakayama. E. Mizutani, T. Wakayama, Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids, Dev. Biol. (2005) in press]. Here, we found that 5-50 nM TSA-treatment for 10 h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells, spleen cells, neural stem cells, and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further, we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus, our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques.

  1. Mouse cloning using a drop of peripheral blood.

    PubMed

    Kamimura, Satoshi; Inoue, Kimiko; Ogonuki, Narumi; Hirose, Michiko; Oikawa, Mami; Yo, Masahiro; Ohara, Osamu; Miyoshi, Hiroyuki; Ogura, Atsuo

    2013-08-01

    Somatic cell nuclear transfer (SCNT) is a unique technology that produces cloned animals from single cells. It is desirable from a practical viewpoint that donor cells can be collected noninvasively and used readily for nuclear transfer. The present study was undertaken to determine whether peripheral blood cells freshly collected from living mice could be used for SCNT. We collected a drop of peripheral blood (15-45 μl) from the tail of a donor. A nucleated cell (leukocyte) suspension was prepared by lysing the red blood cells. Following SCNT using randomly selected leukocyte nuclei, cloned offspring were born at a 2.8% birth rate. Fluorescence-activated cell sorting revealed that granulocytes/monocytes and lymphocytes could be roughly distinguished by their sizes, the former being significantly larger. We then cloned putative granulocytes/monocytes and lymphocytes separately and obtained 2.1% and 1.7% birth rates, respectively (P > 0.05). Because the use of lymphocyte nuclei inevitably results in the birth of offspring with DNA rearrangements, we applied granulocyte/monocyte cloning to two genetically modified strains and two recombinant inbred strains. Normal-looking offspring were obtained from all four strains tested. The present study clearly indicated that genetic copies of mice could be produced using a drop of peripheral blood from living donors. This strategy will be applied to the rescue of infertile founder animals or a "last-of-line" animal possessing invaluable genetic resources.

  2. Introduction of cloned human papillomavirus genomes into mouse cells and expression at the RNA level.

    PubMed

    Brackmann, K H; Green, M; Wold, W S; Rankin, A; Loewenstein, P M; Cartas, M A; Sanders, P R; Olson, K; Orth, G; Jablonska, S; Kremsdorf, D; Favre, M

    1983-08-01

    The entire DNA genomes of five different human papillomaviruses (HPVs) were cloned into the BamHI site of pBR322 (HPV-1a, HPV-3, HPV-4, and HPV-9) or the EcoRI site of pBR325 (HPV-2), using as starting materials virus preparations isolated from papillomas of individual patients. Under stringent hybridization conditions (Tm-28 degrees), the five cloned HPVs exhibited less than 10% homology with one another. To establish model cell systems that may be useful for the identification of HPV genes and HPV gene products, mouse thymidine kinase negative (tk-) cells were cotransformed to the tk+ phenotype with the herpesvirus thymidine kinase gene and each of the five HPV cloned DNAs (either as intact recombinants or excised HPV DNA without removal of pBR). In most tk+ cell clones, a complex pattern of multiple high molecular weight inserts of HPV DNA were present in high copy number. Most of the HPV DNA sequences in the cotransformed cells were not present as unit-length episomal viral DNA. Analyses of the integration pattern (DNA blot) and RNA expression (RNA blot) of several HPV-1a and HPV-3 transformed cell lines suggest that some copies of the viral genome are integrated in a similar manner in different cell lines leading to the expression of identical viral RNA-containing species. Two of the cell lines transformed by the intact HPV-1a/pBR322 recombinant synthesized substantial amounts of four discrete viral polyadenylated cytoplasmic RNA species of 1.9, 3.2, 3.8, and 4.5 kb. Two cell lines transformed by the intact HPV-3/pBR322 recombinant synthesized 4-5 polyadenylated cytoplasmic viral RNA species ranging from 0.8 to 4.6 kb. The analysis shows that each viral RNA species appears to be a hybrid RNA molecule containing both HPV and pBR322 sequences. Based on these findings and the molecular organization of the HPV-1a genome (O. Danos, M. Katinka, and M. Yaniv (1982). EMBO J. 1, 231-237), it is possible that transcription of each of the HPV-1a RNA species is initiated

  3. Cloning and chromosome localization of the mouse Ews gene

    SciTech Connect

    Plougastel, B.; Thomas, G.; Delattre, O.; Mattei, M.G.

    1994-09-01

    The human EWS gene encodes a putative RNA binding protein. As a result of acquired chromosome rearrangement, the N-terminal portion of the EWS protein is fused to the DNA binding domain of either FLI1-or ERG in the Ewing family of tumors and to the DNA binding domain of ATF1 in malignant melanoma of soft parts. We have determined the cDNA sequence of the mouse Ews gene. Its nucleotide sequence and its translation product demonstrate 93 and 98% homology with the human EWS cDNA and protein, respectively. The murine Ews locus lies within a conserved synteny segment between human chromosome 22q12 and mouse chromosome 11A1-A3.

  4. Cloning, characterization, and tissue expression pattern of mouse Nma/BAMBI during odontogenesis.

    PubMed

    Knight, C; Simmons, D; Gu, T T; Gluhak-Heinrich, J; Pavlin, D; Zeichner-David, M; MacDougall, M

    2001-10-01

    Degenerate oligonucleotides to consensus serine kinase functional domains previously identified a novel, partial rabbit tooth cDNA (Zeichner-David et al., 1992) that was used in this study to identify a full-length mouse clone. A 1390-base-pair cDNA clone was isolated encoding a putative 260-amino-acid open reading frame containing a hydrophobic 25-amino-acid potential transmembrane domain. This clone shares some homology with the TGF-beta type I receptor family, but lacks the intracellular kinase domain. DNA database analysis revealed that this clone has 86% identity to a newly isolated human gene termed non-metastatic gene A and 80% identity to a Xenopus cDNA clone termed BMP and activin membrane bound inhibitor. Here we report the mouse Nma/BAMBI cDNA sequence, the tissue expression pattern, and confirmed expression in dental cell lines. This study demonstrates that Nma/BAMBI is a highly conserved protein across species and is expressed at high levels during odontogenesis.

  5. The neuronal nicotinic acetylcholine receptor {alpha}7 subunit gene: Cloning, mapping, structure, and targeting in mouse

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1994-09-01

    The neuronal nicotinic acetylcholine receptor {alpha}7 subunit is a member of a family of ligand-gated ion channels, and is the only subunit know to bind {alpha}-bungarotoxin in mammalian brain. {alpha}-Bungarotoxin binding sites are known to be more abundant in the hippocampus of mouse strains that are particularly sensitive to nicotine-induced seizures. The {alpha}7 receptor is highly permeable to calcium, which could suggest a role in synaptic plasticity in the nervous system. Auditory gating deficiency, an abnormal response to a second auditory stimulus, is characteristic of schizophrenia. Mouse strains that exhibit a similar gating deficit have reduced hippocampal expression of the {alpha}7 subunit. We have cloned and sequenced the full length cDNA for the mouse {alpha}7 gene (Acra-7) and characterized its gene structure. The murine {alpha}7 shares amino acid identity of 99% and 93% with the rat and human {alpha}7 subunits, respectively. Using an interspecies backcross panel, the murine gene was mapped to chromosome 7 near the p locus, a region syntenic with human chromosome 15; the human gene (CHRNA7) was confirmed to map to 15q13-q14 by FISH. To generate a mouse {alpha}7 mutant by homologous recombination, we have constructed a replacement vector which will delete transmembrane domains II-IV and the cytoplasmic domain from the gene product. Recombinant embryonic stem (ES) cell clones were selected and used to develop mouse chimeras that are currently being bred to obtain germline transmission.

  6. Cloning of a cGMP-gated cation channel from mouse kidney inner medullary collecting duct.

    PubMed

    Karlson, K H; Ciampolillo-Bates, F; McCoy, D E; Kizer, N L; Stanton, B A

    1995-05-24

    The cDNA sequence coding for the cGMP-gated cation channel expressed in the mouse kidney inner medullary collecting duct has been determined. The kidney cGMP-gated cation channel cDNA has an open reading frame of 2055 nucleotides and encodes a 685 amino acid protein. One cDNA clone is alternatively spliced thereby producing a deletion of 107 bp. Two differentially spliced 5' untranslated regions were determined by 5' RACE.

  7. Aberrant epigenetic reprogramming of imprinted microRNA-127 and Rtl1 in cloned mouse embryos

    SciTech Connect

    Cui Xiangshun; Zhang Dingxiao; Ko, Yoeung-Gyu; Kim, Nam-Hyung

    2009-02-06

    The microRNA (miRNA) genes mir-127 and mir-136 are located near two CpG islands in the imprinted mouse retrotransposon-like gene Rtl1, a key gene involved in placenta formation. These miRNAs appear to be involved in regulating the imprinting of Rtl1. To obtain insights into the epigenetic reprogramming of cloned embryos, we compared the expression levels of mir-127 and mir-136 in fertilized mouse embryos, parthenotes, androgenotes and cloned embryos developing in vitro. We also examined the DNA methylation status of the promoter regions of Rtl1 and mir-127 in these embryos. Our data showed that mir-127 and mir-136 were highly expressed in parthenotes, but rarely expressed in androgenotes. Interestingly, the expression levels of mir-127 and mir-136 in parthenotes were almost twice that seen in the fertilized embryos, but were much lower in the cloned embryos. The Rtl1 promoter region was hyper-methylated in blastocyst stage parthenotes (75.0%), moderately methylated (32.4%) in the fertilized embryos and methylated to a much lower extent ({approx}10%) in the cloned embryos. Conversely, the promoter region of mir-127 was hypo-methylated in parthenogenetically activated embryos (0.4%), moderately methylated (30.0%) in fertilized embryos and heavily methylated in cloned blastocysts (63-70%). These data support a role for mir-127 and mir-136 in the epigenetic reprogramming of the Rtl1 imprinting process. Analysis of the aberrant epigenetic reprogramming of mir-127 and Rtl1 in cloned embryos may help to explain the nuclear reprogramming procedures that occur in donor cells following somatic cell nuclear transfer (SCNT)

  8. Transgenic Mouse Models Enabling Photolabeling of Individual Neurons In Vivo

    PubMed Central

    Peter, Manuel; Bathellier, Brice; Fontinha, Bruno; Pliota, Pinelopi; Haubensak, Wulf; Rumpel, Simon

    2013-01-01

    One of the biggest tasks in neuroscience is to explain activity patterns of individual neurons during behavior by their cellular characteristics and their connectivity within the neuronal network. To greatly facilitate linking in vivo experiments with a more detailed molecular or physiological analysis in vitro, we have generated and characterized genetically modified mice expressing photoactivatable GFP (PA-GFP) that allow conditional photolabeling of individual neurons. Repeated photolabeling at the soma reveals basic morphological features due to diffusion of activated PA-GFP into the dendrites. Neurons photolabeled in vivo can be re-identified in acute brain slices and targeted for electrophysiological recordings. We demonstrate the advantages of PA-GFP expressing mice by the correlation of in vivo firing rates of individual neurons with their expression levels of the immediate early gene c-fos. Generally, the mouse models described in this study enable the combination of various analytical approaches to characterize living cells, also beyond the neurosciences. PMID:23626779

  9. Molecular cloning of the mouse CCK gene: expression in different brain regions and during cortical development.

    PubMed Central

    Vitale, M; Vashishtha, A; Linzer, E; Powell, D J; Friedman, J M

    1991-01-01

    In this paper we describe experiments that address specific issues concerning the regulation of the mouse cholecystokinin gene in brain and intestine. The mouse cholecystokinin gene was cloned and sequenced. Extensive homology among the mouse, man and rat genes was noted particularly in the three exons and the regions upstream of the RNA start site. RNAse protection assays for each of the three exons were used to demonstrate that CCK is expressed in only a subset of tissues and that the same cap site and splice choices are used in brain, intestine as well as in cerebellum, cortex, midbrain, hypothalamus and hippocampus. CCK RNA was also noted to be detectable in kidney. Thus the same gene using the same promoter is expressed in subsets of cells that differ in their biochemical, morphologic and functional characteristics. The level of expression of CCK was also monitored during mouse cortical development and the appearance of CCK RNA was compared to glutamate decarboxylase (GAD), enkephalin and somatostatin. It was noted that each of these cortical markers was first expressed at different times during cortical development. The appearance of CCK RNA during intestinal development was also measured and found to precede appearance in cortex by several days. Images PMID:2011497

  10. DNA Methylation Errors in Cloned Mouse Sperm by Germ Line Barrier Evasion.

    PubMed

    Koike, Tasuku; Wakai, Takuya; Jincho, Yuko; Sakashita, Akihiko; Kobayashi, Hisato; Mizutani, Eiji; Wakayama, Sayaka; Miura, Fumihito; Ito, Takashi; Kono, Tomohiro

    2016-06-01

    The germ line reprogramming barrier resets parental epigenetic modifications according to sex, conferring totipotency to mammalian embryos upon fertilization. However, it is not known whether epigenetic errors are committed during germ line reprogramming that are then transmitted to germ cells, and consequently to offspring. We addressed this question in the present study by performing a genome-wide DNA methylation analysis using a target postbisulfite sequencing method in order to identify DNA methylation errors in cloned mouse sperm. The sperm genomes of two somatic cell-cloned mice (CL1 and CL7) contained significantly higher numbers of differentially methylated CpG sites (P = 0.0045 and P = 0.0116). As a result, they had higher numbers of differentially methylated CpG islands. However, there was no evidence that these sites were transmitted to the sperm genome of offspring. These results suggest that DNA methylation errors resulting from embryo cloning are transmitted to the sperm genome by evading the germ line reprogramming barrier. PMID:27103445

  11. DNA Methylation Errors in Cloned Mouse Sperm by Germ Line Barrier Evasion.

    PubMed

    Koike, Tasuku; Wakai, Takuya; Jincho, Yuko; Sakashita, Akihiko; Kobayashi, Hisato; Mizutani, Eiji; Wakayama, Sayaka; Miura, Fumihito; Ito, Takashi; Kono, Tomohiro

    2016-06-01

    The germ line reprogramming barrier resets parental epigenetic modifications according to sex, conferring totipotency to mammalian embryos upon fertilization. However, it is not known whether epigenetic errors are committed during germ line reprogramming that are then transmitted to germ cells, and consequently to offspring. We addressed this question in the present study by performing a genome-wide DNA methylation analysis using a target postbisulfite sequencing method in order to identify DNA methylation errors in cloned mouse sperm. The sperm genomes of two somatic cell-cloned mice (CL1 and CL7) contained significantly higher numbers of differentially methylated CpG sites (P = 0.0045 and P = 0.0116). As a result, they had higher numbers of differentially methylated CpG islands. However, there was no evidence that these sites were transmitted to the sperm genome of offspring. These results suggest that DNA methylation errors resulting from embryo cloning are transmitted to the sperm genome by evading the germ line reprogramming barrier.

  12. [Cloning of mouse adam10 gene promoter and construction and identification of dual luciferase reporter system].

    PubMed

    Chen, Wei; Chen, Chong; Zhang, Huan-Xin; Cao, Jiang; Sang, Wei; Wu, Qing-Yun; Zhao, Kai; Zang, Yu; Zeng, Ling-Yu; Xu, Kai-Lin

    2012-06-01

    This study was aimed to clone mouse adam10 gene promoter and construct its dual luciferase report vector, and to investigate its transcriptional activity. Total DNA was extracted from mouse brain and used for amplifying the fragment containing adam10 gene promoter by PCR. The amplified product was inserted into pGL-4.10 vector to construct pGL4.10-adam10. The pGL4.10-adam10 and control plasmid pGL4.74 were co-transfected into HEK293 FT cells by lipofectamine 2000. The activity of adam10 gene promoter was assayed by luciferase system. The results showed that the recombinant plasmid pGL4.10-adam10 containing promoter of mouse adam10 was correctly constructed. The method was optimized by changing ratio of two plasmids. Moreover, the transcriptional activity of pGL4.10-adam10 stimulated by ionomycin increased. It is concluded that the dual luciferase reporter system is successfully established, which is useful in bioluminescence imaging technology in vitro. The effect of ionomycin can enhance the transcriptional activity of adam10 gene promoter.

  13. Cloning and mapping of the mouse {alpha}7-neuronal nicotinic acetylcholine receptor

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1995-03-20

    We report the isolation of cDNA clones for the mouse {alpha}7 neuronal nicotinic acetylcholine receptor subunit (gene symbol Acra7), the only nicotinic receptor subunit known to bind a-bungarotoxin in mammalian brain. This gene may have relevance to nicotine sensitivity and to some electrophysiologic findings in schizophrenia. The mouse {alpha}7 subunit gene encodes a protein of 502 amino acids with substantial identity to the rat (99.6%), human (92.8%), and chicken (87.5%) amino acid sequences. The {alpha}7 gene was mapped to mouse chromosome 7 near the p locus with the following gene order from proximal to distal: Myod1-3.5 {+-}1.7 cM-Gas2-0.9 cM {+-} 0.9 cM-D7Mit70-1.8 {+-} 1.2 cM- Acra7-4.4 {+-}1.0 cM-Hras1-ps11/Igf1r/Snrp2a. The human gene was confirmed to map to the homologous region of human chromosome 15q13-q14. 26 refs., 3 figs.

  14. Cloning

    MedlinePlus

    ... copies of whole animals Therapeutic cloning, which creates embryonic stem cells. Researchers hope to use these cells to grow healthy tissue to replace injured or diseased tissues in the human body. NIH: National Human Genome Research Institute

  15. A cDNA clone containing the entire coding sequence of a mouse H-2Kd histocompatibility antigen

    PubMed Central

    Lalanne, Jean-Louis; Delarbre, Christiane; Gachelin, Gabriel; Kourilsky, Philippe

    1983-01-01

    We have isolated a cDNA clone carrying a 1560 bp long insert which contains the entire coding and 3′ untranslated regions of an H-2Kd mouse histocompatibility antigen. Its sequence and overal features are described. They point to the existence of unique properties of DNA sequences associated with the H-2Kd antigen. PMID:6298749

  16. Cloning of the mouse GABA-benzodiazepine receptor. alpha. 1 subunit in a study of alcohol neurosensitivity

    SciTech Connect

    Keir, W.J.; Deitrich, R.A.; Sikela, J.M. )

    1989-02-09

    The inhibitory action of gamma amino butyric acid (GABA) is mediated by its binding to the benzodiazepine (BDZ) receptor and opening of a chloride channel. This receptor contains a variety of binding sites for several behavorially active drugs. Recent studies with SS and LS mice which were selected for differential neurosensitivity to ethanol, suggest that the GABAergic system plays a role in this differential sensitivity. Thus genes controlling the GABAergic system may also influence the acute hypnotic actions of ethanol. As a fist step towards verifying this hypothesis we have cloned and partially sequenced the mouse GABA-BDZ {alpha}1 subunit cDNA using a 40 bp oligonucleotide derived from the N terminus of a published bovine {alpha} subunit cDNA. A positive clone from a mouse brain cDNA library was identified and contains an insert of approximately 2.5 Kb. Partial sequence analysis indicates that this clone corresponds to the mouse homolog of the {alpha}1 subunit of the GABA-BDZ receptor. This clone is being used as a probe to identify restriction fragment length polymorphisms in several mouse genotypes which differ in their neurosensitivity to ethanol in an attempt to identify molecular genetic changes in the GABA-BDZ receptor that are related to differential ethanol neurosensitivity.

  17. Cloning and optimal Gaussian individual attacks for a continuous-variable quantum key distribution using coherent states and reverse reconciliation

    SciTech Connect

    Namiki, Ryo; Koashi, Masato; Imoto, Nobuyuki

    2006-03-15

    We investigate the security of continuous-variable quantum key distribution using coherent states and reverse reconciliation against Gaussian individual attacks based on an optimal Gaussian 1{yields}2 cloning machine. We provide an implementation of the optimal Gaussian individual attack. We also find a Bell-measurement attack which works without delayed choice of measurements and has better performance than the cloning attack.

  18. [Analysis of individual changes in heat resistance of clones of Daphnia magna at the initial stages of cultivation].

    PubMed

    Mironova, A P

    2012-01-01

    30 clones of water fleas have been studied on the basis of heat resistance (HR) of these organisms at the initial stages of cultivation. Ten clones died before the appearance of the offspring in F2; the remaining clones were multiplied successfully and produced subsequent generations. Based on the change in the individual HR level of the clones in F1 as compared with ancestors, it was concluded a violation of the mechanisms of physiological homeostasis in the population of dead clones. In the remaining clones, there were a high negative correlation between the initial individual resistance level and its change in F1 and F2 and a pronounced narrowing of the variability diapason in F1. In this animal group, the significant lability of individual-level resistance was observed as well as its ability to regulate the shift of its resistance, which provided an efficient work of the mechanisms of physiological homeostasis. PMID:23461033

  19. Cloning and expression of a murine fascin homolog from mouse brain.

    PubMed

    Edwards, R A; Herrera-Sosa, H; Otto, J; Bryan, J

    1995-05-01

    The fascins are a widely distributed family of proteins that organize filamentous actin into bundles. We have cloned, sequenced, and expressed the murine homolog. Fascin is most abundant in brain and is found in other tissues including uterus and spleen. The deduced open reading frame encodes a protein of 493 amino acids with a molecular mass of 54,412 Da. Previous solubility problems with bacterially expressed fascins were overcome by producing the mouse protein as a fusion with Escherichia coli thioredoxin. A method for cleaving the fusion protein and for purifying active recombinant fascin is described. The N-terminal sequence and molecular mass estimated on SDS gels indicate that recombinant fascin is full-length. Two-dimensional gel electrophoresis suggests that recombinant fascin is post-translationally modified in a manner similar to that observed in mouse brain. Recombinant fascin and the fusion protein are recognized by monoclonal anti-fascin antibodies and will bundle rabbit skeletal muscle F-actin in vitro at a stoichiometry of 4.1:1 actin to fascin. Electron cryomicroscopy images show that the reconstituted bundles are highly ordered. However, their fine structure differs from that of echinoid fascin-actin bundles. This structural difference can be attributed to fascin.

  20. Cloning and partial characterization of the mouse glutamine:fructose-6-phosphate amidotransferase (GFAT) gene promoter.

    PubMed Central

    Sayeski, P P; Wang, D; Su, K; Han, I O; Kudlow, J E

    1997-01-01

    Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the enzyme that is rate limiting in the synthesis of glucosamine and hexosamines. Glucosamine has been proposed to contribute to the glucotoxicity of diabetes. Evidence that the gene encoding GFAT is transcriptionally regulated prompted us to clone and characterize its promoter. The position of the mouse GFAT promoter relative to the translational start site was located by primer extension and found to be 149 bp upstream of the translational start site. A 1.9 kb SacI fragment of the GFAT gene was found to contain the promoter and 88 bp of sequence downstream of the transcriptional start site. This promoter segment could drive expression of a luciferase reporter gene, could confer correct transcriptional initiation to the reporter and could confer the EGF-responsiveness previously observed in the native gene. The mouse GFAT promoter lacks a canonical TATA box and has several GC boxes within a highly GC-rich region. Deletional analysis of the promoter indicated that a proximal element extending to -120 relative to the transcriptional start site could confer reporter expression at a level of 57% of the 1.9 kb construct. Detailed analysis of this proximal region by DNase I footprinting, electrophoretic mobility shift assays and site-directed mutagenesis indicated that Sp1 binds to three elements in this proximal promoter segment and plays a vital role in regulation of transcription from this gene. PMID:9060444

  1. Molecular cloning, functional expression, and chromosomal localization of mouse hepatocyte nuclear factor 1.

    PubMed Central

    Kuo, C J; Conley, P B; Hsieh, C L; Francke, U; Crabtree, G R

    1990-01-01

    The homeodomain-containing transcription factor hepatocyte nuclear factor 1 (HNF-1) most likely plays an essential role during liver organogenesis by transactivating a family of greater than 15 predominantly hepatic genes. We have isolated cDNA clones encoding mouse HNF-1 and expressed them in monkey COS cells and in the human T-cell line Jurkat, producing HNF-1 DNA-binding activity as well as transactivation of reporter constructs containing multimerized HNF-1 binding sites. In addition, the HNF-1 gene was assigned by somatic cell hybrids and recombinant inbred strain mapping to mouse chromosome 5 near Bcd-1 and to human chromosome 12 region q22-qter, revealing a homologous chromosome region in these two species. The presence of HNF-1 mRNA in multiple endodermal tissues (liver, stomach, intestine) suggests that HNF-1 may constitute an early marker for endodermal, rather than hepatocyte, differentiation. Further, that HNF-1 DNA-binding and transcriptional activity can be conferred by transfecting the HNF-1 cDNA into several cell lines indicates that it is sufficient to activate transcription in the context of ubiquitously expressed factors. Images PMID:2263635

  2. CLoNe is a new method to target single progenitors and study their progeny in mouse and chick.

    PubMed

    García-Moreno, Fernando; Vasistha, Navneet A; Begbie, Jo; Molnár, Zoltán

    2014-04-01

    Cell lineage analysis enables us to address pivotal questions relating to: the embryonic origin of cells and sibling cell relationships in the adult body; the contribution of progenitors activated after trauma or disease; and the comparison across species in evolutionary biology. To address such fundamental questions, several techniques for clonal labelling have been developed, each with its shortcomings. Here, we report a novel method, CLoNe that is designed to work in all vertebrate species and tissues. CLoNe uses a cocktail of labelling, targeting and transposition vectors that enables targeting of specific subpopulations of progenitor types with a combination of fluorophores resulting in multifluorescence that describes multiple clones per specimen. Furthermore, transposition into the genome ensures the longevity of cell labelling. We demonstrate the robustness of this technique in mouse and chick forebrain development, and show evidence that CLoNe will be broadly applicable to study clonal relationships in different tissues and species.

  3. CLoNe is a new method to target single progenitors and study their progeny in mouse and chick.

    PubMed

    García-Moreno, Fernando; Vasistha, Navneet A; Begbie, Jo; Molnár, Zoltán

    2014-04-01

    Cell lineage analysis enables us to address pivotal questions relating to: the embryonic origin of cells and sibling cell relationships in the adult body; the contribution of progenitors activated after trauma or disease; and the comparison across species in evolutionary biology. To address such fundamental questions, several techniques for clonal labelling have been developed, each with its shortcomings. Here, we report a novel method, CLoNe that is designed to work in all vertebrate species and tissues. CLoNe uses a cocktail of labelling, targeting and transposition vectors that enables targeting of specific subpopulations of progenitor types with a combination of fluorophores resulting in multifluorescence that describes multiple clones per specimen. Furthermore, transposition into the genome ensures the longevity of cell labelling. We demonstrate the robustness of this technique in mouse and chick forebrain development, and show evidence that CLoNe will be broadly applicable to study clonal relationships in different tissues and species. PMID:24644261

  4. CLoNe is a new method to target single progenitors and study their progeny in mouse and chick

    PubMed Central

    García-Moreno, Fernando; Vasistha, Navneet A.; Begbie, Jo; Molnár, Zoltán

    2014-01-01

    Cell lineage analysis enables us to address pivotal questions relating to: the embryonic origin of cells and sibling cell relationships in the adult body; the contribution of progenitors activated after trauma or disease; and the comparison across species in evolutionary biology. To address such fundamental questions, several techniques for clonal labelling have been developed, each with its shortcomings. Here, we report a novel method, CLoNe that is designed to work in all vertebrate species and tissues. CLoNe uses a cocktail of labelling, targeting and transposition vectors that enables targeting of specific subpopulations of progenitor types with a combination of fluorophores resulting in multifluorescence that describes multiple clones per specimen. Furthermore, transposition into the genome ensures the longevity of cell labelling. We demonstrate the robustness of this technique in mouse and chick forebrain development, and show evidence that CLoNe will be broadly applicable to study clonal relationships in different tissues and species. PMID:24644261

  5. Cloning of DNA from double minutes of Y1 mouse adrenocortical tumor cells: evidence for gene amplification.

    PubMed

    George, D L; Powers, V E

    1981-04-01

    We have isolated a metaphase chromosome fraction highly enriched in double minutes (dm) from a mouse adrenocortical tumor cell line (Y1-DM). We have cloned DNA from this dm-enriched fraction in the lambda vector Charon 4A, and have characterized two randomly chosen recombinant bacteriophage clones from this dm DNA library. When 32 P-labeled DNA from each recombinant was hybridized to Southern blots of restriction endonuclease-digested DNA from different mouse cell lines, large differences were seen in the intensity of the resulting autoradiographic images, depending on the source of the genomic DNA. A very strong signal was obtained with DNA from the Y1-DM cells and with DNA from a related Y1 subline that lacks dm but contains a marker chromosome bearing a large homogeneously staining region (HSR). Hybridization to DNA from parental inbred mice and from two unrelated mouse cell lines produced a significantly weaker signal than that obtained with DNA from the Y1 cells, but the DNA fragments from the sources were of similar size. Based on results from filter hybridization analysis, we estimate that sequences homologous to the cloned fragments are approximately 100- to 200-fold more abundant in the genome of the Y1-DM cells than in the parental mouse cells. The data are consistent with the hypothesis that dm and HSRs in these cells contain amplified genes.

  6. Uroporphyrinogen-III synthase: Molecular cloning, nucleotide sequence, expression of a mouse full-length cDNA, and its localization on mouse chromosome 7

    SciTech Connect

    Xu, W.; Desnick, R.J.; Kozak, C.A.

    1995-04-10

    Uroporphyrinogen-III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for the conversion of hydroxymethylbilane to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-S is the enzymatic defect in congenital erythropoietic porphyria (CEP), an autosomal recessive disorder. For the generation of a mouse model of CEP, the human URO-S cDNA was used to screen 2 X 10{sup 6} recombinants from a mouse adult liver cDNA library. Ten positive clones were isolated, and dideoxy sequencing of the entire 1.6-kb insert of clone pmUROS-1 revealed 5{prime} and 3{prime} untranslated sequences of 144 and 623 bp, respectively, and an open reading frame of 798 bp encoding a 265-amino-acid polypeptide with a predicted molecular mass of 28,501 Da. The mouse and human coding sequences had 80.5 and 77.8% nucleotide and amino acid identity, respectively. The authenticity of the mouse cDNA was established by expression of the active monomeric enzyme in Escherichia coli. In addition, the analysis of two multilocus genetic crosses localized the mouse gene on chromosome 7, consistent with the mapping of the human gene to a position of conserved synteny on chromosome 10. The isolation, expression, and chromosomal mapping of this full-length cDNA should facilitate studies of the structure and organization of the mouse genomic sequence and the development of a mouse model of CEP for characterization of the disease pathogenesis and evaluation of gene therapy. 38 refs., 1 tab.

  7. Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation.

    PubMed

    Su, W; Shmukler, B E; Chernova, M N; Stuart-Tilley, A K; de Franceschi, L; Brugnara, C; Alper, S L

    1999-11-01

    Although K-Cl cotransporter (KCC1) mRNA is expressed in many tissues, K-Cl cotransport activity has been measured in few cell types, and detection of endogenous KCC1 polypeptide has not yet been reported. We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flanking genomic regions and mapped the mKCC1 gene to chromosome 8. Three anti-peptide antibodies raised against recombinant mKCC1 function as immunoblot and immunoprecipitation reagents. The tissue distributions of mKCC1 mRNA and protein are widespread, and mKCC1 RNA is constitutively expressed during erythroid differentiation of ES cells. KCC1 polypeptide or related antigen is present in erythrocytes of multiple species in which K-Cl cotransport activity has been documented. Erythroid KCC1 polypeptide abundance is elevated in proportion to reticulocyte counts in density-fractionated cells, in bleeding-induced reticulocytosis, in mouse models of sickle cell disease and thalassemia, and in the corresponding human disorders. mKCC1-mediated uptake of (86)Rb into Xenopus oocytes requires extracellular Cl(-), is blocked by the diuretic R(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2, 3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling, N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.

  8. Cloning of immunoglobulin kappa light chain genes from mouse liver and myeloma MOPC 173.

    PubMed

    Steinmetz, M; Zachau, H G; Mach, B

    1979-07-25

    The organization of the kappa chain constant region gene was compared in DNA from an immunoglobulin-producing mouse myeloma (MOPC 173) and from liver. In situ hybridization using the Southern blotting technique revealed constant region gene-containing EcoRI-DNA fragments of 14 and 20 kb in the myeloma tissue whereas one EcoRI-DNA fragment with a length of 15 kb was found in liver DNA. After enrichment by RPC-5 chromatography and preparative electrophoresis the 14 kb fragment from MOPC 173 DNA and the 15 kb fragment from liver DNA were cloned in the bacteriophage lambda vector Charon 4A using in vitro packaging. Extensive characterization of the two fragments by restriction endonuclease mapping, in situ hybridization, and electron microscopy (R-loop and heteroduplex) showed that both fragments contain the constant region but no MOPC 173 variable region gene. Both fragments are homologous over a length of 12.5 kb including the constant region but differ from one another starting about 2.7 kb from the 5' end of the constant region gene. This indicates that the 14 kb EcoRI-DNA fragment from the myeloma tissue clearly resulted from somatic DNA rearrangement although it does not seem to carry the MOPC 173 variable region gene. These observations suggest that somatic DNA rearrangement of immunoglobulin light chain genes can involve both homologous chromosomes.Images

  9. Transgenic mouse model of hemifacial microsomia: Cloning and characterization of insertional mutation region on chromosome 10

    SciTech Connect

    Naora, Hiroyuki; Otani, Hiroki; Tanaka, Osamu

    1994-10-01

    The 643 transgenic mouse line carries an autosomal dominant insertional mutation that results in hemifacial microsomia (HFM), including microtia and/or abnormal biting. In this paper, we characterize the transgene integration site in transgenic mice and preintegration site of wildtype mice. The locus, designated Hfm (hemifacial microsomia-associated locus), was mapped to chromosome 10, B1-3, by chromosome in situ hybridization. We cloned the transgene insertion site from the transgenic DNA library. By using the 5{prime} and 3{prime} flanking sequences, the preintegration region was isolated. The analysis of these regions showed that a deletion of at least 23 kb DNA occurred in association with the transgene integration. Evolutionarily conserved regions were detected within and beside the deleted region. The result of mating between hemizygotes suggests that the phenotype of the homozygote is lethality in the prenatal period. These results suggests that the Hfm locus is necessary for prenatal development and that this strain is a useful animal model for investigating the genetic predisposition to HFM in humans.

  10. Isolation of cDNA clones specifying the fourth component of mouse complement and its isotype, sex-limited protein.

    PubMed Central

    Nonaka, M; Takahashi, M; Natsuume-Sakai, S; Nonaka, M; Tanaka, S; Shimizu, A; Honjo, T

    1984-01-01

    cDNA clones specific for the fourth component of mouse complement (C4) and its hormonally regulated isotype, sex-linked protein (Slp), were isolated using as a probe a 20-mer synthetic oligonucleotide corresponding to a known sequence of human C4 cDNA. Two types of clones, one specific for C4 (pFC4/10, with a 3.7 kilobase insert) and one specific for Slp (pFSlp/1, with a 4.7 kilobase insert), were isolated from liver cDNA libraries constructed from the Slp-producing FM mouse strain. The cDNA inserts of these clones shared 70% of the restriction sites determined. Only one type of clone was isolated from the Slp-negative DBA/1 strain; this type showed restriction maps indistinguishable from that of pFC4/10. pFC4/10 and pFSlp/1 displayed extensive homology: 94% nucleotide homology and 89% derived amino acid homology in the C4a region and 92% nucleotide homology and 89% derived amino acid homology in the thiol-ester region. An Arg-Gln-Lys-Arg sequence in the beta-alpha junction and a Cys-Ala-Glu-Gln sequence in the thiol-ester site were identified for both proteins. A remarkable divergency between C4 and Slp sequences was recognized in the region immediately following the C4a sequence. PMID:6208559

  11. Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells.

    PubMed

    Ono, Yukiko; Kono, Tomohiro

    2006-08-01

    Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as

  12. Cloning, structure and expression of cDNA for mouse contrapsin and a related protein.

    PubMed Central

    Ohkubo, K; Ogata, S; Misumi, Y; Takami, N; Sinohara, H; Ikehara, Y

    1991-01-01

    A cDNA clone (lambda MC-2) for contrapsin, a serine-proteinase inhibitor, was isolated from a lambda ZAP mouse liver cDNA library. The 1.6 kb cDNA insert of lambda MC-2 contained an open reading frame that encodes a 418-residue polypeptide (46,970 Da), in which a signal peptide of 21 residues was identified by comparison with the N-terminal sequence of the purified protein. The predicted structure (MC-2) also contained other peptide sequences determined by Edman degradation. Four potential sites for N-linked glycosylation were found in the molecule, accounting for the difference in molecular mass between the predicted form and the purified protein (63 kDa). Further screening of the cDNA library with an EcoRI-EcoRI fragment (510 bp) of lambda MC-2 as a probe yielded another cDNA clone (lambda MC-7), which encodes a 418-residue polypeptide (MC-7) with a calculated mass of 47,010 Da. MC-2 showed 83% similarity at the amino acid level to MC-7, in contrast with 44% similarity to alpha 1-proteinase inhibitor. The possible reactive site (P1-P'1) for serine proteinase is suggested to be Lys-Ala for MC-2 and Ser-Arg for MC-7. Northern-blot analysis revealed that both MC-2 and MC-7 mRNAs have the same size of 1.8 kb and are markedly induced in response to acute inflammation. Construction of the expression plasmids pSVMC-2 and pSVMC-7 and their transfection into COS-1 cells demonstrated that pSVMC-2 directs the synthesis of a 63 kDa form whereas pSVMC-7 expresses a 56 kDa form. The difference in molecular mass between the two may be explained by the fact that the MC-7 sequence contains three potential sites for N-glycosylation, one site less than that of MC-2. Images Fig. 4. Fig. 5. PMID:2049065

  13. Cloning, characterisation and bacterial expression of full length cDNA for the mouse liver microsomal glutathione S-transferase.

    PubMed

    Raza, H; Mullick, J; John, A; Bhagwat, S V; Avadhani, N G

    2000-01-01

    We have isolated a cDNA encoding full length microsomal glutathione S-transferase (MGST) from mouse liver. The cDNA was isolated by RT-PCR using primers designed from published cDNA sequence of rat MGST with the addition of 5' Nde-1 and 3' HindIII sites, and cloned into bacterial expression vector pSP19T7LT. Deduced amino acid sequence (155 amino acids, calculated mol.mass 17512 Dalton) confirmed the identity of microsomal GST from mouse liver which has sequence homology with that of rat and human liver MGST1. Recombinant GST cDNA (Genbank accession # 159050) was expressed in BL21(DE3) in the presence of 1 mM IPTG at 30 degrees C. The expressed GST protein was found to be localised in the bacterial membrane as determined by measuring catalytic activity using CDNB and cumene hydroperoxide substrates, SDS-PAGE and Western blot analysis. We have demonstrated the cloning and expression of full length cDNA for MGST from mouse liver and have characterised the functionally active product as MGST protein. These results should facilitate studies on the role of MGST in the regulation of chemical carcinogenesis and in the prevention of oxidative stress caused by endogenous and exogenous chemicals.

  14. Physical map of mouse Chromosome 17 in the region relevant for positional cloning of the hybrid sterility 1 gene

    SciTech Connect

    Trachtulec, Z.; Vincek, V.; Hamvas, R.M.J.

    1994-09-01

    Hybrid sterility 1 (Hst1) is the major gene responsible for sterility of male hybrids between Mus musculus and certain laboratory strains. Thus, Hst1 is of importance in studying both postreproductive isolation of closely related species and male fertility. It has been mapped to mouse chromosome 17 in the region corresponding to the third inversion of the t haplotypes. The aim of the present study was to construct a physical map of the Hst1 region as the first step in an effort to clone the gene. Three yeast artificial chromosome (YAC) libraries (Princeton, Whitehead, and ICRF) were screened with polymerase chain reaction (PCR) oligonucleotide primers and DNA probes specific for loci previously mapped into the region of the third inversion. The isolated YAC clones were restriction mapped and arranged into contigs. Sixteen YAC clones were arranged into a single contig encompassing a region approximately 2000 kb long based on restriction mapping of highly overlapping but independently derived YAC clones. Five new loci in the region of the third inversion were mappd and the order and approximate physical distances of 12 loci established in this contig. The Hst1 gene maps approximately 0.2 cM proximal to the D17Ph1 locus encompassed by the YAC contig. Since the contig extends at least 1200 kb proximal to D17Ph1, it should contain the Hst1 gene.

  15. Molecular cloning, functional expression and pharmacological characterization of a mouse melanocortin receptor gene.

    PubMed Central

    Desarnaud, F; Labbe, O; Eggerickx, D; Vassart, G; Parmentier, M

    1994-01-01

    We describe the cloning of the mouse HGMP01A gene that encodes a melanocortin receptor functionally distinct from the adrenal cortex corticotropin (adrenocorticotrophic hormone; ACTH) receptor and the melanocyte-stimulating hormone (MSH) receptor expressed in melanoma. The gene encodes a protein of 323 amino acids with a calculated molecular mass of 35,800 Da, displaying potential sites for N-linked glycosylation and phosphorylation by protein kinase C. An RNAase protection assay detected weak expression in the brain, but not in adrenal gland, skin, or any of the other tissues tested. Stable CHO cell lines expressing over 100,000 receptors per cell were generated. The recombinant receptor binds iodinated [Nle4,D-Phe7]alpha-MSH (NDP-MSH) with an apparent Kd of 700 pM. Displacement of the ligand by a variety of pro-opiomelanocortin-derived peptides revealed a pharmacological profile distinct from that of the classical ACTH and MSH receptors. NDP-MSH was the most powerful competitor (IC50 1.4 nM), followed by gamma-MSH (IC50 7 nM). alpha-MSH, beta-MSH and ACTH-(1-39) were significantly less potent, with IC50 values of 30, 19 and 21 nM respectively. ACTH-(4-10) was poorly active (IC50 2.4 microM), while corticotropin-like intermediate lobe peptide (CLIP) and beta-endorphin were totally ineffective. The recombinant receptor was found to stimulate adenylate cyclase. The potency order of the agonists in this assay was consistent with that of the binding displacement assays. This receptor represents the orthologue of the human melanocortin 3 receptor reported recently. The growing family of melanocortin receptors constitute the molecular basis for the variety of actions of melanocortins that have been described over the years. The availability of functionally expressed receptors from the melanocortin family will allow the development of a specific pharmacology, and a better understanding of the function of the pro-opiomelanocortin-derived peptides. Images Figure 6 PMID

  16. One Mouse per Child: Interpersonal Computer for Individual Arithmetic Practice

    ERIC Educational Resources Information Center

    Alcoholado, C.; Nussbaum, M.; Tagle, A.; Gomez, F.; Denardin, F.; Susaeta, H.; Villalta, M.; Toyama, K.

    2012-01-01

    Single Display Groupware (SDG) allows multiple people in the same physical space to interact simultaneously over a single communal display through individual input devices that work on the same machine. The aim of this paper is to show how SDG can be used to improve the way resources are used in schools, allowing students to work simultaneously on…

  17. Molecular cloning and characterization of human WINS1 and mouse Wins2, homologous to Drosophila segment polarity gene Lines (Lin).

    PubMed

    Katoh, Masaru

    2002-08-01

    WNT signaling molecules play key roles in carcinogenesis and embryogenesis. Drosophila segment polarity gene Lines (Lin) is essential for Wnt/Wingless-dependent patterning in dorsal epidermis and also for hindgut development. With Wnt signaling, Lin accumulates in the nucleus to modulate transcription of Wnt target genes through association with beta-catenin/Armadillo and TCF/Pangolin. Here, human WINS1 and mouse Wins2, encoding proteins with Drosophila Lin homologous domain, were isolated using bioinformatics and cDNA-PCR. Human WINS1 encoded 757-amino-acid protein, and mouse Wins2 encoded 498-amino-acid protein. Human WINS1 and mouse Wins2 showed 60.0% total-amino-acid identity. Lin homologous domain of WINS1 and Wins2 showed 29.4% and 27.2% amino-acid identity with that of Drosphila Lin, respectively. In the human chromosome 15q26 region, WINS1 gene was clustered with ASB7 gene encoding ankyrin repeat and SOCS box-containing protein 7. Human WINS1 mRNA of 2.8-kb in size was expressed in adult testis, prostate, spleen, thymus, skeletal muscle, fetal kidney and brain. This is the first report on molecular cloning and initial characterization of human WINS1 and mouse Wins2 PMID:12119551

  18. Individual mouse taste cells respond to multiple chemical stimuli

    PubMed Central

    Caicedo, Alejandro; Kim, Kyung-Nyun; Roper, Stephen D

    2002-01-01

    Sensory organs are specialized to detect and decode stimuli in terms of intensity and quality. In the gustatory system, the process of identifying and distinguishing taste qualities (e.g. bitter versus sweet) begins in taste buds. A central question in gustatory research is how information about taste quality is extracted by taste receptor cells. For instance, whether and how individual taste cells respond to multiple chemical stimuli is still a matter for debate. A recent study showed that taste cells expressing bitter-responsive taste receptors do not also express sweet-responsive taste receptors and vice versa. These results suggest that the gustatory system may use separate cellular pathways to process bitter and sweet signals independently. Results from electrophysiological studies, however, reveal that individual taste receptor cells respond to stimuli representing multiple taste qualities. Here we used non-invasive Ca2+ imaging in slices of lingual tissue containing taste buds to address the issue of quality detection in murine taste receptor cells. We recorded calcium transients elicited by chemical stimuli representing different taste qualities (sweet, salty, sour and bitter). Many receptor cells (38 %) responded to multiple taste qualities, with some taste cells responding to both appetitive (‘sweet’) and aversive (‘bitter’) stimuli. Thus, there appears to be no strict and separate detection of taste qualities by distinct subpopulations of taste cells in peripheral gustatory sensory organs in mice. PMID:12381822

  19. Mouse embryos and chimera cloned from neural cells in the postnatal cerebral cortex.

    PubMed

    Makino, Hatsune; Yamazaki, Yukiko; Hirabayashi, Takahiro; Kaneko, Ryosuke; Hamada, Shun; Kawamura, Yoshimi; Osada, Tomoharu; Yanagimachi, Ryuzo; Yagi, Takeshi

    2005-01-01

    Cloning of mice has been achieved by transferring nuclei of various types of somatic cell nuclei into enucleated oocytes. However, all attempts to produce live cloned offspring using the nuclei of neurons from adult cerebral cortex have failed. Previously we obtained cloned mice using the nuclei of neural cells collected from fetal cerebral cortex. Here, we attempted to generate cloned mice using differentiated neurons from the cerebral cortex of postnatal (day 0-4) mice. Although we were unable to obtain live cloned pups, many fetuses reached day 10.5 days of development. These fetuses showed various abnormalities such as spherical omission of the neuroepithelium, collapsed lumen of neural tube, and aberrant expressions of marker proteins of neurons. We produced chimeric mice in which some hair cells and kidney cells were originated from differentiated neurons. In chimeric fetuses, LacZ-positive donor cells were in all three germ cell layers. However, chimeras with large contribution of donor-derived cells were not obtained. These results indicate that nuclei of differentiated neurons have lost their developmental totipotency. In other words, the conventional nuclear transfer technique does not allow nuclei of differentiated neurons to undergo complete genomic reprogramming required for normal embryonic development.

  20. Molecular Cloning and Analysis of a DNA Repetitive Element from the Mouse Genome

    ERIC Educational Resources Information Center

    Geisinger, Adriana; Cossio, Gabriela; Wettstein, Rodolfo

    2006-01-01

    We report the development of a 3-week laboratory activity for an undergraduate molecular biology course. This activity introduces students to the practice of basic molecular techniques such as restriction enzyme digestion, agarose gel electrophoresis, cloning, plasmid DNA purification, Southern blotting, and sequencing. Students learn how to carry…

  1. A spare or an individual? Cloning and the implications of monozygotic twinning.

    PubMed

    Bryan, E M

    1998-01-01

    The creation of Dolly, the cloned sheep, raises the scenario of cloning in humans. Neither the case for, nor against, the ethics of cloning in humans is discussed in this paper. Instead, it considers the neglected issue of the likely happiness or otherwise of the resulting children if they are born as monozygotic twins or triplets. The advantages and disadvantages of twinship are discussed in detail, and it is concluded that recognized medical risks, and incompletely understood psychological effects, should be given serious consideration.

  2. [Molecular cloning and expression in cryptorchid testis of SRG2 from a mouse testis spermatocyte apoptosis-related gene].

    PubMed

    Liu, Shang-Feng; Li, Lu-Yun; Mo, Ya-Qin; Fu, Jun-Jiang; Liu, Gang; Xing, Xiao-Wei; Lu, Guang-Xiu

    2003-10-01

    It was observed that the spermatogenic cells apoptosis dramatically increased in infertile man. Cloning of novel spermatogenic cell-specific gene related to apoptosis is of momentous physiological and pathological significance to illustrate the apoptosis mechanism and the biology process of spermatogenic cells. A novel mouse gene full-length cDNA sequence-SRG2 was identified (GenBank accession number AF395083), which was significantly changed in cryptorchidism, from a mouse testis cDNA library using a cDNA fragment (GenBank accession number BE644542) as an electronic probe. SRG2 was 1,088 bp in length. The putative protein encoded by this gene was 295 amino acids with a theoretical molecular weight of 33,579 kDa and isoelectric point of 9.64. The sequence shared no significant homology with any known protein in databases except TSARG2, with which its homology was 78%. RT-PCR showed that SRG2 was expressed significantly in testis. Using molecular beacon probe to examine the mRNA expression level of SRG2 gene in cryptorchid testis of various stages, we found that the gene was up-regulated distinctly. Therefore, we conclude that this gene plays an important role in cryptorchid testis.

  3. [Cloning and identification of a mouse zinc finger protein gene ZF-12-related pseudogene].

    PubMed

    Li, Jian Zhong; Zhang, Ya Zhou; Wang, Shui Liang; Yang, Hua; Li, Jian; Yu, Long; Fu, Ji Liang

    2002-06-01

    The mouse zinc finger protein ZF-12 gene is homologous to human gene and encodes a protein of 368 amino acids, which contains four tandem C2H2-type zinc finger motifs in the N-terminal and one SCAN domain in the C-terminal. Some recent studies suggest that ZNF191 might be a hepatocarcinogenesis-associated gene. We screened a mouse lambda genomic library with a human ZNF191 cDNA probe and isolated a ZF-12-like gene, named ZF12p (GenBank AY040222). This intronless gene closely resembles ZF-12 but displays several mutations, suggesting that ZF12p represents a ZF-12-related pseudogene. RT-PCR analysis on total RNA from mouse tissue and bioinformatis analysis on promoter region of ZF12p gene, suggest the transcripts of ZF12p may be not synthesized. BLAST on the data of the human genome in the GenBank with ZNF191 cDNA and Southern blotting show there is no any psedogene related to ZNF191 gene in the human genome. The high similarity of ZF12p to ZF-12 might be of considerable importance for mutation and evolution analysis of ZF-12.

  4. Do individually ventilated cage systems generate a problem for genetic mouse model research?

    PubMed

    Logge, W; Kingham, J; Karl, T

    2014-09-01

    Technological developments over recent decades have produced a novel housing system for laboratory mice, so-called 'individually ventilated cage' (IVC) systems. IVCs present a cage environment which is different to conventional filter-top cages (FILTER). Nothing is known about the consequences of IVC housing on genetic mouse models, despite studies reporting IVC-mediated changes to the phenotypes of inbred mouse strains. Thus, in this study, we systematically compared the established behavioural phenotype of a validated mouse model for the schizophrenia risk gene neuregulin 1 (TM Nrg1 HET) kept in FILTER housing with Nrg1 mutant mice raised in IVC systems. We found that particular schizophrenia-relevant endophenotypes of TM Nrg1 HETs which had been established and widely published using FILTER housing were altered when mice were raised in IVC housing. IVCs diminished the schizophrenia-relevant prepulse inhibition deficit of Nrg1 mutant males. Furthermore, IVC housing had a sex-dependent moderate effect on the locomotive phenotype of Nrg1 mice across test paradigms. Behavioural effects of IVC housing were less prominent in female mice. Thus, transferring the breeding colony of mouse mutants from FILTER to IVC systems can shift disease-relevant behaviours and therefore challenge the face validity of these mice. Researchers facing an upgrade of their mouse breeding or holding facilities to IVC systems must be aware of the potential impact this upgrade might have on their genetic mouse models. Future publications should provide more details on the cage system used to allow appropriate data comparison across research sites. PMID:24920375

  5. Molecular cloning, genomic organization, chromosomal mapping and subcellular localization of mouse PAP7: a PBR and PKA-RIalpha associated protein.

    PubMed

    Liu, Jun; Cavalli, Luciane R; Haddad, Bassem R; Papadopoulos, Vassilios

    2003-04-10

    A mouse protein that interacts with the peripheral-type benzodiazepine receptor (PBR) and the cAMP-dependent protein kinase A (PKA) regulatory subunit RIalpha (PKA-RIalpha), named PBR and PKA associated protein 7 (PAP7) was identified and shown to be involved in hormone-induced steroid biosynthesis in testicular Leydig cells. In the present study, mouse PAP7 cDNA was extended by 5'-rapid amplification of cDNA ends; and a 3432 bp sequence, encoding a 525-amino-acid protein with a calculated molecular weight of 60 kDa, was re-assembled. Mouse and human PAP7 share an 85% amino acid identity and contain a conserved acyl-CoA-binding protein/diazepam binding inhibitor (ACBP/DBI) motif. ACBP/DBI has been identified as the endogenous PBR ligand able to stimulate mitochondrial steroid formation in all steroidogenic cells. The full-length mouse PAP7 gene was cloned and assembled by screening a BAC clone, polymerase chain reaction and searching the mouse genome database. The gene is approximately 29 kb in length and includes eight exons and seven introns. Although it is shorter than the human PAP7 gene, all exons are conserved between the mouse and human. The mouse PAP7 gene was mapped to chromosome 1H3-5 by fluorescence in situ hybridization in agreement with in silico search of the mouse genome database that mapped the PAP7 cDNA sequence to the 1H4 area. Immunofluorescence confocal microscopy demonstrated that PAP7 is mainly localized in the trans-Golgi apparatus and mitochondria in mouse tumor Leydig cells, in agreement with its proposed function in targeting the PKA isoenzyme to organelles rich in PBR, i.e. mitochondria, where phosphorylation of specific protein substrates mediates the hormone-induced steroid formation.

  6. Individual variability in finger-to-finger transmission efficiency of Enterococcus faecium clones

    PubMed Central

    del Campo, Rosa; Sánchez-Díaz, Ana María; Zamora, Javier; Torres, Carmen; Cintas, Luis María; Franco, Elvira; Cantón, Rafael; Baquero, Fernando

    2014-01-01

    A fingertip-to-fingertip intraindividual transmission experiment was carried out in 30 healthy volunteers, using four MLST-typed Enterococcus faecium clones. Overall results showed an adequate fit goodness to a theoretical exponential model, whereas four volunteers (13%) exhibited a significantly higher finger-to-finger bacterial transmission efficiency. This observation might have deep consequences in nosocomial epidemiology. PMID:24382843

  7. Individual variability in finger-to-finger transmission efficiency of Enterococcus faecium clones.

    PubMed

    del Campo, Rosa; Sánchez-Díaz, Ana María; Zamora, Javier; Torres, Carmen; Cintas, Luis María; Franco, Elvira; Cantón, Rafael; Baquero, Fernando

    2014-02-01

    A fingertip-to-fingertip intraindividual transmission experiment was carried out in 30 healthy volunteers, using four MLST-typed Enterococcus faecium clones. Overall results showed an adequate fit goodness to a theoretical exponential model, whereas four volunteers (13%) exhibited a significantly higher finger-to-finger bacterial transmission efficiency. This observation might have deep consequences in nosocomial epidemiology. PMID:24382843

  8. Molecular cloning and functional characterization of a mouse gene upregulated by lipopolysaccharide treatment reveals alternative splicing

    SciTech Connect

    Du, Kejun; Chen, Yaoming; Dai, Zongming; Bi, Yuan; Cai, Tongjian; Hou, Lichao; Chai, Yubo; Song, Qinghe; Chen, Sumin; Luo, Wenjing; Chen, Jingyuan

    2010-01-01

    Treatment of mouse cells with lipopolysaccharide (LPS) potently initiates an inflammatory response, but the underlying mechanisms are unclear. We therefore sought to characterize cDNA sequences of a new mouse LPS-responsive gene, and to evaluate the effects of MLrg. Full-length cDNAs were obtained from LPS-treated NIH3T3 cells. We report that the MLrg gene produces two alternative splice products (GenBank Accession Nos. (DQ316984) and (DQ320011)), respectively, encoding MLrgW and MLrgS polypeptides. Both proteins contain zinc finger and leucine zipper domains and are thus potential regulators of transcription. Expression of MLrgW and MLrgS were robustly upregulated following LPS treatment, and the proteins were localized predominantly in the nuclear membrane and cytoplasm. In stable transfectants over-expressing MLrgW the proportion of cells in G1 phase was significantly reduced, while in cells over-expressing MLrgS the proportion of cells in G2 was significantly increased; both proteins are thus potential regulators of cell cycle progression. Upregulation of MLrgW and MLrgS may be an important component of the LPS inflammatory pathway and of the host response to infection with GNB.

  9. Extreme individual flexibility of heterothermy in free-ranging Malagasy mouse lemurs (Microcebus griseorufus).

    PubMed

    Kobbe, Susanne; Ganzhorn, Jörg U; Dausmann, Kathrin H

    2011-01-01

    Flexibility in physiological processes is essential to adequately respond to changes in environmental conditions. Madagascar is a particularly challenging environment because climatic conditions seem less predictable than in comparative ecosystems in other parts of the world. We used the reddish-gray mouse lemur (Microcebus griseorufus) from the most unpredictable environment in Madagascar as a model to investigate the flexibility of energy saving strategies to cope with the unpredictability of their habitat. For this we measured T (sk) of free-ranging mouse lemurs throughout the year using temperature data loggers. M. griseorufus showed a very strong seasonal as well as an individual flexibility in thermoregulation. During the rainy season all M. griseorufus remained normothermic. At the beginning of the dry season individuals started to exhibit different energy saving strategies: irregular short torpor bouts, regular daily torpor, prolonged torpor of a few days, and hibernation over several weeks. The accumulation of sufficient seasonal body fat was the crucial factor determining the thermal behavior of individuals. The observed intraspecific and sex independent variation in thermoregulatory patterns within one population inhabiting the same small geographical area is exceptional and gives M. griseorufus the ability to respond to current environmental as well as individual conditions. This thermal plasticity might be seen as a key to success and survival for M. griseorufus in an extremely unpredictable environment. PMID:20717683

  10. Individual and combined effects of deoxynivalenol and zearalenone on mouse kidney.

    PubMed

    Liang, Zhen; Ren, Zhihua; Gao, Shuang; Chen, Yun; Yang, Yanyi; Yang, Dan; Deng, Junliang; Zuo, Zhicai; Wang, Ya; Shen, Liuhong

    2015-11-01

    This study was performed to investigate the individual and combined toxic effects of deoxynivalenol (DON) and zearalenone (ZEA) on mouse kidney. A total of 360 female mice were divided into nine groups. Each group received intraperitoneal injection of solvent (control), DON, ZEA, or DON+ZEA four times for 12d. Results showed that ZEA and/or DON increased the apoptosis rate in the kidney, as well as the levels of serum creatinine and blood urea nitrogen. DON and/or ZEA also induced renal oxidative stress as indicated by increased malondialdehyde concentration and nitric oxide level and reduced superoxide dismutase enzyme activity and hydroxyl radical inhibiting capacity. The observed changes were dose and time dependent. This study reports that DON and/or ZEA induced apoptosis, dysfunction, and oxidative stress in mouse kidney. Furthermore, the combination of DON+ZEA exhibited a sub-additive nephrotoxic effect.

  11. The human and mouse homologs of the yeat RAD52 gene: cDNA cloning, sequence analysis, assignment to human chromosome 12p12.2-p13, and mRNA expression in mouse tissues

    SciTech Connect

    Shen, Z.; Chen, D.J.; Denison, K.

    1995-01-01

    The yeast Saccharomyces cerevisiae RAD52 gene is involved in DNA double-strand break repair and mitotic/meiotic recombination. The N-terminal amino acid sequence of yeast S. cerevisiae, Schizosaccharomyces pombe, and Kluyveromyces lactis and chicken is highly conserved. Using the technology of mixed oligonucleotide primed amplification of cDNA (MOPAC), two mouse RAD52 homologous cDNA fragments were amplified and sequenced. Subsequently, we have cloned the cDNA of the human and mouse homologs of yeast RAD52 gene by screening cDNA libraries using the identified mouse cDNA fragments. Sequence analysis of cDNA derived amino acid revealed a highly conserved N-terminus among human, mouse, chicken, and yeast RAD52 genes. The human RAD52 gene was assigned to chromosome 12p12.2-p13 by fluorescence in situ hybridization, R-banding, and DNA analysis of somatic cell hybrids. Unlike chicken RAD52 and mouse RAD51, no significant difference in mouse RAD52 mRNA level was found among mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis. In addition to an {approximately}1.9-kb RAD52 mRNA band that is present in all of the tested tissues, an extra mRNA species of {approximately}0.85 kb was detectable in mouse testis. 40 refs., 7 figs., 1 tab.

  12. The transcriptomic architecture of mouse Sertoli cell clone embryos reveals temporal–spatial-specific reprogramming.

    PubMed

    Cao, Feng; Fukuda, Atsushi; Watanabe, Hiroshi; Kono, Tomohiro

    2013-03-01

    Somatic cell nuclear transfer, a technique used to generate clone embryos by transferring the nucleus of a somatic cell into an enucleated oocyte, is an excellent approach to study the reprogramming of the nuclei of differentiated cells. Here, we conducted a transcriptomic study by performing microarray analysis on single Sertoli cell nuclear transfer (SeCNT) embryos throughout preimplantation development. The extensive data collected from the oocyte to the blastocyst stage helped to identify specific genes that were incorrectly reprogrammed at each stage, thereby providing a novel perspective for understanding reprogramming progression in SeCNT embryos.This attempt provided an opportunity to discuss the possibility that ectopic gene expression could be involved in the developmental failure of SeCNT embryos. Network analysis at each stage suggested that in total, 127 networks were involved in developmental and functional disorders in SeCNT embryos. Furthermore, chromosome mapping using our time-lapse expression data highlighted temporal–spatial changes of the abnormal expression, showing the characteristic distribution of the genes on each chromosome.Thus, the present study revealed that the preimplantation development of SeCNT embryos appears normal; however, the progression of incorrect reprogramming is concealed throughout development.

  13. Molecular cloning of the gene encoding the mouse parathyroid hormone/parathyroid hormone-related peptide receptor.

    PubMed Central

    McCuaig, K A; Clarke, J C; White, J H

    1994-01-01

    The parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) is a G-protein-coupled receptor containing seven predicted transmembrane domains. We have isolated and characterized recombinant bacteriophage lambda EMBL3 genomic clones containing the mouse PTHR gene, including 10 kilobases of the promoter region. The gene spans > 32 kilobases and is divided into 15 exons, 8 of which contain the transmembrane domains. The PTHR exons containing the predicted membrane-spanning domains are heterogeneous in length and three of the exon-intron boundaries fall within putative transmembrane sequences, suggesting that the exons did not arise from duplication events. This arrangement is closely related to that of the growth hormone releasing factor receptor gene, particularly in the transmembrane region, providing strong evidence that the two genes evolved from a common precursor. Transcription is initiated principally at a series of sites over a 15-base-pair region. The proximal promoter region is highly (G+C)-rich and lacks an apparent TATA box or initiator element homologies but does contain CCGCCC motifs. The presumptive amino acid sequence of the encoded receptor is 99%, 91%, and 76% identical to those of the rat, human, and opossum receptors, respectively. There is no consensus polyadenylation signal in the 3' untranslated region. The poly(A) tail of the PTHR transcript begins 32 bases downstream of a 35-base-long A-rich sequence, suggesting that this region directs polyadenylylation. Images PMID:8197183

  14. Effects of nuclear transfer procedures on ES cell cloning efficiency in the mouse.

    PubMed

    Yabuuchi, Akiko; Yasuda, Yoshiko; Kato, Yoko; Tsunoda, Yukio

    2004-04-01

    Enucleated oocytes receiving mouse embryonic stem (ES) cells develop into fertile young. The developmental potential to young is low, however, and the rate of postnatal death is high. We examined the effect of various nuclear transfer procedures on the in vitro and in vivo developmental potential of nuclear-transferred oocytes. The potential of oocytes receiving ES cells at M phase to develop into blastocysts after fusion by Sendai virus was high compared with that after direct injection (67% vs. 30%). The developmental potential of oocytes receiving ES cells at the M phase is higher than that of oocytes receiving ES cells at the G(1) phase (30-67% vs. 2-5%). Developmental ability to live young was low in all groups (0-4%). Different activation protocols affected the potential to develop into blastocysts to a different extent (27-62%), but did not affect the potential to develop into live young (0-3%). The present study demonstrated that the various conditions examined did not affect the potential of nuclear-transferred oocytes receiving ES cells to develop into live young or the incidence of postnatal death.

  15. Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter

    PubMed Central

    Liu, Dan; Liu, Xin; Wu, Ye; Wang, Wen; Ma, Xinliang; Liu, Huirong

    2016-01-01

    HtrA serine peptidase 2 (HtrA2), also named Omi, is a pro-apoptotic protein that exhibits dramatic changes in expression levels in a variety of disorders, including ischemia/reperfusion injury, cancer, and neurodegeneration. In our study, Omi/HtrA2 protein levels were high in the heart, brain, kidney and liver, with elevated heart/brain expression in aging mice. A similar expression pattern was observed at the mRNA level, which suggests that the regulation of Omi/HtrA2 is predominately transcriptional. Promoter binding by transcription factors is the main influencing factor of transcription, and to identify specific promoter elements that contribute to the differential expression of mouse Omi/HtrA2, we constructed truncated Omi/HtrA2 promoter/luciferase reporter vectors and analyzed their relative luciferase activity; it was greatest in the promoter regions at −1205~−838 bp and −146~+93 bp, with the −838~−649 bp region exhibiting negative regulatory activity. Bioinformatics analysis suggested that the Omi/HtrA2 gene promoter contains a CpG island at −709~+37 bp, and eight heat shock transcription factor 1 (HSF1) sites, two Sp1 transcription factor (SP1)sites, one activator protein (AP) site, seven p53 sites, and four YY1 transcription factor(YY1) sites were predicted in the core areas. Furthermore, we found that p53 and HSF1 specifically binds to the Omi/HtrA2 promoter using chromatin immunoprecipitation analysis. These results provide a foundation for understanding Omi/HtrA2 regulatory mechanisms, which could further understanding of HtrA-associated diseases. PMID:26784188

  16. cDNA cloning and chromosomal mapping of the mouse type VII collagen gene (Col7a1): Evidence for rapid evolutionary divergence of the gene

    SciTech Connect

    Li, Kehua; Christiano, A.M.; Chu, Mon Li; Uitto, J. Thomas Jefferson Univ., Philadelphia, PA ); Copeland, N.G.; Gilbert, D.J. )

    1993-06-01

    Type VII collagen is the major component of anchoring fibrils, critical attachment structures at the dermal-epidermal basement membrane zone. Genetic linkage analyses with recently cloned human type VII collagen cDNAs have indicated that the corresponding gene, COL7A1, is the candidate gene in the dystrophic forms of epidermolysis bullosa. To gain insight into the evolutionary conservation of COL7A1, in this study the authors have isolated mouse type VII collagen cDNAs by screening a mouse epidermal keratinocyte cDNA library with a human COL7A1 cDNA. Two overlapping mouse cDNAs were isolated, and Northern hybridization of mouse epidermal keratinocyte RNA with one of them revealed the presence of a mRNA transcript of [approximately]9.5 kb, the approximate size of the human COL7A1 mRNA. Nucleotide sequencing of the mouse cDNAs revealed a 2760-bp open reading frame that encodes the 5[prime] half of the collagenous domain and a segment of the NC-1, the noncollagenous amino-terminal domain of type VII collagen. Comparison of the mouse amino acid sequences with the corresponding human sequences deduced from cDNAs revealed 82.5% identity. The evolutionary divergence of the gene was relatively rapid in comparison to other collagen genes. Despite the high degree of sequence variation, several sequences, including the size and the position of noncollagenous imperfections and interruptions within the Gly-X-Y repeat sequence, were precisely conserved. Finally, the mouse Col7a1 gene was located by interspecific backcross mapping to mouse Chromosome 9, a region that corresponds to human chromosome 3p21, the position of human COL7Al. This assignment confirms and extends the relationship between the mouse and the human chromosomes in this region of the genome. 33 refs., 5 figs., 1 tab.

  17. Individual strains of Lactobacillus paracasei differentially inhibit human basophil and mouse mast cell activation

    PubMed Central

    Cassard, Lydie; Lalanne, Ana Inés; Garault, Peggy; Cotillard, Aurélie; Chervaux, Christian; Wels, Michiel; Smokvina, Tamara

    2016-01-01

    Abstract Introduction The microbiota controls a variety of biological functions, including immunity, and alterations of the microbiota in early life are associated with a higher risk of developing allergies later in life. Several probiotic bacteria, and particularly lactic acid bacteria, were described to reduce both the induction of allergic responses and allergic manifestations. Although specific probiotic strains were used in these studies, their protective effects on allergic responses also might be common for all lactobacilli. Methods To determine whether allergic effector cells inhibition is a common feature of lactobacilli or whether it varies among lactobacilli strains, we compared the ability of 40 strains of the same Lactobacillus paracasei species to inhibit IgE‐dependent mouse mast cell and human basophil activation. Results We uncovered a marked heterogeneity in the inhibitory properties of the 40 Lactobacillus strains tested. These segregated into three to four clusters depending on the intensity of inhibition. Some strains inhibited both mouse mast cell and human basophil activation, others strains inhibited only one cell type and another group induced no inhibition of activation for either cell type. Conclusions Individual Lactobacillus strains of the same species differentially inhibit IgE‐dependent activation of mouse mast cells and human basophils, two cell types that are critical in the onset of allergic manifestations. Although we failed to identify specific bacterial genes associated with inhibition by gene‐trait matching analysis, our findings demonstrate the complexity of the interactions between the microbiota and the host. These results suggest that some L. paracasei strains might be more beneficial in allergies than others strains and provide the bases for a rational screening of lactic acid bacteria strains as next‐generation probiotics in the field of allergy.

  18. Individual strains of Lactobacillus paracasei differentially inhibit human basophil and mouse mast cell activation

    PubMed Central

    Cassard, Lydie; Lalanne, Ana Inés; Garault, Peggy; Cotillard, Aurélie; Chervaux, Christian; Wels, Michiel; Smokvina, Tamara

    2016-01-01

    Abstract Introduction The microbiota controls a variety of biological functions, including immunity, and alterations of the microbiota in early life are associated with a higher risk of developing allergies later in life. Several probiotic bacteria, and particularly lactic acid bacteria, were described to reduce both the induction of allergic responses and allergic manifestations. Although specific probiotic strains were used in these studies, their protective effects on allergic responses also might be common for all lactobacilli. Methods To determine whether allergic effector cells inhibition is a common feature of lactobacilli or whether it varies among lactobacilli strains, we compared the ability of 40 strains of the same Lactobacillus paracasei species to inhibit IgE‐dependent mouse mast cell and human basophil activation. Results We uncovered a marked heterogeneity in the inhibitory properties of the 40 Lactobacillus strains tested. These segregated into three to four clusters depending on the intensity of inhibition. Some strains inhibited both mouse mast cell and human basophil activation, others strains inhibited only one cell type and another group induced no inhibition of activation for either cell type. Conclusions Individual Lactobacillus strains of the same species differentially inhibit IgE‐dependent activation of mouse mast cells and human basophils, two cell types that are critical in the onset of allergic manifestations. Although we failed to identify specific bacterial genes associated with inhibition by gene‐trait matching analysis, our findings demonstrate the complexity of the interactions between the microbiota and the host. These results suggest that some L. paracasei strains might be more beneficial in allergies than others strains and provide the bases for a rational screening of lactic acid bacteria strains as next‐generation probiotics in the field of allergy. PMID:27621812

  19. Molecular cloning and functional characterization of the mouse organic-anion-transporting polypeptide 1 (Oatp1) and mapping of the gene to chromosome X.

    PubMed

    Hagenbuch, B; Adler, I D; Schmid, T E

    2000-01-01

    We have cloned a murine member of the organic-anion-transporting polypeptide (Oatp) family of membrane-transport proteins from mouse liver. The cloned cDNA insert of 2783 bp with an open reading frame of 2011 bp codes for a 12-transmembrane 670-amino-acid protein with highest amino acid identity with the rat Oatp1. When expressed in Xenopus laevis oocytes, the mouse Oatp exhibited the same substrate specificity as the rat Oatp1. Besides the common Oatp substrates bromosulphophthalein, taurocholate, oestrone 3-sulphate and ouabain, the new mouse Oatp also mediates transport of the Oatp1-specific magnetic-resonance-imaging agent gadoxetate. The Oatp2-specific cardiac glycoside digoxin, however, is not transported. Kinetic analyses performed for taurocholate and oestrone 3-sulphate revealed apparent K(m) values of 12 microM and 5 microM respectively. Northern-blot analysis demonstrated a predominant expression in the liver with an additional moderate expression in the kidney. Taken together, the amino acid identity, the functional characteristics and the tissue distribution suggest that we have isolated the murine orthologue of the rat Oatp1, and consequently the identified protein will be called Oatp1. Using fluorescence in situ hybridization, the murine Oatp1 gene was mapped to chromosome XA3-A5.

  20. Molecular cloning and functional characterization of the mouse organic-anion-transporting polypeptide 1 (Oatp1) and mapping of the gene to chromosome X.

    PubMed Central

    Hagenbuch, B; Adler, I D; Schmid, T E

    2000-01-01

    We have cloned a murine member of the organic-anion-transporting polypeptide (Oatp) family of membrane-transport proteins from mouse liver. The cloned cDNA insert of 2783 bp with an open reading frame of 2011 bp codes for a 12-transmembrane 670-amino-acid protein with highest amino acid identity with the rat Oatp1. When expressed in Xenopus laevis oocytes, the mouse Oatp exhibited the same substrate specificity as the rat Oatp1. Besides the common Oatp substrates bromosulphophthalein, taurocholate, oestrone 3-sulphate and ouabain, the new mouse Oatp also mediates transport of the Oatp1-specific magnetic-resonance-imaging agent gadoxetate. The Oatp2-specific cardiac glycoside digoxin, however, is not transported. Kinetic analyses performed for taurocholate and oestrone 3-sulphate revealed apparent K(m) values of 12 microM and 5 microM respectively. Northern-blot analysis demonstrated a predominant expression in the liver with an additional moderate expression in the kidney. Taken together, the amino acid identity, the functional characteristics and the tissue distribution suggest that we have isolated the murine orthologue of the rat Oatp1, and consequently the identified protein will be called Oatp1. Using fluorescence in situ hybridization, the murine Oatp1 gene was mapped to chromosome XA3-A5. PMID:10600646

  1. Cloning, analysis, and chromosomal localization of myoxin (MYH12), the human homologue to the mouse dilute gene

    SciTech Connect

    Engle, L.J.; Kennett, R.H. )

    1994-02-01

    The mouse dilute gene encodes a novel type of non-muscle myosin that structurally combines elements from both nonmuscle myosin type I and nonmuscle myosin type II. Phenotypically, mutations in the mouse dilute gene result not only in the lightening of coat color, but also in the onset of severe neurological defects shortly after birth. This may indicate that the mouse dilute gene is important in maintaining the normal neuronal function in the mouse. The authors report the isolation and sequencing of [open quotes]myoxin[close quotes] (MYH12), the human homologue of the mouse dilute gene, and its assignment to human chromosome 15. 35 refs., 6 figs.

  2. Ability of Antibiotic-Resistant Nonvaccine-Type Pneumococcal Clones to Cause Otitis Media in an Infant Mouse Model of Pneumococcal-Influenza Virus Coinfection.

    PubMed

    Frazão, Nelson; Hermans, Peter; van Selm, Saskia; Sá-Leão, Raquel; de Lencastre, Hermínia; Tomasz, Alexander; Diavatopoulos, Dimitri

    2016-01-01

    The introduction of the 7-valent pneumococcal conjugate vaccine in Portugal resulted in reduced carriage in children by vaccine-type strains and an increased carriage of three major antibiotic-resistant clones, ST2191, ST276, and ST63 expressing capsules 6A, 19A, and 15A, respectively. Pneumococcal otitis media (OM), a frequent infection among preschool age children, is often associated with viral coinfection. To evaluate the ability of these three antibiotic-resistant clones to cause disease, we used an infant mouse model of influenza virus pneumococcal coinfection. The 6A and 19A clonal types induced OM, while 15A induced pneumococcal pneumonia and bloodstream infection, suggesting potential for invasive disease.

  3. Antiviral Activity of a Cloned Peptide RC28 Isolated from the Higher Basidiomycetes Mushroom Rozites caperata in a Mouse Model of HSV-1 Keratitis.

    PubMed

    Yan, Naihong; He, Fen; Piraino, Frank F; Xiang, Haotian; Chen, Jun; Wang, Yun; Liu, Xuyang

    2015-01-01

    An Escherichia coli-expressed peptide with a molecular weight of 28.26, derived from the complementary DNA of antiviral protein RC28 isolated from the mushroom Rozites caperata (=Cortinarius caperatus), demonstrated potent antiviral activity against herpes simplex virus-1 in Vero cells and in a herpes simplex virus-1 mouse keratitis model. Plaque assays in Vero cells showed that the peptide reduced viral yields by at least 1.2 logs; in the animal model the cloned peptide delayed the occurrence of stromal keratitis and alleviated the severity of the disease. We believe this is the first report of a cloned mushroom peptide with antiviral activity for the prevention and treatment of a viral disease.

  4. Molecular cloning of a cDNA coding for mouse liver xanthine dehydrogenase. Regulation of its transcript by interferons in vivo.

    PubMed Central

    Terao, M; Cazzaniga, G; Ghezzi, P; Bianchi, M; Falciani, F; Perani, P; Garattini, E

    1992-01-01

    The cDNA coding for xanthine dehydrogenase (XD) is isolated from mouse liver mRNA by cross-hybridization with a DNA fragment of the Drosophila melanogaster homologue. Two lambda bacteriophage overlapping clones represent the copy of a 4538-nucleotide-residue-long transcript with an open reading frame of 4005 nucleotide residues, coding for a putative polypeptide of 1335 amino acid residues. Comparison of the deduced amino acid sequence of the mouse XD with those of the Drosophila and the rat homologues shows a high conservation of this protein (55% identity between mouse and Drosophila, and 94% identity between mouse and rat). RNA blotting analysis demonstrates that interferon-alpha (IFN-alpha) and its inducers, i.e. poly(I).poly(C), bacterial lipopolysaccharide (LPS) and tilorone (2,7-bis-[2-(diethylamino)ethoxy]fluoren-9-one), increase the expression of XD mRNA in liver. Poly(I).poly(C) also induces XD mRNA in several other tissues in vivo. Protein synthesis de novo is not required for the elevation of XD mRNA after IFN-alpha treatment, since cycloheximide does not block the induction. The elevation of XD mRNA concentration is relatively fast and precedes the induction of both XD and xanthine oxidase (XO) enzymic activities. Images Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1590774

  5. 3T3 fibroblasts induce cloned interleukin 3-dependent mouse mast cells to resemble connective tissue mast cells in granular constituency

    SciTech Connect

    Dayton, E.T.; Pharr, P.; Ogawa, M.; Serafin, W.E.; Austen, K.F.; Levi-Schaffer, F.; Stevens, R.L.

    1988-01-01

    As assessed by ultrastructure, histochemical staining, and T-cell dependency, in vitro-differentiated interleukin 3-dependent mouse mast cells are comparable to the mast cells that reside in the gastrointestinal mucosa but not in the skin or the serosal cavity of the mouse. The authors now demonstrate that when cloned interleukin 3-dependent mast cells are cocultured with mouse skin-derived 3T3 fibroblasts in the presence of WEHI-3 conditioned medium for 28 days, the mast cells acquire the ability to stain with safranin, increase their histamine content approx. 50-fold and their carboxypeptidase. A content approx. 100-fold, and augment approx. their biosynthesis of proteoglycans bearing /sup 35/S-labeled haparin relative to /sup 35/S-labeled chondroitin sulfate glycosaminoglycans. Thus, fibroblasts induce interleukin 3-dependent mouse mast cells to change phenotype from mucosal-like to connective tissue-like, indicating that the biochemical and functional characteristics of this mast cell type are strongly influenced by the connective tissue microenvironment.

  6. A partial genomic DNA clone for the alpha subunit of the mouse complement receptor type 3 and cellular adhesion molecule Mac-1.

    PubMed Central

    Sastre, L; Roman, J M; Teplow, D B; Dreyer, W J; Gee, C E; Larson, R S; Roberts, T M; Springer, T A

    1986-01-01

    A genomic clone coding for the alpha subunit of the mouse complement receptor type 3 and the cellular adhesion molecule Mac-1 has been isolated directly from a genomic library using synthetic oligonucleotide probes based on the amino-terminal amino acid sequence of the protein. The identity of the clone has been established by DNA sequencing and in vitro translation of hybrid-selected mRNA. The gene is present in a single copy in the murine genome. The region containing the amino-terminal exon has been sequenced. RNA gel blotting shows that the Mac-1 alpha-subunit mRNA is 6 kilobases in length. Mac-1 alpha-subunit mRNA is present in macrophages but not T lymphoma or L cells. During gamma interferon-stimulated maturation of the mouse premyelocytic cell line M1, Mac-1 alpha-subunit mRNA is induced. This corresponds with the tissue distribution of the Mac-1 alpha subunit, showing expression is regulated at least partially at the message level. Images PMID:2942940

  7. Recognition of HLA-A2 and -B7 antigens by cloned cytotoxic T lymphocytes after gene transfer into human and monkey, but not mouse, cells.

    PubMed Central

    Barbosa, J A; Mentzer, S J; Minowada, G; Strominger, J L; Burakoff, S J; Biro, P A

    1984-01-01

    The genes that code for the human major histocompatibility class I antigens, HLA-A2 and HLA-B7, were introduced into human, monkey, and mouse cell lines by cotransfection with suitable biochemical markers and the fluorescence-activated cell sorter was used to identify and/or select stable cell populations expressing high surface levels of these antigens. Levels of expression obtained were similar to those observed for endogenous HLA antigens on various human cell lines and were 25-80% of those observed on the human B-lymphoblastoid cell line JY. Serologically defined HLA-A2 and HLA-B7 polymorphic determinants remained intact on all transfected recipient cells analyzed. Cloned human allospecific cytotoxic T lymphocytes (CTL) specific for HLA-A2 or HLA-B7 were capable of lysing appropriate HLA-transfected human cells with comparable efficiency to JY cell lysis. Two of 10 CTL clones lysed appropriate monkey cell transfectants with approximately equal to 20% the efficiency of human cell transfectants. No specific lysis of any HLA-transfected mouse cell lines, including a B cell lymphoma, was observed despite comparable levels of surface antigen expression or after induction of higher levels by mouse gamma-interferon. Furthermore, L cells expressing human beta 2-microglobulin in addition to HLA-A2 or -B7 were not lysed by these CTL. Thus, an additional species-specific component may be involved in lysis by allogeneic CTL--possibly related to the function(s) of other surface proteins on target cells. PMID:6390442

  8. Differences in susceptibility among mouse strains to infection with Plasmodium berghei (ANKA clone) sporozoites and its relationship to protection by gamma-irradiated sporozoites

    SciTech Connect

    Jaffe, R.I.; Lowell, G.H.; Gordon, D.M. )

    1990-04-01

    Three inbred mouse strains, C57BL/6 (H-2b), A/J (H-2a), and BALB/c (H-2d), and 1 outbred strain, CD-1, demonstrated differences in susceptibility to iv challenge with the ANKA clone of Plasmodium berghei. Mice were challenged with 100, 1,000, or 10,000 sporozoites, then evaluated daily beginning on day 4 for patency. CD-1 mice were further evaluated at challenge doses of 12,500, 25,000, and 50,000 sporozoites. C57BL/6 mice were the easiest to infect, with 90% becoming infected with 100 sporozoites. The outbred strain CD-1 was the most difficult to infect, requiring a challenge dose of 25,000 sporozoites/mouse in order to achieve a 100% infection rate. Mouse strains also demonstrated differences in their ability to be protected by intravenous immunization with gamma-irradiated sporozoites. A/J mice needed a minimum of 3 doses of irradiated sporozoites for protection against a challenge with 10,000 sporozoites. In contrast, BALB/c mice immunized with a single dose of 1,000 irradiated sporozoites are protected against a 10,000 sporozoite challenge. These data suggest that both infectivity and protection are genetically restricted and that susceptibility to infection may be inversely related to protection.

  9. Identical T Cell Clones Are Located within the Mouse Gut Epithelium and Lamina Propria and Circulate in the Thoracic Duct Lymph

    PubMed Central

    Arstila, Tuula; Arstila, T. Petteri; Calbo, Sébastien; Selz, Françoise; Malassis-Seris, Michèle; Vassalli, Pierre; Kourilsky, Philippe; Guy-Grand, Delphine

    2000-01-01

    Murine gut intraepithelial (IEL) T cell receptor (TCR)-α/β1 lymphocytes bearing CD8α/β or CD8α/α coreceptors have been shown previously to express different oligoclonal TCR β chain repertoires in the same mouse, in agreement with other evidence indicating that these two populations belong to different ontogenic lineages, with only CD8α/β1 IELs being fully thymus dependent. CD8α/β1, but not CD8α/α1, T lymphocytes are also present in the lamina propria. Here, we show that CD8α/β+ lymphocytes from the lamina propria and the epithelium are both oligoclonal, and that they share the same TCR-β clonotypes in the same mouse, as is also the case for CD4+ T cells. Furthermore, identical T cell clones were detected among CD8α/β1 IELs and CD8α/β1 blasts circulating into the thoracic duct (TD) lymph of the same mouse, whereas TD small lymphocytes are polyclonal. These findings must be considered in light of previous observations showing that T blasts, but not small T lymphocytes, circulating in the TD lymph have the capacity of homing into the gut epithelium and lamina propria. These combined observations have interesting implications for our understanding of the recirculation of gut thymus-dependent lymphocytes and their precursors, and of the events leading up to the selection of their restricted TCR repertoire. PMID:10755885

  10. Improvement of mouse cloning using nuclear transfer-derived embryonic stem cells and/or histone deacetylase inhibitor.

    PubMed

    Wakayama, Sayaka; Wakayama, Teruhiko

    2010-01-01

    Nuclear transfer-derived ES (ntES) cell lines can be established from somatic cell nuclei with a relatively high success rate. Although ntES cells have been shown to be equivalent to ES cells, there are ethical objections concerning human cells, such as the use of fresh oocyte donation from young healthy woman. In contrast, the use of induced pluripotent stem (iPS) cells for cloning poses few ethical problems and is a relatively easy technique compared with nuclear transfer. Therefore, although there are several reports proposing the use of ntES cells as a model of regenerative medicine, the use of these cells in preliminary medical research is waning. However, in theory, 5 to 10 donor cells can establish one ntES cell line and, once established, these cells will propagate indefinitely. These cells can be used to generate cloned animals from ntES cell lines using a second round of NT. Even in infertile and "unclonable" strains of mice, we can generate offspring from somatic cells by combining cloning with ntES technology. Moreover, cloned offspring can be generated potentially even from the nuclei of dead bodies or freeze-dried cells via ntES cells, such as from an extinct frozen animal. Currently, only the ntES technology is available for this purpose, because all other techniques, including iPS cell derivation, require significant numbers of living donor cells. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.

  11. Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

    PubMed

    Fregien, N; Dolecki, G J; Mandel, M; Humphreys, T

    1983-06-01

    Five developmentally regulated sea urchin mRNA sequences which increase in abundance between the blastula and pluteus stages of development were isolated by molecular cloning of cDNA. The regulated sequences all appeared in moderately abundant mRNA molecules of pluteus cells and represented 4% of the clones tested. There were no regulated sequences detected in the 40% of the clones which hybridized to the most abundant mRNA, and the screening procedures were inadequate to detect possible regulation in the 20 to 30% of the clones presumably derived from rare-class mRNA. The reaction of 32P[cDNA] from blastula and pluteus mRNA to dots of the cloned DNAs on nitrocellulose filters indicated that the mRNAs complementary to the different cloned pluteus-specific sequences were between 3- and 47-fold more prevalent at the pluteus stage than at the blastula stage. Polyadenylated RNA from different developmental stages was transferred from electrophoretic gels to nitrocellulose filters and reacted to the different cloned sequences. The regulated mRNAs were undetectable in the RNA of 3-h embryos, became evident at the hatching blastula stage, and reached a maximum in abundance by the gastrula or pluteus stage. Certain of the clones reacted to two sizes of mRNA which did not vary coordinately with development. Transfers of RNA isolated from each of the three cell layers of pluteus embryos that were reacted to the cloned sequences revealed that two of the sequences were found in the mRNA of all three layers, two were ectoderm specific, and one was endoderm specific. Four of the regulated sequences were complementary to one or two major bands and one to at least 50 bands on Southern transfers of restriction endonuclease-digested total sea urchin DNA. PMID:6688291

  12. The nucleotide sequence of the mouse embryonic beta-like y-globin messenger RNA as determined from cloned cDNA.

    PubMed

    Vanin, E F; Farace, M G; Gambari, R; Fantoni, A

    1981-12-01

    We have determined the nucleotide sequence of two cloned cDNAs corresponding to the mRNA of mouse embryonic y2 globin. The combined overlapping sequences span a total of 480 bp, beginning at the codon corresponding to amino acido residue 21 and extending to the AATAAA sequence in the 3' untranslated region. Therefore, when the amino acid sequence encoded by the cDNA is combined with the available amino acid sequence, a complete y2 protein sequence can be obtained. Comparisons, at the nucleotide level, between the known beta- and beta-like globin sequences and the y2 sequence show that the embryonic, fetal-adult duplication occurred approx. 160 million years (MY) ago and that the embryonic-fetal duplication occurred approx. 100 MY ago.

  13. Molecular cloning of a highly conserved mouse and human integral membrane protein (Itm1) and genetic mapping to mouse chromosome 9

    SciTech Connect

    Hong, Guizhu; Tylzanowski, P.; Deleersnijder, W.

    1996-02-01

    We have isolated and characterized a novel cDNA coding for a highly hydrophobic protein (B5) from a fetal mouse mandibular condyle cDNA library. The full-length mouse B5 cDNA is 3095 nucleotides long and contains a potential open reading frame coding for a protein of 705 amino acids with a calculated molecular weight of 80.5 kDa. The B5 mRNA is differentially polyadenylated, with the most abundant transcript having a length of 2.7 kb. The human homolog of B5 was isolated from a cDNA testis library. The predicted amino acid sequence of the human B5 is 98.5% identical to that of mouse. The most striking feature of the B5 protein is the presence of numerous (10-14) potential transmembrane domains, characteristic of an integral membrane protein. Similarity searches in public databanks reveal that B5 is 58% similar to the T12A2.2 gene of Caenorhabditis elegans and 60% similar to the STT3 gene of Saccharomyces cerevisiae. Futhermore, the report of an EST sequence (Accession No. Z13858) related to the human B5, but identical to the STT3 gene, indicates that B5 belongs to a larger gene family coding for novel putative transmembrane proteins. This family exhibits a remarkable degree of conservation in different species. The gene for B5, designated Itm1 (Integral membrane protein 1), is located on mouse chromosome 9. 28 refs., 4 figs.

  14. Recapitulation of the ovum mutant (Om) phenotype and loss of Om locus polarity in cloned mouse embryos.

    PubMed

    Gao, Shaorong; Wu, Guangming; Han, Zhiming; de la Casa-Esperón, Elena; Sapienza, Carmen; Latham, Keith E

    2005-02-01

    The ovum mutant (Om) locus in mice affects early interactions between sperm and egg that in turn affect viability of embryos beyond the morula stage. Crosses of DDK females to males of many other inbred strains are 95% lethal around the morula stage, whereas reciprocal crosses are fully viable. Available data indicate that the early lethality is the result of an interaction between a factor in the ooplasm and the paternal genome. In this study, we examined whether this lethal interaction would likewise occur in cloned embryos produced by somatic cell nuclear transfer. We find that the Om effect is recapitulated but that the parental origin effect at the Om locus is no longer evident in cloned embryos.

  15. Positional cloning of the nude locus: Genetic, physical, and transcription maps of the region and mutations in the mouse and rat

    SciTech Connect

    Segre, J.A.; Lander, E.S. |; Taylor, B.A.

    1995-08-10

    Mutations in the nude locus in mice and rats produce the pleiotropic phenotype of hairlessness and athymia, resulting in severely compromised immune system. To identify the causative gene, we utilized modern tools and techniques of positional cloning. Specifically, spanning the region in which the nude locus resides, we constructed a genetic map of polymorphic markers, a physical map of yeast artificial chromosomes and bacteriophage P1 clones, and a transcription map of genes obtained by direct cDNA selection and exon trapping. We identified seven novel transcripts with similarity to genes from Drosophila, Caenorhabditis elegans, rat or human and three previously identified mouse genes. Based on our transcription mapping results, we present a novel approach to estimate that the nude locus resides in a region approximately threefold enriched for genes. We confirm a recently published report that the nude phenotype is caused by mutations in a gene encoding a novel winged helix or fork head domain transcription factor, whn. We report as well as the mutations in the rat rnu allele and the complete coding sequence of the rat whn mRNA. 42 refs., 4 figs., 1 tab.

  16. Direct cDNA selection with DNA microdissected from mouse chromosome 16: isolation of novel clones and construction of a partial transcription map of the C3-C4 region.

    PubMed

    Wei, J; Hodes, M E; Wang, Y; Feng, Y; Ghetti, B; Dlouhy, S R

    1996-08-01

    A group of cDNA segments was selected by direct hybridization of mouse cerebellar cDNAs against genomic DNA pools generated by microdissection of the mouse chromosome 16 (MMU16) C3-C4 region. After elimination of repetitive sequences and adjustment for redundancy among clones, 34 novel cDNA fragments were isolated. The MMU16 origin of clones was confirmed by genetic linkage mapping. Reverse transcription PCR indicated that approximately 68% of the cDNAs represent transcripts that are expressed in adult mouse cerebellum. Northern blotting showed that some of these are predominantly or solely expressed in brain. This work demonstrates that DNA microdissected from banded MMU16 can be used for direct cDNA selection, thus enabling construction of a new, region-specific partial transcription map. This selected cDNA library should be a useful reagent for further molecular neurobiological studies.

  17. The identity of clones.

    PubMed

    Evers, K

    1999-02-01

    A common concern with respect to cloning is based on the belief that cloning produces identical individuals. This is a fundamental misunderstanding of what type of identity-relation cloning involves. The concept "identity" is ambiguous, and the statement that cloning produces "identical" individuals is not meaningful unless the notion of identity is clarified. This paper distinguishes between numerical and qualitative; relational and intrinsic: logical and empirical identity, and discusses the empirical individuation of clones in terms of genetics, physiology, perception, cognition and personality. I argue that the only relation of identity cloning involves is qualitative, intrinsic and empirical: genetic indiscernibility, unlikely to include identity under other aspects mentioned. A popular argument against cloning claims our "right" to a "unique identity". This objection either implies (absurdly) the right not to be an identical twin, or assumes (incorrectly) that cloning involves identity other than genetic. Either way, the argument is untenable.

  18. Molecular cloning and characterization of the mouse carboxyl ester lipase gene and evidence for expression in the lactating mammary gland

    SciTech Connect

    Lidmer, A.S.; Lundberg, L.; Kannius, M.; Bjursell, G.

    1995-09-01

    DNA hybridization was used to isolate a 2.04-kb cDNA encoding carboxyl ester lipase (CEL) from a mouse lactating mammary gland, {lambda}gt10 cDNA library. The cDNA sequence translated into a protein of 599 amino acids, including 20 amino acids of a putative signal peptide. Comparison of the deduced amino acid sequence of the mouse CEL with CEL from five other species revealed that there is a high degree of a homology between the different species. The mouse CEL gene was also isolated and found to span approximately 7.2 kb and to include 11 exons. This organization is similar to those of the recently reported human and rat CEL genes. We have also analyzed expression of the CEL gene in the mammary glands from other species by performing a Northern blot analysis with RNA from goat and cow. The results show that the gene is expressed in both species. 36 refs., 6 figs., 1 tab.

  19. 3-Hydroxy-3-methylglutaryl CoA lyase (HL): Mouse and human HL gene (HMGCL) cloning and detection of large gene deletions in two unrelated HL-deficient patients

    SciTech Connect

    Wang, S.P.; Robert, M.F.; Mitchell, G.A.

    1996-04-01

    3-hydroxy-3-methylglutaryl CoA lyase (HL, EC 4.1.3.4) catalyzes the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetic acid and acetyl CoA, the final reaction of both ketogenesis and leucine catabolism. Autosomal-recessive HL deficiency in humans results in episodes of hypoketotic hypoglycemia and coma. Using a mouse HL cDNA as a probe, we isolated a clone containing the full-length mouse HL gene that spans about 15 kb of mouse chromosome 4 and contains nine exons. The promoter region of the mouse HL gene contains elements characteristic of a housekeeping gene: a CpG island containing multiple Sp1 binding sites surrounds exon 1, and neither a TATA nor a CAAT box are present. We identified multiple transcription start sites in the mouse HL gene, 35 to 9 bases upstream of the translation start codon. We also isolated two human HL genomic clones that include HL exons 2 to 9 within 18 kb. The mouse and human HL genes (HGMW-approved symbol HMGCL) are highly homologous, with identical locations of intron-exon junctions. By genomic Southern blot analysis and exonic PCR, was found 2 of 33 HL-deficient probands to be homozygous for large deletions in the HL gene. 26 refs., 4 figs., 2 tabs.

  20. Molecular cloning of mouse liver flavin containing monooxygenase (FMO1) cDNA and characterization of the expression product: metabolism of the neurotoxin, 1,2,3,4-tetrahydroisoquinoline (TIQ).

    PubMed

    Itoh, K; Nakamura, K; Kimura, T; Itoh, S; Kamataki, T

    1997-02-01

    A mouse liver cDNA clone, MFMO1, coding for a flavin-containing monooxygenase (FMO) was isolated. This cDNA clone encoded a protein of 532 amino acids. Based upon its predicted amino acid sequence, this clone was assumed to belong to the FMO1 subfamily. The deduced amino acid sequence showed 94, 84, 83, and 83% identity with FMO1s of rats, pigs, rabbits and humans, respectively, while it showed only 50-59% identity with human FMO3 and FMO4, rabbit FMO2, FMO3, FMO4 and FMO5, and guinea-pig FMO2. RNA blot analysis showed that the mouse FMO1 was also expressed in the lung and kidney and to lesser extents in the heart, spleen, testis and brain. Mouse FMO1 expressed in yeast showed activities of thiobenzamide S-oxidation, and NADPH oxidation associated with the S- or N-oxidation of chlorpromazine, N,N-dimethylaniline, N,N-dimethyl-hydrazine, imipramine, nicotine, thioacetamide, thiourea and trimethylamine. Moreover, 1,2,3,4-tetrahydroisoquinoline (TIQ), a substance known to induce a parkinsonism-like syndrome in monkeys, was also metabolized by the mouse FMO1. The K(m) values for chlorpromazine, imipramine and TIQ were determined to be 2,4, 16.0, 435 mM, respectively. This is the first report to show that an expressed FMO can metabolize a neurotoxin, TIQ. PMID:9076656

  1. Stable yeast transformants that secrete functional. cap alpha. -amylase encoded by cloned mouse pancreatic cDNA

    SciTech Connect

    Filho, S.A.; Galembeck, E.V.; Faria, J.B.; Frascino, A.C.S.

    1986-04-01

    Mouse pancreatic ..cap alpha..-amylase complementary DNA was inserted into a yeast shuttle vector after the Saccharomyces cerevisiae MF..cap alpha..1 promoter and secretion signals coding sequences. When transformed with the recombinant plasmid, S. cerevisiae cells were able to synthesize and secrete functional ..cap alpha..-amylase, efficiently hydrolyzing starch present in the culture medium. Stable amylolytic cells were obtained from different yeast strains. This work represents a significant step towards producing yeast that can convert starchy materials directly to ethanol.

  2. The Voice of Bats: How Greater Mouse-eared Bats Recognize Individuals Based on Their Echolocation Calls

    PubMed Central

    Franz, Matthias O.; Denzinger, Annette; Schnitzler, Hans-Ulrich

    2009-01-01

    Echolocating bats use the echoes from their echolocation calls to perceive their surroundings. The ability to use these continuously emitted calls, whose main function is not communication, for recognition of individual conspecifics might facilitate many of the social behaviours observed in bats. Several studies of individual-specific information in echolocation calls found some evidence for its existence but did not quantify or explain it. We used a direct paradigm to show that greater mouse-eared bats (Myotis myotis) can easily discriminate between individuals based on their echolocation calls and that they can generalize their knowledge to discriminate new individuals that they were not trained to recognize. We conclude that, despite their high variability, broadband bat-echolocation calls contain individual-specific information that is sufficient for recognition. An analysis of the call spectra showed that formant-related features are suitable cues for individual recognition. As a model for the bat's decision strategy, we trained nonlinear statistical classifiers to reproduce the behaviour of the bats, namely to repeat correct and incorrect decisions of the bats. The comparison of the bats with the model strongly implies that the bats are using a prototype classification approach: they learn the average call characteristics of individuals and use them as a reference for classification. PMID:19503606

  3. Variation in Taxonomic Composition of the Fecal Microbiota in an Inbred Mouse Strain across Individuals and Time

    PubMed Central

    Hoy, Yana Emmy; Bik, Elisabeth M.; Lawley, Trevor D.; Holmes, Susan P.; Monack, Denise M.

    2015-01-01

    Genetics, diet, and other environmental exposures are thought to be major factors in the development and composition of the intestinal microbiota of animals. However, the relative contributions of these factors in adult animals, as well as variation with time in a variety of important settings, are still not fully understood. We studied a population of inbred, female mice fed the same diet and housed under the same conditions. We collected fecal samples from 46 individual mice over two weeks, sampling four of these mice for periods as long as 236 days for a total of 190 samples, and determined the phylogenetic composition of their microbial communities after analyzing 1,849,990 high-quality pyrosequencing reads of the 16S rRNA gene V3 region. Even under these controlled conditions, we found significant inter-individual variation in community composition, as well as variation within an individual over time, including increases in alpha diversity during the first 2 months of co-habitation. Some variation was explained by mouse membership in different cage and vendor shipment groups. The differences among individual mice from the same shipment group and cage were still significant. Overall, we found that 23% of the variation in intestinal microbiota composition was explained by changes within the fecal microbiota of a mouse over time, 12% was explained by persistent differences among individual mice, 14% by cage, and 18% by shipment group. Our findings suggest that the microbiota of controlled populations of inbred laboratory animals may not be as uniform as previously thought, that animal rearing and handling may account for some variation, and that as yet unidentified factors may explain additional components of variation in the composition of the microbiota within populations and individuals over time. These findings have implications for the design and interpretation of experiments involving laboratory animals. PMID:26565698

  4. Interspecies diversity of the occludin sequence: cDNA cloning of human, mouse, dog, and rat-kangaroo homologues.

    PubMed

    Ando-Akatsuka, Y; Saitou, M; Hirase, T; Kishi, M; Sakakibara, A; Itoh, M; Yonemura, S; Furuse, M; Tsukita, S

    1996-04-01

    Occludin has been identified from chick liver as a novel integral membrane protein localizing at tight junctions (Furuse, M., T. Hirase, M. Itoh, A. Nagafuchi, S. Yonemura, Sa. Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 123:1777-1788). To analyze and modulate the functions of tight junctions, it would be advantageous to know the mammalian homologues of occludin and their genes. Here we describe the nucleotide sequences of full length cDNAs encoding occludin of rat-kangaroo (potoroo), human, mouse, and dog. Rat-kangaroo occludin cDNA was prepared from RNA isolated from PtK2 cell culture, using a mAb against chicken occludin, whereas the others were amplified by polymerase chain reaction based on the sequence found around the human neuronal apoptosis inhibitory protein gene. The amino acid sequences of the three mammalian (human, murine, and canine) occludins were very closely related to each other (approximately 90% identity), whereas they diverged considerably from those of chicken and rat-kangaroo (approximately 50% identity). Implications of these data and novel experimental options in cell biological research are discussed.

  5. Gastrointestinal microbiota of wild and inbred individuals of two house mouse subspecies assessed using high-throughput parallel pyrosequencing.

    PubMed

    Kreisinger, Jakub; Cížková, Dagmar; Vohánka, Jaroslav; Piálek, Jaroslav

    2014-10-01

    The effects of gastrointestinal tract microbiota (GTM) on host physiology and health have been the subject of considerable interest in recent years. While a variety of captive bred species have been used in experiments, the extent to which GTM of captive and/or inbred individuals resembles natural composition and variation in wild populations is poorly understood. Using 454 pyrosequencing, we performed 16S rDNA GTM barcoding for 30 wild house mice (Mus musculus) and wild-derived inbred strain mice belonging to two subspecies (M. m. musculus and M. m. domesticus). Sequenced individuals were selected according to a 2 × 2 experimental design: wild (14) vs. inbred origin (16) and M. m. musculus (15) vs. M. m. domesticus (15). We compared alpha diversity (i.e. number of operational taxonomic units - OTUs), beta diversity (i.e. interindividual variability) and microbiota composition across the four groups. We found no difference between M. m. musculus and M. m. domesticus subspecies, suggesting low effect of genetic differentiation between these two subspecies on GTM structure. Both inbred and wild populations showed the same level of microbial alpha and beta diversity; however, we found strong differentiation in microbiota composition between wild and inbred populations. Relative abundance of ~ 16% of OTUs differed significantly between wild and inbred individuals. As laboratory mice represent the most abundant model for studying the effects of gut microbiota on host metabolism, immunity and neurology, we suggest that the distinctness of laboratory-kept mouse microbiota, which differs from wild mouse microbiota, needs to be considered in future biomedical research. PMID:25204516

  6. Gastrointestinal microbiota of wild and inbred individuals of two house mouse subspecies assessed using high-throughput parallel pyrosequencing.

    PubMed

    Kreisinger, Jakub; Cížková, Dagmar; Vohánka, Jaroslav; Piálek, Jaroslav

    2014-10-01

    The effects of gastrointestinal tract microbiota (GTM) on host physiology and health have been the subject of considerable interest in recent years. While a variety of captive bred species have been used in experiments, the extent to which GTM of captive and/or inbred individuals resembles natural composition and variation in wild populations is poorly understood. Using 454 pyrosequencing, we performed 16S rDNA GTM barcoding for 30 wild house mice (Mus musculus) and wild-derived inbred strain mice belonging to two subspecies (M. m. musculus and M. m. domesticus). Sequenced individuals were selected according to a 2 × 2 experimental design: wild (14) vs. inbred origin (16) and M. m. musculus (15) vs. M. m. domesticus (15). We compared alpha diversity (i.e. number of operational taxonomic units - OTUs), beta diversity (i.e. interindividual variability) and microbiota composition across the four groups. We found no difference between M. m. musculus and M. m. domesticus subspecies, suggesting low effect of genetic differentiation between these two subspecies on GTM structure. Both inbred and wild populations showed the same level of microbial alpha and beta diversity; however, we found strong differentiation in microbiota composition between wild and inbred populations. Relative abundance of ~ 16% of OTUs differed significantly between wild and inbred individuals. As laboratory mice represent the most abundant model for studying the effects of gut microbiota on host metabolism, immunity and neurology, we suggest that the distinctness of laboratory-kept mouse microbiota, which differs from wild mouse microbiota, needs to be considered in future biomedical research.

  7. Ammonia Levels and Urine-Spot Characteristics as Cage-Change Indicators for High-Density Individually Ventilated Mouse Cages.

    PubMed

    Washington, Ida M; Payton, Mark E

    2016-01-01

    Mouse cage and bedding changes are potentially stressful to mice and are also labor- and resource-intensive. These changes are often performed on a calendar-based schedule to maintain a clean microenvironment and limit the concentrations of ammonia to which mice and workers are exposed. The current study sought to establish a performance-based approach to mouse cage-changing that uses urine spot characteristics as visual indicators of intracage ammonia levels. Colorimetric ammonia indicators were used to measure ammonia levels in individually-ventilated cages (IVC) housing male or female mice (n =5 per cage) of various strains at 1 to 16 d after cage change. Urine spot characteristics were correlated with ammonia levels to create a visual indicator of the cage-change criterion of 25 ppm ammonia. Results demonstrated a consistent increase in ammonia levels with days since cage change, with cages reaching the cage-change criterion at approximately 10 d for IVC containing male mice and 16 d for those with female mice. Ammonia levels were higher for male than female mice but were not correlated with mouse age. However, urine spot diameter, color, and edge characteristics were strongly correlated with ammonia levels. Husbandry practices based on using urine spot characteristics as indicators of ammonia levels led to fewer weekly cage changes and concomitant savings in labor and resources. Therefore, urine spot characteristics can be used as visual indicators of intracage ammonia levels for use of a performance (urine spot)-based approach to cage-changing frequency that maintains animal health and wellbeing. PMID:27177558

  8. Structure of the FBJ murine osteosarcoma virus genome: molecular cloning of its associated helper virus and the cellular homolog of the v-fos gene from mouse and human cells.

    PubMed Central

    Curran, T; MacConnell, W P; van Straaten, F; Verma, I M

    1983-01-01

    The 8.2-kilobase (kb) unintegrated circular DNA form of the FBJ murine leukemia virus (FBJ-MLV) was linearized by cleavage at the single HindIII site, molecularly cloned into bacteriophage Charon 30, and subsequently subcloned into pBR322 (pFBJ-MLV-1). Both FBJ-MLV virion RNA and pFBJ-MLV-1 DNA were used to investigate the arrangement of helper virus sequences in the FBJ murine osteosarcoma virus genome (FBJ-MSV) by heteroduplex formation with cloned FBJ-MSV proviral DNA. The results showed that the FBJ-MSV genome contained 0.8 kb of helper virus sequence at its 5' terminus and 0.98 kb at its 3' terminus. Approximately 6.8 kb of helper virus sequence had been deleted, and 1.7 kb of unrelated sequence was inserted into the FBJ-MSV genome. This substituted region contains v-fos, the transforming gene of FBJ-MSV. Using a probe specific for v-fos, we have cloned homologous sequences (c-fos) from mouse and human chromosomal DNA. Heteroduplex analysis of FBJ-MSV DNA with these recombinant clones showed that both the c-fos(mouse) and the c-fos(human) sequences hybridized to the entire 1.7-kb v-fos region. However, five regions of homology of 0.27, 0.26, 0.14, 0.5, and 0.5 kb were separated by four regions of nonhomology of 0.76, 0.55, 0.1, and 0.1 kb from 5' to 3' with respect to the FBJ-MSV genome. The size of these sequences showed striking similarity in both c-fos(mouse) and c-fos(human). Images PMID:6306448

  9. Visualizing the Distribution of Synapses from Individual Neurons in the Mouse Brain

    PubMed Central

    Li, Ling; Tasic, Bosiljka; Micheva, Kristina D.; Ivanov, Vsevolod M.; Spletter, Maria L.; Smith, Stephen J.; Luo, Liqun

    2010-01-01

    Background Proper function of the mammalian brain relies on the establishment of highly specific synaptic connections among billions of neurons. To understand how complex neural circuits function, it is crucial to precisely describe neuronal connectivity and the distributions of synapses to and from individual neurons. Methods and Findings In this study, we present a new genetic synaptic labeling method that relies on expression of a presynaptic marker, synaptophysin-GFP (Syp-GFP) in individual neurons in vivo. We assess the reliability of this method and use it to analyze the spatial patterning of synapses in developing and mature cerebellar granule cells (GCs). In immature GCs, Syp-GFP is distributed in both axonal and dendritic regions. Upon maturation, it becomes strongly enriched in axons. In mature GCs, we analyzed synapses along their ascending segments and parallel fibers. We observe no differences in presynaptic distribution between GCs born at different developmental time points and thus having varied depths of projections in the molecular layer. We found that the mean densities of synapses along the parallel fiber and the ascending segment above the Purkinje cell (PC) layer are statistically indistinguishable, and higher than previous estimates. Interestingly, presynaptic terminals were also found in the ascending segments of GCs below and within the PC layer, with the mean densities two-fold lower than that above the PC layer. The difference in the density of synapses in these parts of the ascending segment likely reflects the regional differences in postsynaptic target cells of GCs. Conclusions The ability to visualize synapses of single neurons in vivo is valuable for studying synaptogenesis and synaptic plasticity within individual neurons as well as information flow in neural circuits. PMID:20634890

  10. Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain.

    PubMed Central

    Kiss, A; Farah, K; Kim, J; Garriock, R J; Drysdale, T A; Hammond, J R

    2000-01-01

    Mammalian cells express at least two subtypes of equilibrative nucleoside transporters, i.e. ENT1 and ENT2, which can be distinguished functionally by their sensitivity and resistance respectively to inhibition by nitrobenzylthioinosine. The ENT1 transporters exhibit distinctive species differences in their sensitivities to inhibition by dipyridamole, dilazep and draflazine (human>mouse>rat). A comparison of the ENT1 structures in the three species would facilitate the identification of the regions involved in the actions of these cardioprotective agents. We now report the molecular cloning and functional expression of the murine (m)ENT1 and mENT2 transporters. mENT1 and mENT2 encode proteins containing 458 and 456 residues respectively, with a predicted 11-transmembrane-domain topology. mENT1 has 88% and 78% amino acid identity with rat ENT1 and human ENT1 respectively; mENT2 is more highly conserved, with 94% and 88% identity with rat ENT2 and human ENT2 respectively. We have also isolated two additional distinct cDNAs that encode proteins similar to mENT1; these probably represent distinct mENT1 isoforms or alternative splicing products. One cDNA encodes a protein with two additional amino acids (designated mENT1b) that adds a potential protein kinase CK2 phosphorylation site in the central intracellular loop of the transporter, and is similar, in this regard, to the human and rat ENT1 orthologues. The other cDNA has a 5'-untranslated region sequence that is distinct from that of full-length mENT1. Microinjection of mENT1, mENT1b or mENT2 cRNA into Xenopus oocytes resulted in enhanced uptake of [(3)H]uridine by the oocytes relative to that seen in water-injected controls. mENT1-mediated, but not mENT2-mediated, [(3)H]uridine uptake was inhibited by nitrobenzylthioinosine and dilazep. Dipyridamole inhibited both mENT1 and mENT2, but was significantly more effective against mENT1. Adenosine inhibited both systems with a similar potency, as did a range of other

  11. Bioluminescence imaging of mitochondrial Ca2+ dynamics in soma and neurites of individual adult mouse sympathetic neurons

    PubMed Central

    Núñez, Lucía; Senovilla, Laura; Sanz-Blasco, Sara; Chamero, Pablo; Alonso, María T; Villalobos, Carlos; García-Sancho, Javier

    2007-01-01

    Changes in the cytosolic Ca2+ concentration ([Ca2+]c) are essential for triggering neurotransmitter release from presynaptic nerve terminals. Calcium-induced Ca2+ release (CICR) from the endoplasmic reticulum (ER) may amplify the [Ca2+]c signals and facilitate neurotransmitter release in sympathetic neurons. In adrenal chromaffin cells, functional triads are formed by voltage-operated Ca2+ channels (VOCCs), CICR sites and mitochondria. In fact, mitochondria take up most of the Ca2+ load entering the cells and are essential for shaping [Ca2+]c signals and exocytosis. Here we have investigated the existence of such functional triads in sympathetic neurons. The mitochondrial Ca2+ concentration ([Ca2+]m) in soma and neurites of individual mouse superior cervical ganglion (SCG) neurons was monitored by bioluminescence imaging of targeted aequorins. In soma, Ca2+ entry through VOCCs evoked rapid, near millimolar [Ca2+]m increases in a subpopulation of mitochondria containing about 40% of the aequorin. Caffeine evoked a similar [Ca2+]m increase in a mitochondrial pool containing about 30% of the aequorin and overlapping with the VOCC-sensitive pool. These observations suggest the existence of functional triads similar to the ones described in chromaffin cells. In neurites, mitochondria were able to buffer [Ca2+]c increases resulting from activation of VOCCs but not those mediated by caffeine-induced Ca2+ release from the ER. The weaker Ca2+ buffering by mitochondria in neurites could contribute to facilitate Ca2+-induced exocytosis at the presynaptic sites. PMID:17234693

  12. Cloning cattle.

    PubMed

    Oback, B; Wells, D N

    2003-01-01

    Over the past six years, hundreds of apparently normal calves have been cloned worldwide from bovine somatic donor cells. However, these surviving animals represent less than 5% of all cloned embryos transferred into recipient cows. Most of the remaining 95% die at various stages of development from a predictable pattern of placental and fetal abnormalities, collectively referred to as the "cloning-syndrome." The low efficiency seriously limits commercial applicability and ethical acceptance of somatic cloning and enforces the development of improved cloning methods. In this paper, we describe our current standard operating procedure (SOP) for cattle cloning using zona-free nuclear transfer. Following this SOP, the output of viable and healthy calves at weaning is about 9% of embryos transferred. Better standardization of cloning protocols across and within research groups is needed to separate technical from biological factors underlying low cloning efficiency.

  13. Why Clone?

    MedlinePlus

    ... How might cloning be used in medicine? Cloning animal models of disease Much of what researchers learn about human disease comes from studying animal models such as mice. Often, animal models are ...

  14. Cloning of a parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) cDNA from a rat osteosarcoma (UMR 106) cell line: Chromosomal assignment of the gene in the human, mouse, and rat genomes

    SciTech Connect

    Pausova, Z.; Bourdon, J.; Clayton, D.; Janicic, N.; Goltzman, D.; Hendy, G.N. ); Mattei, M.G. ); Seldin, M.F. ); Riviere, M.; Szpirer, J. )

    1994-03-01

    Complementary DNAs spanning the entire coding region of the rat parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) were isolated from a rat osteosarcoma (UMR 106) cell-line cDNA library. The longest of these clones (rPTHrec4) was used to chromosomally assign the PTHR gene in the human, rat, and mouse genomes. By somatic cell hybrid analysis, the gene was localized to human chromosome 3 and rat chromosome 8; by in situ hybridization, the gene was mapped to human chromosome 3p21.1-p22 and to mouse chromosome 9 band F; and by interspecific backcross analysis, the Pthr gene segregated with the transferrin (Trf) gene in chromosome 9 band F. Mouse chromosome 9 and rat chromosome 8 are known to be highly homologous and to also show synteny conservation with human chromosome 3. These three chromosomes share the transferrin gene (TF), the myosin light polypeptide 3 gene (MYL3), and the acelpeptide hydrolase gene (APEH). These results add a fourth gene, the PTHR gene, to the synteny group conserved in these chromosomes. 34 refs., 7 figs. 1 tab.

  15. Ethical issues in livestock cloning.

    PubMed

    Thompson, P B

    1999-01-01

    Although cloning may eventually become an important technology for livestock production, four ethical issues must be addressed before the practice becomes widespread. First, researchers must establish that the procedure is not detrimental to the health or well-being of affected animals. Second, animal research institutions should evaluate the net social benefits to livestock producers by weighing the benefits to producers against the opportunity cost of research capacity lost to biomedical projects. Third, scientists should consider the indirect effects of cloning research on the larger ethical issues surrounding human cloning. Finally, the market structure for products of cloned animals should protect individual choice, and should recognize that many individuals find the prospect of cloning (or consuming cloned animals) repugnant. Analysis of these four issues is complicated by spurious arguments alleging that cloning will have a negative impact on environment and genetic diversity.

  16. Ethical issues in livestock cloning.

    PubMed

    Thompson, P B

    1999-01-01

    Although cloning may eventually become an important technology for livestock production, four ethical issues must be addressed before the practice becomes widespread. First, researchers must establish that the procedure is not detrimental to the health or well-being of affected animals. Second, animal research institutions should evaluate the net social benefits to livestock producers by weighing the benefits to producers against the opportunity cost of research capacity lost to biomedical projects. Third, scientists should consider the indirect effects of cloning research on the larger ethical issues surrounding human cloning. Finally, the market structure for products of cloned animals should protect individual choice, and should recognize that many individuals find the prospect of cloning (or consuming cloned animals) repugnant. Analysis of these four issues is complicated by spurious arguments alleging that cloning will have a negative impact on environment and genetic diversity. PMID:15719505

  17. [Cloning - controversies].

    PubMed

    Twardowski, T; Michalska, A

    2001-01-01

    Cloning of the human being is not only highly controversial; in the opinion of the authors it is impossible - we are not able to reproduce human behaviour and character traits. Reproduction through cloning is limited to personal genome resources. The more important is protection of genomic characteristics as private property and taking advantage of cloning for production of the human organs directly or through xenotransplants. In this paper we present the legislation related to cloning in Poland, in the European Union and other countries. We also indicate who and why is interested in cloning.

  18. T-cell receptor heterogeneity of gamma delta T-cell clones from human female reproductive tissues.

    PubMed

    Christmas, S E; Brew, R; Deniz, G; Taylor, J J

    1993-03-01

    gamma delta T cells were isolated from human decidua parietalis, decidua basalis and cervix and cloned in the presence of interleukin-2 (IL-2). T-cell receptor (TcR) expression was then analysed and compared with that of a panel of gamma delta T-cell clones from peripheral blood. Only 17/40 (42.5%) clones from decidua parietalis were V gamma 9+/V delta 2+ as compared to 68/94 (72%) of peripheral blood clones (P < 0.005). Conversely, 50% of clones from decidua parietalis but only 15% of clones from peripheral blood were V delta 1+ (P < 0.001). At least seven distinct TcR types were identified among the panel of clones from decidua parietalis and at least six different types were expressed by the panel of 17 clones from cervix. This receptor heterogeneity was not a result of interdonor variation as in all instances where more than one clone was obtained from a single sample, individual clones having between two and five receptor types were identified. However, 23/24 (95.8%) of clones from decidua basalis were V gamma 9+/V delta 2+. Most clones from decidua parietalis and cervix, whether V gamma 9+/V delta 2+ or V delta 1+, were positive for the mucosal lymphocyte marker, HML-1, but expression was often heterogeneous within a single clone. In contrast, almost all gamma delta T-cell clones from peripheral blood were HML-1-. Thus, unlike the mouse, gamma delta T cells within these human female reproductive tissues have a diverse TcR repertoire which, in decidua parietalis, is distinct from that of peripheral blood.

  19. Comparative mapping on the mouse and human X chromosomes of a human cDNA clone encoding the vasopressin renal-type receptor (AVP2R)

    SciTech Connect

    Faust, C.J.; Gonzales, J.C.; Seibold, A.; Birnbaumer, M.; Herman, G.E. )

    1993-02-01

    Mutation in the gene for the human renal-type vasopressin receptor (V2R) have recently been identified in patients with nephrogenic diabetes insipidus (NDI). Both V2R and NDI have been independently mapped to Xq28. Using a combination of genetic and physical mapping, we have localized the murine V2r locus to within 100 kb of L1Cam on the mouse X chromosome in a region syntenic with human Xq28. Based on conserved gene order of mouse and human loci in this region, physical mapping using DNA derived form human lymphoblasts has established that the corresponding human loci V2R and L1CAM are linked within 210 kb. The efficiency and precision of genetic mapping of V2r and other loci in the mouse suggest that it might be easier to map additional human genes in the mouse first and infer the corresponding human location. More precise physical mapping in man could then be performed using pulsed-field gel electrophoresis and/or yeast artificial chromosomes. 16 refs., 1 fig. 1 tab.

  20. Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus

    SciTech Connect

    Kwon, B.S.; Haq, A.K.; Pomerantz, S.H.; Halaban, R.

    1987-11-01

    Screening of a lambdagt11 human melanocyte cDNA library with antibodies against hamster tyrosinase resulted in the isolation of 16 clones. The cDNA inserts from 13 of the 16 clones cross-hybridized with each other, indicating that they were form related mRNA species. One of the cDNA clones, Pmel34, detected one mRNA species with an approximate length of 2.4 kilobases that was expressed preferentially in normal and malignant melanocytes but not in other cell types. The amino acid sequence deduced from the nucleotide sequence showed that the putative human tyrosinase is composed of 548 amino acids with a molecular weight of 62,610. The deduced protein contains glycosylation sites and histidine-rich sites that could be used for copper binding. Southern blot analysis of DNA derived from newborn mice carrying lethal albino deletion mutations revealed that Pmel34 maps near or at the c-albino locus, the position of the structural gene for tyrosinase.

  1. Imperfect Cloning Operations in Algebraic Quantum Theory

    NASA Astrophysics Data System (ADS)

    Kitajima, Yuichiro

    2015-01-01

    No-cloning theorem says that there is no unitary operation that makes perfect clones of non-orthogonal quantum states. The objective of the present paper is to examine whether an imperfect cloning operation exists or not in a C*-algebraic framework. We define a universal -imperfect cloning operation which tolerates a finite loss of fidelity in the cloned state, and show that an individual system's algebra of observables is abelian if and only if there is a universal -imperfect cloning operation in the case where the loss of fidelity is less than . Therefore in this case no universal -imperfect cloning operation is possible in algebraic quantum theory.

  2. Twins: A cloning experience.

    PubMed

    Prainsack, Barbara; Spector, Tim D

    2006-11-01

    Drawing upon qualitative interviews with monozygotic (identical) twins sharing 100% of their genes, and with dizygotic (fraternal) twins and singletons as control groups, this paper explores what it means to be genetically identical. (The twins interviewed were from the TwinsUK register in London.) In the context of the ongoing debate on human reproductive cloning, it examines questions such as: To what extent do identical twins perceive their emotional and physical bond to be a result of their genetic makeup? What would they think if they had been deliberately created genetically identical? How would they feel about being genetically identical to a person who was born a few years earlier or later? First, our respondents ascribed no great significance to the role of genes in their understanding of what it means to be identical twins. Second, the opinion that human reproductive cloning would "interfere with nature", or "contradict God's will", was expressed by our respondents exclusively on the abstract level. The more our respondents were able to relate a particular invented cloning scenario to their own life-worlds, the lower the prevalence of the argument. Third, for all three groups of respondents, the scenario of having been born in one of the other groups was perceived as strange. Fourth, the aspect that our respondents disliked about cloning scenarios was the potential motives of the cloners. Without equating monozygotic twins directly with "clones", these results from "naturally" genetically identical individuals add a new dimension to what a future cloning situation could entail: The cloned person might possibly (a) perceive a close physical and emotional connection to the progenitor as a blessing; (b) suffer from preconceptions of people who regard physical likeness as a sign of incomplete individuality; and (c) perceive the idea of not having been born a clone of a particular person as unpleasant.

  3. Cloning of cDNAs that encode human mast cell carboxypeptidase A, and comparison of the protein with mouse mast cell carboxypeptidase A and rat pancreatic carboxypeptidases

    SciTech Connect

    Reynolds, D.S.; Gurley, D.S.; Stevens, R.L.; Austen, K.F.; Serafin, W.E. Brigham and Women's Hospital, Boston, MA ); Sugarbaker, D.J. )

    1989-12-01

    Human skin and lung mast cells and rodent peritoneal cells contain a carboxypeptidase in their secretory granules. The authors have screened human lung cDNA libraries with a mouse mast cell carboxypeptidase A (MC-CPA) cDNA probe to isolate a near-full-length cDNA that encodes human MC-CPA. The 5{prime} end of the human MC-CPA transcript was defined by direct mRNA sequencing and by isolation and partial sequencing of the human MC-CPA gene. Human MC-CPA is predicted to be translated as a 417 amino acid preproenzyme which includes a 15 amino acid signal peptide and a 94-amino acid activation peptide. The mature human MC-CPA enzyme has a predicted size of 36.1 kDa, a net positive charge of 16 at neutral pH, and 86% amino acid sequence identity with mouse MC-CPA. DNA blot analyses showed that human MC-CPA mRNA is transcribed from a single locus in the human genome. Comparison of the human MC-CPA with mouse MC-CPA and with three rat pancreatic carboxypeptidases shows that these enzymes are encoded by distinct but homologous genes.

  4. Academic Cloning.

    ERIC Educational Resources Information Center

    Sikula, John P.; Sikula, Andrew F.

    1980-01-01

    The authors define "cloning" as an integral feature of all educational systems, citing teaching practices which reward students for closely reproducing the teacher's thoughts and/or behaviors and administrative systems which tend to promote like-minded subordinates. They insist, however, that "academic cloning" is not a totally negative practice.…

  5. Mitochondrial HMG to CoA synthase (mHS): cDNA cloning in human, mouse and C. elegans, mapping to human chromosome 1p12-13 and partial human genomic cloning

    SciTech Connect

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A. |

    1994-09-01

    mHS catalyzes the rate-limiting first step of ketogenesis in the liver. A cytoplasmic HS isozyme, encoded by another gene, catalyzes an early step in cholesterol synthesis. Starting from a rat mHS cDNA obtained by RT-PCR from the published rat cDNA sequence, we obtained and sequenced human and mouse cDNAs spanning the entire coding sequence of natural human and mouse mHS, as well as sequencing C. elegans HS-like cDNA. Consensus sequences for 3 mitochondrial and 4 cytoplasmic HSs were created and compared to invertebrate HS sequences. We found high conversation in the active site and at other regions presumably important for HS function. We mapped the mHS locus, HMGCS2 by in situ hybridization to chromosome 1P12-13, in contrast to the human cHS locus (HMGCS1) known to be on chromosome 5p13. Comparative mapping results suggest that these two chromosomal regions may be contiguous in other species, constant with a recent gene duplication event. Furthermore, we have characterized a human genomic mHS subclone containing 4 mHS exons, and found the position of all splice junctions to be identical to that of the hamster cHS gene except for one site in the 3{prime} nontranslated region. We calculate that the mHS and cHS genes were derived from a common ancestor 400-700 Myrs ago, implying that ketogenesis from fat may have become possible around the time of emergence of vertebrates ({approximately}500 Myr ago). Ketogenesis has evolved into an important pathway of energy metabolism, and we predict the mHS deficiency may prove to be responsible for some as yet explained cases of Reye-like syndromes in humans. This hypothesis can now be tested at the molecular level without the necessity of obtaining hepatic tissue.

  6. Cloning-free CRISPR

    PubMed Central

    Arbab, Mandana; Srinivasan, Sharanya; Hashimoto, Tatsunori; Geijsen, Niels; Sherwood, Richard I.

    2015-01-01

    Summary We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis. PMID:26527385

  7. Stable isotopes complement focal individual observations and confirm dietary variability in reddish-gray mouse lemurs (Microcebus griseorufus) from southwestern Madagascar.

    PubMed

    Crowley, Brooke E; Rasoazanabary, Emilienne; Godfrey, Laurie R

    2014-09-01

    We examine the ecology of reddish-gray mouse lemurs from three habitats at Beza Mahafaly Special Reserve using focal follows and stable carbon and nitrogen isotope data. Focal observations indicate dietary differences among habitats as well as sexes and seasons. Both sexes consume more arthropods during the rainy season but overall, females consume more sugar-rich exudates and fruit than males, and individuals from riparian forest consume fewer arthropods and more fruit than those in xeric or dry forest. We ask whether these observations are isotopically detectable. Isotope data support differences between seasons and sexes. Nitrogen isotope values are higher during the rainy season when lemurs consume more arthropods, and higher in males than females, particularly during the dry season. However, differences among populations inferred from focal observations are not fully supported. Lemurs from riparian forest have lower isotope values than those in xeric scrub, but isotope data suggest that lemurs from the dry forest eat the least animal matter and that focal observations overestimated dry forest arthropod consumption. Overall, our results suggest that observational and isotopic data are complementary. Isotope data can be obtained from a larger number of individuals and can quantify ingestion of animal matter, but they apparently cannot quantify the relative consumption of different sugar-rich foods. Combined focal and isotope data provide valuable insight into the dietary constraints of reddish-grey mouse lemurs, with implications for their vulnerability to future habitat change.

  8. The mouse glucocorticoid receptor: mapping of functional domains by cloning, sequencing and expression of wild-type and mutant receptor proteins.

    PubMed Central

    Danielsen, M; Northrop, J P; Ringold, G M

    1986-01-01

    We have isolated mouse glucocorticoid receptor (GR) cDNAs which, when expressed in transfected mammalian cells, produce a fully functional GR protein. Sequence analysis reveals an open reading frame of 2349 bp which could encode a protein of approximately 86,000 daltons. We have also isolated two receptor cDNAs from mouse S49 nuclear transfer-deficient (nt-) cells which encode mutant forms of the receptor protein. One cDNA encodes a protein that is unable to bind hormone and represents the endogenous hormone binding deficient receptor recently discovered in S49 cells. The lesion in this receptor is due to a single amino acid substitution (Glu-546 to Gly). The second cDNA from nt- cells produces a receptor protein that is able to bind hormone but has reduced nuclear binding. This cDNA, therefore, encodes for the S49 nt- receptor which has been shown to have reduced affinity for DNA. The lesion maps to a single amino acid substitution (Arg-484 to His) located in a highly Cys, Lys, Arg-rich region of the protein previously implicated in DNA binding. Our studies provide unambiguous identification of receptor domains and specific amino acids critical for the hormone and DNA binding properties of this transcriptional regulatory protein. Contained within the first 106 amino acids of the mouse GR is a stretch of nine glutamines with two prolines which are related to the family of transcribed repetitive elements, opa, found in Drosophila melanogaster. A truncated receptor lacking these 106 amino acids is functionally indistinguishable from the wild-type receptor. Images Fig. 2. Fig. 5. Fig. 6. Fig. 7. PMID:3780669

  9. Cloning of mouse telomerase reverse transcriptase gene promoter and identification of proximal core promoter sequences essential for the expression of transgenes in cancer cells.

    PubMed

    Si, Shao-Yan; Song, Shu-Jun; Zhang, Jian-Zhong; Liu, Jun-Li; Liang, Shuang; Feng, Kai; Zhao, Gang; Tan, Xiao-Qing

    2011-08-01

    Telomerase is a ribonucleoprotein complex, whose function is to add motif-specific nucleotides to the end of chromosomes. Telomerase consists of three major subunits, the telomerase RNA template (hTR), the telomerase-associated protein (TEP1) and telomerase reverse transcriptase (TERT). TERT is the most important component responsible for the catalytic activity of telomerase and a rate-limiting determinant of the activity. Telomerase activities were at high levels in approximately 90% of mouse cancers or tumor-derived cell lines through TERT transcriptional up-regulation. Unlike human telomerase, telomerase activity exists in colon, liver, ovary and testis but not in brain, heart, stomach and muscle in normal mouse tissues. In this study, we prepared 5' truncations of 1086 bp fragments upstream of the initiating ATG codon of the mTERT gene to construct luciferase reporter gene plasmids, and transfected these plasmids into a normal mouse cell line and several cancer lines to identify the core promoter region essential for transcriptional activation in cancer cells by a luciferase assay. We constructed a eukaryotic expression vector of membrane-expressing staphylococcal endotoxin A (SEA) gene driven by the core promoter region of the mTERT gene and observed if the core promoter region could express the SEA gene in these cancer cells, but not in normal cells following transfection with the construct. The results showed that the transcriptional activities of each fragment of the mTERT gene promoter in the cancer cell lines Hepa1-6, B16 and CT26 were higher than those in NIH3T3 cells, and the proximal 333-bp fragment was the core promoter of the mTERT gene in the cancer cells. The proximal 333-bp fragment was able to make the SEA express on the surface of the cancer cells, but not in NIH3T3 cells. It provides a foundation for cancer targeting gene therapy by using the mTERT gene promoter. PMID:21567104

  10. Mouse Curve Biometrics

    SciTech Connect

    Schulz, Douglas A.

    2007-10-08

    A biometric system suitable for validating user identity using only mouse movements and no specialized equipment is presented. Mouse curves (mouse movements with little or no pause between them) are individually classied and used to develop classication histograms, which are representative of an individual's typical mouse use. These classication histograms can then be compared to validate identity. This classication approach is suitable for providing continuous identity validation during an entire user session.

  11. Cloning and partial nucleotide sequence of human immunoglobulin mu chain cDNA from B cells and mouse-human hybridomas.

    PubMed Central

    Dolby, T W; Devuono, J; Croce, C M

    1980-01-01

    Purified mRNAs coding for mu and kappa human immunoglobulin polypeptides were translated in vitro and their products were characterized. The mu-specific mRNAs, derived from both human lymphoblastoid cells (GM607) and from a mouse-human somatic cell hybrid secreting human mu chains (alpha D5-H11-BC11), were copied into cDNAs and inserted into the plasmid pBR322. Several recombinant cDNAs that were obtained were identified by a combination of colony hybridization with labeled probes, in vitro translation of plasmid-selected mu mRNAs, and DNA nucleotide sequence determination. One recombinant DNA, for which the sequence has been partially determined, contains the codons for part of the C3 constant region domain through the carboxy-terminal piece (155 amino acids total) as well as the entire 3' noncoding sequence up to the poly(A) site of the human mu mRNA. The sequence A-A-U-A-A occurs 12 nucleotides prior to the poly(A) addition site in the human mu mRNA. Considerable sequence homology is observed in the mouse and human mu mRNA 3' coding and noncoding sequences. Images PMID:6777778

  12. Molecular cloning of mouse glycolate oxidase. High evolutionary conservation and presence of an iron-responsive element-like sequence in the mRNA.

    PubMed

    Kohler, S A; Menotti, E; Kühn, L C

    1999-01-22

    Iron regulatory proteins (IRPs) control the synthesis of several proteins in iron metabolism by binding to iron-responsive elements (IREs), a hairpin structure in the untranslated region (UTR) of corresponding mRNAs. Binding of IRPs to IREs in the 5' UTR inhibits translation of ferritin heavy and light chain, erythroid aminolevulinic acid synthase, mitochondrial aconitase, and Drosophila succinate dehydrogenase b, whereas IRP binding to IREs in the 3' UTR of transferrin receptor mRNA prolongs mRNA half-life. To identify new targets of IRPs, we devised a method to enrich IRE-containing mRNAs by using recombinant IRP-1 as an affinity matrix. A cDNA library established from enriched mRNA was screened by an RNA-protein band shift assay. This revealed a novel IRE-like sequence in the 3' UTR of a liver-specific mouse mRNA. The newly identified cDNA codes for a protein with high homology to plant glycolate oxidase (GOX). Recombinant protein expressed in bacteria displayed enzymatic GOX activity. Therefore, this cDNA represents the first vertebrate GOX homologue. The IRE-like sequence in mouse GOX exhibited strong binding to IRPs at room temperature. However, it differs from functional IREs by a mismatch in the middle of its upper stem and did not confer iron-dependent regulation in cells. PMID:9891009

  13. Lessons learned from cloning dogs.

    PubMed

    Kim, M J; Oh, H J; Kim, G A; Park, J E; Park, E J; Jang, G; Ra, J C; Kang, S K; Lee, B C

    2012-08-01

    The aim of this article is to review dog cloning research and to suggest its applications based on a discussion about the normality of cloned dogs. Somatic cell nuclear transfer was successfully used for production of viable cloned puppies despite limited understanding of in vitro dog embryo production. Cloned dogs have similar growth characteristics to those born from natural fertilization, with no evidence of serious adverse effects. The offspring of cloned dogs also have similar growth performance and health to those of naturally bred puppies. Therefore, cloning in domestic dogs can be applied as an assisted reproductive technique to conserve endangered species, to treat sterile canids or aged dogs, to improve reproductive performance of valuable individuals and to generate disease model animals.

  14. Development of dry skin in the NOA mouse under individual housing conditions: a potentially useful animal model for evaluating moisturizing effects.

    PubMed

    Kondo, Taizo; Ohno, Hitoshi; Kondo, Toshio; Shiomoto, Yasuhisa; Momii, Akira

    2005-10-01

    In a previous study, we reported the development of grossly observable dry skin in all of the Naruto Research Institute Otsuka Atrichia (NOA) mice that were housed individually. In the present study, dermal physiological function tests were conducted and the usefulness of this dry skin model for evaluating the efficacy of topical moisturizers was assessed. As a result, we have confirmed a marked reduction in the water content of the stratum corneum in these animals. Therefore, the development of dry skin in the NOA mouse strain under individual housing conditions may be expected to serve as a useful animal model for evaluating topical moisturizers. Specifically, the water content of the stratum corneum was restored in proportion to the oil content of the ointment base used to treat the animals, and the moisturizing effects of urea were confirmed in animals treated with urea-containing ointment. In addition, when the animals that had been housed individually were returned to group housing conditions, the water content of the stratum corneum was restored, with a corresponding improvement in dry skin. This finding suggests that socio-psychological factors are involved in the etiology of dry skin in individually housed NOA mice. PMID:16365520

  15. Chorioallantoic placenta defects in cloned mice

    SciTech Connect

    Wakisaka-Saito, Noriko; Kohda, Takashi . E-mail: tkhoda.epgn@tmd.ac.jp; Inoue, Kimiko; Ogonuki, Narumi; Miki, Hiromi; Hikichi, Takafusa; Mizutani, Eiji; Wakayama, Teruhiko; Kaneko-Ishino, Tomoko; Ogura, Atsuo; Ishino, Fumitoshi

    2006-10-13

    Somatic cell nuclear transfer technology has been applied to produce live clones successfully in several mammalian species, but the success rates are very low. In mice, about half of the nuclear transfer embryos undergo implantation, but very few survive to term. We undertook detailed histological analyses of placentas from cloned mouse embryos generated from cumulus cells at 10.5 dpc of pregnancy, by which stage most clones have terminated their development. At 10.5 dpc, the extraembryonic tissues displayed several defined histological patterns, each reflecting their stage of developmental arrest. The most notable abnormality was the poor development of the spongiotrophoblast layer of diploid cells. This is in contrast to the placental hyperplasia frequently observed in somatic clones at 12.5 dpc or later stages. A variety of structural abnormalities were also observed in the embryos. Both placental and embryonic defects likely contribute to the low success rate of the mouse clones.

  16. A mouse model for ulcerative colitis based on NOD-scid IL2R γnull mice reconstituted with peripheral blood mononuclear cells from affected individuals.

    PubMed

    Palamides, Pia; Jodeleit, Henrika; Föhlinger, Michael; Beigel, Florian; Herbach, Nadja; Mueller, Thomas; Wolf, Eckhard; Siebeck, Matthias; Gropp, Roswitha

    2016-09-01

    Animal models reflective of ulcerative colitis (UC) remain a major challenge, and yet are crucial to understand mechanisms underlying the onset of disease and inflammatory characteristics of relapses and remission. Mouse models in which colitis-like symptoms are induced through challenge with toxins such as oxazolone, dextran sodium sulfate (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) have been instrumental in understanding the inflammatory processes of UC. However, these neither reflect the heterogeneous symptoms observed in the UC-affected population nor can they be used to test the efficacy of inhibitors developed against human targets where high sequence and structural similarity of the respective ligands is lacking. In an attempt to overcome these problems, we have developed a mouse model that relies on NOD-scid IL2R γ(null) mice reconstituted with peripheral blood mononuclear cells derived from UC-affected individuals. Upon challenge with ethanol, mice developed colitis-like symptoms and changes in the colon architecture, characterized by influx of inflammatory cells, edema, crypt loss, crypt abscesses and epithelial hyperplasia, as previously observed in immune-competent mice. TARC, TGFβ1 and HGF expression increased in distal parts of the colon. Analysis of human leucocytes isolated from mouse spleen revealed an increase in frequencies of CD1a+, CD64+, CD163+ and TSLPR+ CD14+ monocytes, and antigen-experienced CD44+ CD4+ and CD8+ T-cells in response to ethanol. Analysis of human leucocytes from the colon of challenged mice identified CD14+ monocytes and CD11b+ monocytes as the predominant populations. Quantitative real-time PCR (RT-PCR) analysis from distal parts of the colon indicated that IFNγ might be one of the cytokines driving inflammation. Treatment with infliximab ameliorated symptoms and pathological manifestations, whereas pitrakinra had no therapeutic benefit. Thus, this model is partially reflective of the human disease and might help

  17. A mouse model for ulcerative colitis based on NOD-scid IL2R γnull mice reconstituted with peripheral blood mononuclear cells from affected individuals

    PubMed Central

    Palamides, Pia; Jodeleit, Henrika; Föhlinger, Michael; Beigel, Florian; Herbach, Nadja; Mueller, Thomas; Wolf, Eckhard; Siebeck, Matthias

    2016-01-01

    ABSTRACT Animal models reflective of ulcerative colitis (UC) remain a major challenge, and yet are crucial to understand mechanisms underlying the onset of disease and inflammatory characteristics of relapses and remission. Mouse models in which colitis-like symptoms are induced through challenge with toxins such as oxazolone, dextran sodium sulfate (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) have been instrumental in understanding the inflammatory processes of UC. However, these neither reflect the heterogeneous symptoms observed in the UC-affected population nor can they be used to test the efficacy of inhibitors developed against human targets where high sequence and structural similarity of the respective ligands is lacking. In an attempt to overcome these problems, we have developed a mouse model that relies on NOD-scid IL2R γnull mice reconstituted with peripheral blood mononuclear cells derived from UC-affected individuals. Upon challenge with ethanol, mice developed colitis-like symptoms and changes in the colon architecture, characterized by influx of inflammatory cells, edema, crypt loss, crypt abscesses and epithelial hyperplasia, as previously observed in immune-competent mice. TARC, TGFβ1 and HGF expression increased in distal parts of the colon. Analysis of human leucocytes isolated from mouse spleen revealed an increase in frequencies of CD1a+, CD64+, CD163+ and TSLPR+ CD14+ monocytes, and antigen-experienced CD44+ CD4+ and CD8+ T-cells in response to ethanol. Analysis of human leucocytes from the colon of challenged mice identified CD14+ monocytes and CD11b+ monocytes as the predominant populations. Quantitative real-time PCR (RT-PCR) analysis from distal parts of the colon indicated that IFNγ might be one of the cytokines driving inflammation. Treatment with infliximab ameliorated symptoms and pathological manifestations, whereas pitrakinra had no therapeutic benefit. Thus, this model is partially reflective of the human disease and might

  18. Undetectable levels of N6-methyl adenine in mouse DNA: Cloning and analysis of PRED28, a gene coding for a putative mammalian DNA adenine methyltransferase.

    PubMed

    Ratel, David; Ravanat, Jean-Luc; Charles, Marie-Pierre; Platet, Nadine; Breuillaud, Lionel; Lunardi, Joël; Berger, François; Wion, Didier

    2006-05-29

    Three methylated bases, 5-methylcytosine, N4-methylcytosine and N6-methyladenine (m6A), can be found in DNA. However, to date, only 5-methylcytosine has been detected in mammalian genomes. To reinvestigate the presence of m6A in mammalian DNA, we used a highly sensitive method capable of detecting one N6-methyldeoxyadenosine per million nucleosides. Our results suggest that the total mouse genome contains, if any, less than 10(3) m6A. Experiments were next performed on PRED28, a putative mammalian N6-DNA methyltransferase. The murine PRED28 encodes two alternatively spliced RNA. However, although recombinant PRED28 proteins are found in the nucleus, no evidence for an adenine-methyltransferase activity was detected. PMID:16684535

  19. Definition of the tumor protein D52 (TPD52) gene family through cloning of D52 homologues in human (hD53) and mouse (mD52)

    SciTech Connect

    Byrne, J.A.; Basset, P.; Mattei, M.G.

    1996-08-01

    Cloning is reported of a cDNA homologue to the breast carcinoma-associated D52 cDNA, termed D53, and of a mouse D52 cDNA (HGMW-approved symbols TPD52L1 and TPD52). Human D53 and mouse D52 proteins are predicted to be 52 and 86% identical to human D52, respectively. Analysis of the three protein sequences identified a coiled-coil domain and N- and C-terminally located PEST domains in each. The conservation of homology between the D52 and the D53 sequences, combined with a lack of homology between these and known proteins, defines a new mammalian gene/protein family, the D52 family. The human D52 locus has been previously mapped to chromosome 8q21, and using in situ mapping in the present study, a human D53 locus was mapped to chromosome 6q22-q23. We observed coexpression of the human D52 and D53 genes in some breast tumors and derivative cell lines and found that maintenance of D52 and D53 transcript levels in estrogen receptor-positive MCF7 breast carcinoma cells depends upon estradiol. However, D52 and D53 genes were specifically expressed in HL-60 and K-562 leukemia cells, respectively, with 12-O-tetradecanoylphorbol-13-acetate treatment decreasing D52 and D53 transcript levels in these cell lines. The presence of a coiled-coil domain, combined with observed codependent or independent expression of the D52 and D53 genes, suggests that D52 and D53 proteins may be capable of hetero- and/or homodimer formation. 41 refs., 5 figs., 1 tab.

  20. Nuclear factor XIIIa staining (clone AC-1A1 mouse monoclonal) is a sensitive and specific marker to discriminate sebaceous proliferations from other cutaneous clear cell neoplasms.

    PubMed

    Uhlenhake, Elizabeth E; Clark, Lindsey N; Smoller, Bruce R; Shalin, Sara C; Gardner, Jerad M

    2016-08-01

    Sebaceous carcinoma is a rare but serious malignancy that may be difficult to diagnose when poorly differentiated. Other epithelial tumors with clear cell change may mimic sebaceous carcinoma. Few useful or specific immunohistochemical markers for sebaceous differentiation are available. Nuclear staining with factor XIIIa (clone AC-1A1) was recently found to be a highly sensitive marker of sebaceous differentiation. We evaluated nuclear factor XIIIa (AC-1A1) staining in sebaceous neoplasms vs. other cutaneous clear cell tumors. We stained 27 sebaceous proliferations: sebaceous hyperplasia (7), sebaceous adenoma (8), sebaceoma (5), sebaceous carcinoma (7). We also stained 67 tumors with clear cell change: basal cell carcinoma (8), squamous cell carcinoma (8), hidradenoma (7), desmoplastic trichilemmoma (2), trichilemmoma (10), trichilemmal carcinoma (3), clear cell acanthoma (9), atypical fibroxanthoma (1), syringoma (8), trichoepithelioma (1), metastatic renal cell carcinoma (2), and nevi with balloon cell change (8). Nuclear factor XIIIa (AC-1A1) staining was present in 100% of sebaceous proliferations; 96% displayed strong staining. Non-sebaceous clear cell tumors were negative or only weakly positive with factor XIIIa (AC-1A1) in 95.5%; only 4.5% showed strong staining. This suggests that strong nuclear factor XIIIa (AC-1A1) staining is a sensitive and specific marker of sebaceous neoplasms vs. other clear cell tumors.

  1. Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha.

    PubMed Central

    Lax, I; Johnson, A; Howk, R; Sap, J; Bellot, F; Winkler, M; Ullrich, A; Vennstrom, B; Schlessinger, J; Givol, D

    1988-01-01

    The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors. Images PMID:3260329

  2. Analysis of Individual Mouse Activity in Group Housed Animals of Different Inbred Strains using a Novel Automated Home Cage Analysis System

    PubMed Central

    Bains, Rasneer S.; Cater, Heather L.; Sillito, Rowland R.; Chartsias, Agisilaos; Sneddon, Duncan; Concas, Danilo; Keskivali-Bond, Piia; Lukins, Timothy C.; Wells, Sara; Acevedo Arozena, Abraham; Nolan, Patrick M.; Armstrong, J. Douglas

    2016-01-01

    Central nervous system disorders such as autism as well as the range of neurodegenerative diseases such as Huntington's disease are commonly investigated using genetically altered mouse models. The current system for characterizing these mice usually involves removing the animals from their home-cage environment and placing them into novel environments where they undergo a battery of tests measuring a range of behavioral and physical phenotypes. These tests are often only conducted for short periods of times in social isolation. However, human manifestations of such disorders are often characterized by multiple phenotypes, presented over long periods of time and leading to significant social impacts. Here, we have developed a system which will allow the automated monitoring of individual mice housed socially in the cage they are reared and housed in, within established social groups and over long periods of time. We demonstrate that the system accurately reports individual locomotor behavior within the group and that the measurements taken can provide unique insights into the effects of genetic background on individual and group behavior not previously recognized. PMID:27375446

  3. Cloning and immunocytochemical localization of a cyclic nucleotide-gated channel alpha-subunit to all cone photoreceptors in the mouse retina.

    PubMed

    Hirano, A A; Hack, I; Wässle, H; Duvoisin, R M

    2000-05-22

    Cyclic nucleotide-gated channels (CNGC) are ligand-gated ion channels that open and close in response to changes in the intracellular concentration of the second messengers, 3;,5;-cyclic adenosine monophosphate and 3;,5;-cyclic guanosine monophosphate. Most notably, they transduce the chemical signal produced by the absorption of light in photoreceptors into a membrane potential change, which is then transmitted to the ascending visual pathway. CNGCs have also been implicated in the signal transduction of other neurons downstream of the photoreceptors, in particular the ON-bipolar cells, as well as in other areas of the central nervous system. We therefore undertook a search for additional cyclic nucleotide-gated channels expressed in the retina. Following a degenerate reverse transcription polymerase chain reaction approach to amplify low-copy number messages, a cDNA encoding a new splice variant of CNGC alpha-subunit was isolated from mouse retina and classified as mCNG3. An antiserum raised against the carboxy-terminal sequence identified the retinal cell type expressing mCNG3 as cone photoreceptors. Preembedding immunoelectron microscopy demonstrated its membrane localization in the outer segments, consistent with its role in phototransduction. Double-labeling experiments with cone-specific markers indicated that all cone photoreceptors in the murid retina use the same or a highly conserved cyclic nucleotide-gated channel. Therefore, defects in this channel would be predicted to severely impair photopic vision.

  4. A Japanese Encephalitis Virus Genotype 5 Molecular Clone Is Highly Neuropathogenic in a Mouse Model: Impact of the Structural Protein Region on Virulence

    PubMed Central

    de Wispelaere, Mélissanne; Frenkiel, Marie-Pascale

    2015-01-01

    ABSTRACT Japanese encephalitis virus (JEV) strains can be separated into 5 genotypes (g1 to g5) based on sequence similarity. JEV g5 strains have been rarely isolated and are poorly characterized. We report here the full characterization of a g5 virus generated using a cDNA-based technology and its comparison with a widely studied g3 strain. We did not observe any major differences between those viruses when their infectious cycles were studied in various cell lines in vitro. Interestingly, the JEV g5 strain was highly pathogenic when inoculated to BALB/c mice, which are known to be largely resistant to JEV g3 infection. The study of chimeric viruses between JEV g3 and g5 showed that there was a poor viral clearance of viruses that express JEV g5 structural proteins in BALB/c mice blood, which correlated with viral invasion of the central nervous system and encephalitis. In addition, using an in vitro model of the blood-brain barrier, we were able to show that JEV g5 does not have an enhanced capacity for entering the central nervous system, compared to JEV g3. Overall, in addition to providing a first characterization of the understudied JEV g5, our work highlights the importance of sustaining an early viremia in the development of JEV encephalitis. IMPORTANCE Genotype 5 viruses are genetically and serologically distinct from other JEV genotypes and can been associated with human encephalitis, which warrants the need for their characterization. In this study, we characterized the in vitro and in vivo properties of a JEV g5 strain and showed that it was more neuropathogenic in a mouse model than a well-characterized JEV g3 strain. The enhanced virulence of JEV g5 was associated with poor viral clearance but not with enhanced crossing of the blood-brain barrier, thus providing new insights into JEV pathogenesis. PMID:25787283

  5. Nuclear transfer technology in mammalian cloning.

    PubMed

    Wolf, D P; Mitalipov, S; Norgren, R B

    2001-01-01

    The past several years have witnessed remarkable progress in mammalian cloning using nuclear transfer (NT). Until 1997 and the announcement of the successful cloning of sheep from adult mammary gland or fetal fibroblast cells, our working assumption was that cloning by NT could only be accomplished with relatively undifferentiated embryonic cells. Indeed, live offspring were first produced by NT over 15 years ago from totipotent, embryonic blastomeres derived from early cleavage-stage embryos. However, once begun, the progression to somatic cell cloning or NT employing differentiated cells as the source of donor nuclei was meteoric, initially involving differentiated embryonic cell cultures in sheep in 1996 and quickly thereafter, fetal or adult somatic cells in sheep, cow, mouse, goat, and pig. Several recent reviews provide a background for and discussion of these successes. Here we will focus on the potential uses of reproductive cloning along with recent activities in the field and a discussion concerning current interests in human reproductive and therapeutic cloning.

  6. A common structure for concepts of individuals, stuffs, and real kinds: more Mama, more milk, and more mouse.

    PubMed

    Millikan, R G

    1998-02-01

    Concepts are highly theoretical entities. One cannot study them empirically without committing oneself to substantial preliminary assumptions. Among the competing theories of concepts and categorization developed by psychologists in the last thirty years, the implicit theoretical assumption that what falls under a concept is determined by description ("descriptionism") has never been seriously challenged. I present a nondescriptionist theory of our most basic concepts, "substances," which include (1) stuffs (gold, milk), (2) real kinds (cat, chair), and (3) individuals (Mama, Bill Clinton, the Empire State Building). On the basis of something important that all three have in common, our earliest and most basic concepts of substances are identical in structure. The membership of the category "cat," like that of "Mama," is a natural unit in nature, to which the concept "cat" does something like pointing, and continues to point despite large changes in the properties the thinker represents the unit as having. For example, large changes can occur in the way a child identifies cats and the things it is willing to call "cat" without affecting the extension of its word "cat." The difficulty is to cash in the metaphor of "pointing" in this context. Having substance concepts need not depend on knowing words, but language interacts with substance concepts, completely transforming the conceptual repertoire. I will discuss how public language plays a crucial role in both the acquisition of substance concepts and their completed structure.

  7. [Ethical considerations on human cloning. A psychoanalytic perspective].

    PubMed

    Alvarez, A

    2000-01-01

    A brief review of ethical issues related to two types of human cloning is presented: cloning embryonic cells not intended to culminate in the birth of a new individual and cloning human beings. Advantages and objections related to both types of human cloning are analyzed from an ethical point of view. Repercussions on individuals born by the technique of cloning are discussed from a psychoanalytical perspective. It can be concluded that cloning embryonic cells could be admissible, while not cloning considered as a reproductive option.

  8. Artificial cloning of domestic animals.

    PubMed

    Keefer, Carol L

    2015-07-21

    Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research.

  9. Artificial cloning of domestic animals.

    PubMed

    Keefer, Carol L

    2015-07-21

    Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research. PMID:26195770

  10. Artificial cloning of domestic animals

    PubMed Central

    Keefer, Carol L.

    2015-01-01

    Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research. PMID:26195770

  11. Human reproductive cloning: a conflict of liberties.

    PubMed

    Havstad, Joyce C

    2010-02-01

    Proponents of human reproductive cloning do not dispute that cloning may lead to violations of clones' right to self-determination, or that these violations could cause psychological harms. But they proceed with their endorsement of human reproductive cloning by dismissing these psychological harms, mainly in two ways. The first tactic is to point out that to commit the genetic fallacy is indeed a mistake; the second is to invoke Parfit's non-identity problem. The argument of this paper is that neither approach succeeds in removing our moral responsibility to consider and to prevent psychological harms to cloned individuals. In fact, the same commitment to personal liberty that generates the right to reproduce by means of cloning also creates the need to limit that right appropriately. Discussion of human reproductive cloning ought to involve a careful and balanced consideration of both the relevant aspects of personal liberty - the parents' right to reproductive freedom and the cloned child's right to self-determination.

  12. Cloning of two individual cDNAS encoding 9-cis-epoxycarotenoid dioxygenase from Gentiana lutea, their tissue-specific expression and physiological effect in transgenic tobacco.

    PubMed

    Zhu, Changfu; Kauder, Friedrich; Römer, Susanne; Sandmann, Gerhard

    2007-02-01

    Two 9-cis-epoxycarotenoid dioxygenase (NCED) cDNAs have been cloned from a petal library of Gentiana lutea. Both cDNAs carry a putative transit sequence for chloroplast import and differ mainly in their length and the 5'-flanking regions. GlNCED1 was evolutionary closely related to Arabidopsis thaliana NCED6 whereas GlNCED2 showed highest homology to tomato NCED1 and A. thaliana NCED3. The amounts of GlNCED2 transcript were below Northern detection in G. lutea. In contrast, GlNCED1 was specifically expressed at higher levels in developing flowers when petals start appearing. By genetic engineering of tobacco with coding regions of either gene under a constitutive promoter, their function was further analyzed. Although mRNA of both genes was detectable in the corresponding transgenic plants, a physiological effect was only found for GlNCED1 but not for GlNCED2. In germination experiments of GlNCED1 transgenic lines, delayed radicle formation and cotyledon appearance were observed. However, the transformants exhibited no improved tolerance against desiccation stress. In contrast to other plants with over-expressed NCEDs, prolonged delay of seed germination is the only abscisic-acid-related phenotypic effect in the GlNCED1 transgenic lines.

  13. Gene expression analysis of the CD4+ T-cell clones derived from gingival tissues of periodontitis patients.

    PubMed

    Ito, H; Honda, T; Domon, H; Oda, T; Okui, T; Amanuma, R; Nakajima, T; Yamazaki, K

    2005-12-01

    The function of T cells infiltrating periodontitis lesions is complex and has not been fully elucidated. Here, we established T-cell clones from the gingival tissues of periodontitis patients and examined their gene expression. A total of 57 and 101 T-cell clones were established by means of immobilized anti-CD3 antibody and IL-2 from gingival tissues and peripheral blood, respectively. The gingival T-cell clones were derived from three patients, and the peripheral blood T-cell clones from two of these patients and a further patient whose gingival T-cell clones were not established. Gingival tissues were also obtained from a further 19 periodontitis patients. The expression of cytokines and molecules related to both regulatory function and tissue destruction were examined by means of reverse-transcription polymerase chain reaction. All the gingival T-cell clones expressed mRNA for TGF-beta1, CTLA-4, and CD25, and all the T-cell clones from peripheral blood expressed IFN-gamma and TGF-beta1 mRNAs. Most but not all the T-cell clones from gingival tissues and peripheral blood expressed mRNA for IFN-gamma and, CD25 and CTLA-4, respectively. The frequency of T-cell clones and gingival tissues expressing FOXP3, a possible master gene for mouse CD4(+)CD25(+) regulatory T cells, was very high (97%, 93%, and 100% for gingival T-cell clones, peripheral blood T-cell clones, and gingival tissues, respectively). Whereas the frequency of IL-4-expressing T-cell clones was lower for gingival T-cell clones (70% vs. 87%), the frequency of the gingival T-cell clones expressing IL-10 and IL-17 was higher than peripheral blood T-cell clones (75% vs. 62% for IL-10, 51% vs. 11% for IL-17). A similar expression profile was observed for gingival T-cell clones compared with gingival tissue samples with the exception of IL-4 expression, where the frequency of positive samples was lower in the gingival tissues (70% vs. 11%). These results suggest that the individual T cells infiltrating

  14. Tissue-specific methylation of individual CpG dinucleotides in the 5{prime} upstream region of the mouse catalase gene (Cas-1)

    SciTech Connect

    Pillay, I.L.; Singh, S.M.

    1994-09-01

    The intracellular antioxidant enzyme, catalase, is encoded by a gene whose level of expression in different organisms, including humans, varies with tissue-type. The {open_quotes}TATA-less{close_quotes} 5{prime} upstream region of the catalase gene, in mice and humans, contains a CpG island. Such CG-rich regions are target sites for cytosine methylation and have been implicated in tissue-specific gene expression. However, the methylation status of individual CpG dinucleotides and their significance in gene expression has not been established. A 275 bp fragment within the 5{prime} region of Cas-1 was evaluated for CpG methylation. HpaII digestion of genomic DNA, followed by polymerase chain reaction amplification (HpaII-PCR), suggests that at least one of three CCGG is not methylated in nine different somatic tissues that express this enzyme at various levels. In contrast, all three CCGG sites are methylated in DNA from sperm and spleen. Further examination of the methylation specificity of individual CCGG sites was conducted using sodium bisulfite modification of genomic DNA followed by HPaII-PCR. Sodium bisulfite modifies non-methylated cytosines to uracils, changing a CG to a TG dinucleotide. This nucleotide substitution eliminates HpaII sites and allows the methylation status of each of the CCGG sites to be assessed. The ability to discern the number and combination of methylated sites within the 5{prime} region of a gene permits the determination of a possible correlation between differential methylation patterns and temporal/spatial gene regulation. Analysis of differential methylation, using the mouse catalase gene as a model, provides further insight into CpG methylation as one mechanism of mammalian gene regulation.

  15. Identifying Differentiation Stage of Individual Primary Hematopoietic Cells from Mouse Bone Marrow by Multivariate Analysis of TOF-Secondary Ion Mass Spectrometry Data

    PubMed Central

    Frisz, Jessica F.; Choi, Ji Sun; Wilson, Robert L.; Harley, Brendan A. C.; Kraft, Mary L.

    2014-01-01

    The ability to self-renew and differentiate into multiple types of blood and immune cells renders hematopoietic stem and progenitor cells (HSPCs) valuable for clinical treatment of hematopoietic pathologies and as models of stem cell differentiation for tissue engineering applications. To study directed HSC differentiation and identify the conditions that recreate the native bone marrow environment, combinatorial biomaterials that exhibit lateral variations in chemical and mechanical properties are employed. New experimental approaches are needed to facilitate correlating cell differentiation stage with location in the culture system. We demonstrate that multivariate analysis of time-of-flight secondary ion mass spectrometry (TOF-SIMS) data can be used to identify the differentiation state of individual hematopoietic cells (HCs) isolated from mouse bone marrow. Here, we identify primary HCs from three distinct stages of B cell lymphopoiesis at the single cell level: HSPCs, common lymphoid progenitors, and mature B cells. The differentiation state of individual HCs in a test set could be identified with a partial least squares discriminant analysis (PLS-DA) model that was constructed with calibration spectra from HCs of known differentiation status. The lowest error of identification was obtained when the intra-population spectral variation between the cells in the calibration and test sets was minimized. This approach complements the traditional methods that are used to identify HC differentiation stage. Further, the ability to gather mass spectrometry data from single HSCs cultured on graded biomaterial substrates may provide significant new insight into how HSPCs respond to extrinsic cues as well as the molecular changes that occur during cell differentiation. PMID:22507202

  16. [Cloning and expression analysis in mature individuals of salmon gonadotropin-releasing hormone (sGnRH) gene in common carp].

    PubMed

    Li, Shuang-Fei; Hu, Wei; Wang, Ya-Ping; Sun, Yong-Hua; Chen, Shang-Ping; Zhu, Zuo-Yani

    2004-10-01

    Two types of complementary DNAs (cDNA) encoding the precursor of salmon gonadotropin-releasing hormone (sGnRH, [Trp7, Leu8] GnRH) are cloned and sequenced from common carp brain using rapid amplification of cDNA ends (RACE). The two cDNAs are referred to sGnRH cDNA1 and cDNA2, and the full-length fragment of cDNA1 and cDNA2 were 393 and 478 bp, respectively. Two sGnRH cDNAs contain an open reading frame of 285 bp, which encodes the sGnRH precursor including 94 amino acid residues. The sGnRH precursors consist of a signal peptide, sGnRH decapeptide and a GnRH-associated peptide (GAP) which is linked by the processing site (Gly-Lys-Arg). Two different sGnRH genes are characterized by intron trapping, and they share a similar structure composed of four exons and three introns. The nucleotide sequences identity of intron 1, intron 2 and intron 3 in sGnRH gene1 and gene2 are 71.1, 76. 1 and 88.0%, respectively. The basic structure and encoding architecture of sGnRH cDNAs and genes are similar with the reported GnRH. It is presumed that all the GnRH variants may evolve from a common ancestral molecular. Southern blot results confirm further the conclusion that there are two differential sGnRH genes in common carp genome. The results of reverse transcription-polymerase chain reaction (RT-PCR) assaying show that the two sGnRH genes co-express in the dissected brain regions, pituitary and ovary with the exception of expression of sGnRH genes in testis. According to the co-existence of two sGnRH genes in distinct brain regions,pituitary and ovary, it is presumed that sGnRH plays an important role in the regulation of hypothalamic-pituitary-gonadal (HPG) axis,and that sGnRH also operates as the neuromodulator, autocrine and/or paracrine regulator.

  17. Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein.

    PubMed

    Timofeeva, Anna M; Buneva, Valentina N; Nevinsky, Georgy A

    2015-10-01

    Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP-Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27 kDa). Seventy-two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty-two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7-9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease-like and three thiol protease-like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca(2+), Mg(2+), Mn(2+), Ni(2+), Zn(2+), Cu(2+), and Co(2+) was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti-MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti-MBP abzymes, which can attack MBP of myelin-proteolipid sheath of axons and play an important role in MS and SLE pathogenesis.

  18. Molecular cloning and analysis of lymphokines. Volume 13

    SciTech Connect

    Webb, D.R.; Goeddel, D.V.

    1987-01-01

    These proceedings collect papers on the subject of lymphokines. Topics include: DNA-cloning of mouse and human lymphokine genes, inteferons, interleukins, gene expression, tumor necrosis factors, and recombinant DNA.

  19. [Human cloning: judicial and legislative framework].

    PubMed

    Shenfield, F

    2000-09-01

    The application, either theoretical or fantastical, of the somatic cloning which resulted in the birth of Dolly the sheep to human reproduction has led to international uproar. A paragraph specifically banning reproductive cloning (defined as "any intervention seeking to produce genetically identical human individuals in the sense of individuals sharing the same nuclear gene set") was added to the Council of Europe Convention on Human Rights and Biomedicine in January 1998. In several responses both at national and international level, reproductive cloning is banned either directly or indirectly. The challenge for the future is the consideration of integrating therapeutic cloning in legislations both nationally and in international declarations.

  20. [Human cloning: judicial and legislative framework].

    PubMed

    Shenfield, F

    2000-09-01

    The application, either theoretical or fantastical, of the somatic cloning which resulted in the birth of Dolly the sheep to human reproduction has led to international uproar. A paragraph specifically banning reproductive cloning (defined as "any intervention seeking to produce genetically identical human individuals in the sense of individuals sharing the same nuclear gene set") was added to the Council of Europe Convention on Human Rights and Biomedicine in January 1998. In several responses both at national and international level, reproductive cloning is banned either directly or indirectly. The challenge for the future is the consideration of integrating therapeutic cloning in legislations both nationally and in international declarations. PMID:11075502

  1. RIKEN mouse genome encyclopedia.

    PubMed

    Hayashizaki, Yoshihide

    2003-01-01

    We have been working to establish the comprehensive mouse full-length cDNA collection and sequence database to cover as many genes as we can, named Riken mouse genome encyclopedia. Recently we are constructing higher-level annotation (Functional ANnoTation Of Mouse cDNA; FANTOM) not only with homology search based annotation but also with expression data profile, mapping information and protein-protein database. More than 1,000,000 clones prepared from 163 tissues were end-sequenced to classify into 159,789 clusters and 60,770 representative clones were fully sequenced. As a conclusion, the 60,770 sequences contained 33,409 unique. The next generation of life science is clearly based on all of the genome information and resources. Based on our cDNA clones we developed the additional system to explore gene function. We developed cDNA microarray system to print all of these cDNA clones, protein-protein interaction screening system, protein-DNA interaction screening system and so on. The integrated database of all the information is very useful not only for analysis of gene transcriptional network and for the connection of gene to phenotype to facilitate positional candidate approach. In this talk, the prospect of the application of these genome resourced should be discussed. More information is available at the web page: http://genome.gsc.riken.go.jp/.

  2. Self-Cloning CRISPR.

    PubMed

    Arbab, Mandana; Sherwood, Richard I

    2016-01-01

    CRISPR/Cas9-gene editing has emerged as a revolutionary technology to easily modify specific genomic loci by designing complementary sgRNA sequences and introducing these into cells along with Cas9. Self-cloning CRISPR/Cas9 (scCRISPR) uses a self-cleaving palindromic sgRNA plasmid (sgPal) that recombines with short PCR-amplified site-specific sgRNA sequences within the target cell by homologous recombination to circumvent the process of sgRNA plasmid construction. Through this mechanism, scCRISPR enables gene editing within 2 hr once sgRNA oligos are available, with high efficiency equivalent to conventional sgRNA targeting: >90% gene knockout in both mouse and human embryonic stem cells and cancer cell lines. Furthermore, using PCR-based addition of short homology arms, we achieve efficient site-specific knock-in of transgenes such as GFP without traditional plasmid cloning or genome-integrated selection cassette (2% to 4% knock-in rate). The methods in this paper describe the most rapid and efficient means of CRISPR gene editing. © 2016 by John Wiley & Sons, Inc. PMID:27532819

  3. Self-Cloning CRISPR.

    PubMed

    Arbab, Mandana; Sherwood, Richard I

    2016-01-01

    CRISPR/Cas9-gene editing has emerged as a revolutionary technology to easily modify specific genomic loci by designing complementary sgRNA sequences and introducing these into cells along with Cas9. Self-cloning CRISPR/Cas9 (scCRISPR) uses a self-cleaving palindromic sgRNA plasmid (sgPal) that recombines with short PCR-amplified site-specific sgRNA sequences within the target cell by homologous recombination to circumvent the process of sgRNA plasmid construction. Through this mechanism, scCRISPR enables gene editing within 2 hr once sgRNA oligos are available, with high efficiency equivalent to conventional sgRNA targeting: >90% gene knockout in both mouse and human embryonic stem cells and cancer cell lines. Furthermore, using PCR-based addition of short homology arms, we achieve efficient site-specific knock-in of transgenes such as GFP without traditional plasmid cloning or genome-integrated selection cassette (2% to 4% knock-in rate). The methods in this paper describe the most rapid and efficient means of CRISPR gene editing. © 2016 by John Wiley & Sons, Inc.

  4. Wildlife conservation and reproductive cloning.

    PubMed

    Holt, William V; Pickard, Amanda R; Prather, Randall S

    2004-03-01

    Reproductive cloning, or the production of offspring by nuclear transfer, is often regarded as having potential for conserving endangered species of wildlife. Currently, however, low success rates for reproductive cloning limit the practical application of this technique to experimental use and proof of principle investigations. In this review, we consider how cloning may contribute to wildlife conservation strategies. The cloning of endangered mammals presents practical problems, many of which stem from the paucity of knowledge about their basic reproductive biology. However, situations may arise where resources could be targeted at recovering lost or under-represented genetic lines; these could then contribute to the future fitness of the population. Approaches of this type would be preferable to the indiscriminate generation of large numbers of identical individuals. Applying cloning technology to non-mammalian vertebrates may be more practical than attempting to use conventional reproductive technologies. As the scientific background to cloning technology was pioneered using amphibians, it may be possible to breed imminently threatened amphibians, or even restore extinct amphibian species, by the use of cloning. In this respect species with external embryonic development may have an advantage over mammals as developmental abnormalities associated with inappropriate embryonic reprogramming would not be relevant.

  5. The effect of two different Individually Ventilated Cage systems on anxiety-related behaviour and welfare in two strains of laboratory mouse.

    PubMed

    Burman, O; Buccarello, L; Redaelli, V; Cervo, L

    2014-01-30

    The environment in which a laboratory animal is housed can significantly influence its behaviour and welfare, acting as a potential confounding factor for those studies in which it is utilised. This study investigated the impact of two Individually Ventilated Cage (IVC) housing systems on anxiety-related behaviour and welfare indicators in two common strains of laboratory mice. Subjects were juvenile female C57BL/6J and BALB/c mice (N=128) housed in groups of four in two different IVC systems for 7weeks. System One had air delivery at the cage 'cover' level at 75 ACH (Air Changes/Hour) and System Two had air delivery at the 'animal' level at 50 ACH. Mice were assessed twice a week (e.g. bodyweight) or at the end of the study (e.g. anxiety tests). Our results showed significant differences in anxiety-related behaviour between strains and housing systems. Mice in System Two, regardless of strain, defecated more in the Elevated Plus Maze (EPM), spent less time in the open arms of the EPM, and less time in the central zone of the Open Field (OF). Strain differences in anxiety-like behaviour were seen in the increased defecation by BALB/c mice in the OF and EPM and less time spent in the open arms of the EPM compared to C57BL/6J mice. These results suggest that different IVC housing systems can influence mouse behaviour in different ways, with mice of both strains studied exhibiting more anxiety-related behaviour when housed in System Two (air entry at the 'animal' level at 50 ACH), which could impact upon experimental data.

  6. Creating humans: an ethical study of human cloning.

    PubMed

    Haman, K H

    2001-01-01

    What does it mean to clone an individual? A clone is created directly from the cells of one individual through several different mechanisms. The most popular of these mechanisms, which created Dolly, is Somatic Cell Nuclear Transfer. It is this type that is predicted to accomplish the feat of cloning a human. With the increasing amount of technology readily available the ethical questions regarding human cloning must be addressed.

  7. Microdissection and visualization of individual hair follicles for lineage tracing studies.

    PubMed

    Sequeira, Inês; Legué, Emilie; Capgras, Suzanne; Nicolas, Jean-François

    2014-01-01

    In vivo lineage tracing is a valuable technique to study cellular behavior. Our lab developed a lineage tracing method, based on the Cre/lox system, to genetically induce clonal labelling of cells and follow their progeny. Here we describe a protocol for temporally controlled clonal labelling and for microdissection of individual mouse hair follicles. We further present staining and visualization techniques used in our lab to analyze clones issued from genetically induced labelling. PMID:24281870

  8. 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.

  9. On classical cloning and no-cloning

    NASA Astrophysics Data System (ADS)

    Teh, Nicholas J.

    2012-02-01

    It is part of information theory folklore that, while quantum theory prohibits the generic (or universal) cloning of states, such cloning is allowed by classical information theory. Indeed, many take the phenomenon of no-cloning to be one of the features that distinguishes quantum mechanics from classical mechanics. In this paper, we argue that pace conventional wisdom, in the case where one does not include a machine system, there is an analog of the no-cloning theorem for classical systems. However, upon adjoining a non-trivial machine system (or ancilla) one finds that, pace the quantum case, the obstruction to cloning disappears for pure states. We begin by discussing some conceptual points and category-theoretic generalities having to do with cloning, and proceed to discuss no-cloning in both the case of (non-statistical) classical mechanics and classical statistical mechanics.

  10. The Clone Factory

    ERIC Educational Resources Information Center

    Stoddard, Beryl

    2005-01-01

    Have humans been cloned? Is it possible? Immediate interest is sparked when students are asked these questions. In response to their curiosity, the clone factory activity was developed to help them understand the process of cloning. In this activity, students reenact the cloning process, in a very simplified simulation. After completing the…

  11. Phenotype of cloned mice: development, behavior, and physiology.

    PubMed

    Tamashiro, Kellie L K; Wakayama, Teruhiko; Yamazaki, Yukiko; Akutsu, Hidenori; Woods, Stephen C; Kondo, Sylvia; Yanagimachi, Ryuzo; Sakai, Randall R

    2003-11-01

    Cloning technology has potential to be a valuable tool in basic research, clinical medicine, and agriculture. However, it is critical to determine the consequences of this technique in resulting offspring before widespread use of the technology. Mammalian cloning using somatic cells was first demonstrated in sheep in 1997 and since then has been extended to a number of other species. We examined development, behavior, physiology, and longevity in B6C3F1 female mice cloned from adult cumulus cells. Control mice were naturally fertilized embryos subjected to the same in vitro manipulation and culture conditions as clone embryos. Clones attained developmental milestones similar to controls. Activity level, motor ability and coordination, and learning and memory skills of cloned mice were comparable with controls. Interestingly, clones gained more body weight than controls during adulthood. Increased body weight was attributable to higher body fat and was associated with hyperleptinemia and hyperinsulinemia indicating that cloned mice are obese. Cloned mice were not hyperphagic as adults and had hypersensitive leptin and melanocortin signaling systems. Longevity of cloned mice was comparable with that reported by the National Institute on Aging and the causes of death were typical for this strain of mouse. These studies represent the first comprehensive set of data to characterize cloned mice and provide critical information about the long-term effects of somatic cell cloning. PMID:14610260

  12. The Riken mouse genome encyclopedia project.

    PubMed

    Hayashizaki, Yoshihide

    2003-01-01

    The Riken mouse genome encyclopedia a comprehensive full-length cDNA collection and sequence database. High-level functional annotation is based on sequence homology search, expression profiling, mapping and protein-protein interactions. More than 1000000 clones prepared from 163 tissues were end-sequenced and classified into 128000 clusters, and 60000 representative clones were fully sequenced representing 24000 clear protein-encoding genes. The application of the mouse genome database for positional cloning and gene network regulation analysis is reported.

  13. Human cloning 2001.

    PubMed

    Healy, David L; Weston, Gareth; Pera, Martin F; Rombauts, Luk; Trounson, Alan O

    2002-05-01

    This review summaries human cloning from a clinical perspective. Natural human clones, that is, monozygotic twins, are increasing in the general community. Iatrogenic human clones have been produced for decades in infertile couples given fertility treatment such as ovulation induction. A clear distinction must be made between therapeutic cloning using embryonic stem cells and reproductive cloning attempts. Unlike the early clinical years of in vitro fertilization, with cloning there is no animal model that is safe and dependable. Until there is such a model, 'Dolly'-style human cloning is medically unacceptable.

  14. Unstable resistance of G mouse fibroblasts to ecotropic murine leukemia virus infection.

    PubMed Central

    Yoshikura, H; Naito, Y; Moriwaki, K

    1979-01-01

    G mouse cells were resistant to N- and NB-tropic Friend leukemia viruses and to B-tropic WN 1802B. Though the cells were resistant to focus formation by the Moloney isolate of murine sarcoma virus, they were relatively sensitive to helper component murine leukemia virus. To amphotropic murine leukemia virus and to focus formation by amphotropic murine sarcoma virus, G mouse cells were fully permissive. When the cell lines were established starting from the individual embryos, most cell lines were not resistant to the murine leukemia viruses. Only one resistant line was established. Cloning of this cell line indicated that the resistant cells constantly segregated sensitive cells during the culture; i.e., the G mouse cell cultures were probably always mixtures of sensitive and resistant cells. Among the sensitive cell clones, some were devoid of Fv-1 restriction. Such dually permissive cells, and also feral mouse-derived SC-1 cells, retained glucose-6-phosphate dehydrogenase-1 and apparently normal number 4 chromosomes. The loss of Fv-1 restriction in these mouse cells was not brought about by any gross structural changes in the vicinity of Fv-1 on number 4 chromosomes. Images PMID:221667

  15. FISH probes for mouse chromosome identification

    SciTech Connect

    Shi, Yu-Ping; Mohapatra, G.; Hanahan, D.; Miller, J.

    1997-10-01

    P1 clones near the telomeres and centromeres of each mouse chromosome except Y have been selected from a mouse genomic library and mapped using fluorescence in situ hybridization (FISH). Each clone was selected to contain a genetically mapped polymorphic DNA sequence as close as possible to the centromere or telomere of a chromosome. The genetic distance from the various P1 clones to the most distal genetically mapped polymorphic sequence ranged from 0 for about half of the clones to 6.7 cM for the probe at the telomere of chromosome 14. The average distance to the most distal or proximal chromosome marker was 1.5 cM. The use of FISH with these probes for mouse chromosome identification during comparative genomic hybridization is illustrated. 17 refs., 2 figs., 2 tabs.

  16. 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.

  17. Analysis of neural crest–derived clones reveals novel aspects of facial development

    PubMed Central

    Kaucka, Marketa; Ivashkin, Evgeny; Gyllborg, Daniel; Zikmund, Tomas; Tesarova, Marketa; Kaiser, Jozef; Xie, Meng; Petersen, Julian; Pachnis, Vassilis; Nicolis, Silvia K.; Yu, Tian; Sharpe, Paul; Arenas, Ernest; Brismar, Hjalmar; Blom, Hans; Clevers, Hans; Suter, Ueli; Chagin, Andrei S.; Fried, Kaj; Hellander, Andreas; Adameyko, Igor

    2016-01-01

    Cranial neural crest cells populate the future facial region and produce ectomesenchyme-derived tissues, such as cartilage, bone, dermis, smooth muscle, adipocytes, and many others. However, the contribution of individual neural crest cells to certain facial locations and the general spatial clonal organization of the ectomesenchyme have not been determined. We investigated how neural crest cells give rise to clonally organized ectomesenchyme and how this early ectomesenchyme behaves during the developmental processes that shape the face. Using a combination of mouse and zebrafish models, we analyzed individual migration, cell crowd movement, oriented cell division, clonal spatial overlapping, and multilineage differentiation. The early face appears to be built from multiple spatially defined overlapping ectomesenchymal clones. During early face development, these clones remain oligopotent and generate various tissues in a given location. By combining clonal analysis, computer simulations, mouse mutants, and live imaging, we show that facial shaping results from an array of local cellular activities in the ectomesenchyme. These activities mostly involve oriented divisions and crowd movements of cells during morphogenetic events. Cellular behavior that can be recognized as individual cell migration is very limited and short-ranged and likely results from cellular mixing due to the proliferation activity of the tissue. These cellular mechanisms resemble the strategy behind limb bud morphogenesis, suggesting the possibility of common principles and deep homology between facial and limb outgrowth. PMID:27493992

  18. Analysis of neural crest-derived clones reveals novel aspects of facial development.

    PubMed

    Kaucka, Marketa; Ivashkin, Evgeny; Gyllborg, Daniel; Zikmund, Tomas; Tesarova, Marketa; Kaiser, Jozef; Xie, Meng; Petersen, Julian; Pachnis, Vassilis; Nicolis, Silvia K; Yu, Tian; Sharpe, Paul; Arenas, Ernest; Brismar, Hjalmar; Blom, Hans; Clevers, Hans; Suter, Ueli; Chagin, Andrei S; Fried, Kaj; Hellander, Andreas; Adameyko, Igor

    2016-08-01

    Cranial neural crest cells populate the future facial region and produce ectomesenchyme-derived tissues, such as cartilage, bone, dermis, smooth muscle, adipocytes, and many others. However, the contribution of individual neural crest cells to certain facial locations and the general spatial clonal organization of the ectomesenchyme have not been determined. We investigated how neural crest cells give rise to clonally organized ectomesenchyme and how this early ectomesenchyme behaves during the developmental processes that shape the face. Using a combination of mouse and zebrafish models, we analyzed individual migration, cell crowd movement, oriented cell division, clonal spatial overlapping, and multilineage differentiation. The early face appears to be built from multiple spatially defined overlapping ectomesenchymal clones. During early face development, these clones remain oligopotent and generate various tissues in a given location. By combining clonal analysis, computer simulations, mouse mutants, and live imaging, we show that facial shaping results from an array of local cellular activities in the ectomesenchyme. These activities mostly involve oriented divisions and crowd movements of cells during morphogenetic events. Cellular behavior that can be recognized as individual cell migration is very limited and short-ranged and likely results from cellular mixing due to the proliferation activity of the tissue. These cellular mechanisms resemble the strategy behind limb bud morphogenesis, suggesting the possibility of common principles and deep homology between facial and limb outgrowth.

  19. [Cloning and law in Hungary].

    PubMed

    Julesz, Máté

    2015-03-01

    Reproductive human cloning is prohibited in Hungary, as in many other countries. Therapeutic human cloning is not prohibited, just like in many other countries. Stem cell therapy is also allowed. Article III, paragraph (3) of the Hungarian basic law (constitution) strictly forbids total human cloning. Article 1 of the Additional Protocol to the Oviedo Convention, on the Prohibition of Cloning Human Beings (1998) stipulates that any intervention seeking to create a human being genetically identical to another human being, whether living or dead, is prohibited. In Hungary, according to Article 174 of the Criminal Code, total human cloning constitutes a crime. Article 180, paragraph (3) of the Hungarian Act on Health declares that embryos shall not be brought about for research purposes; research shall be conducted only on embryos brought about for reproductive purposes when this is authorized by the persons entitled to decide upon its disposal, or when the embryo is damaged. Article 180, paragraph (5) of the Hungarian Act on Health stipulates that multiple individuals who genetically conform to one another shall not be brought about. According to Article 181, paragraph (1) of the Hungarian Act on Health, an embryo used for research shall be kept alive for not longer than 14 days, not counting the time it was frozen for storage and the time period of research.

  20. Mapping individual cosmid DNAs by direct AFM imaging.

    PubMed

    Allison, D P; Kerper, P S; Doktycz, M J; Thundat, T; Modrich, P; Larimer, F W; Johnson, D K; Hoyt, P R; Mucenski, M L; Warmack, R J

    1997-05-01

    Individual cosmid clones have been restriction mapped by directly imaging, with the atomic force microscope (AFM), a mutant EcoRI endonuclease site-specifically bound to DNA. Images and data are presented that locate six restriction sites, predicted from gel electrophoresis, on a 35-kb cosmid isolated from mouse chromosome 7. Measured distances between endonuclease molecules bound to lambda DNA, when compared to known values, demonstrate the accuracy of AFM mapping to better than 1%. These results may be extended to identify other important site-specific protein-DNA interactions, such as transcription factor and mismatch repair enzyme binding, difficult to resolve by current techniques.

  1. Social behavior and kin discrimination in a mixed group of cloned and non cloned heifers (Bos taurus).

    PubMed

    Coulon, M; Baudoin, C; Abdi, H; Heyman, Y; Deputte, B L

    2010-12-01

    For more than ten years, reproductive biotechnologies using somatic cell nuclear transfer have made possible the production of cloned animals in various domestic and laboratory species. The influence of the cloning process on offspring characteristics has been studied in various developmental aspects, however, it has not yet been documented in detail for behavioral traits. Behavioral studies of cloned animals have failed to show clear inter-individual differences associated with the cloning process. Preliminary results showed that clones favor each other's company. Preferential social interactions were observed among cloned heifers from the same donor in a mixed herd that also included cloned heifers and control heifers produced by artificial insemination (AI). These results suggest behavioral differences between cloned and non-cloned animals and similarities between clones from the same donor. The aim of the present study was to replicate and to extend these previous results and to study behavioral and cognitive mechanisms of this preferential grouping. We studied a group composed of five cloned heifers derived from the same donor cow, two cloned heifers derived from another donor cow, and AI heifers. Cloned heifers from the same donor were more spatially associated and interacted more between themselves than with heifers derived from another donor or with the AI individuals. This pattern indicates a possible kin discrimination in clones. To study this process, we performed an experiment (using an instrumental conditioning procedure with food reward) of visual discrimination between images of heads of familiar heifers, either related to the subjects or not. The results showed that all subjects (AI and cloned heifers) discriminated between images of familiar cloned heifers produced from the same donor and images of familiar unrelated heifers. Cattle discriminated well between images and used morphological similarities characteristic of cloned related heifers. Our

  2. Inhibition of class IIb histone deacetylase significantly improves cloning efficiency in mice.

    PubMed

    Ono, Tetsuo; Li, Chong; Mizutani, Eiji; Terashita, Yukari; Yamagata, Kazuo; Wakayama, Teruhiko

    2010-12-01

    Since the first mouse clone was produced by somatic cell nuclear transfer, the success rate of cloning in mice has been extremely low. Some histone deacetylase inhibitors, such as trichostatin A and scriptaid, have improved the full-term development of mouse clones significantly, but the mechanisms allowing for this are unclear. Here, we found that two other specific inhibitors, suberoylanilide hydroxamic acid and oxamflatin, could also reduce the rate of apoptosis in blastocysts, improve the full-term development of cloned mice, and increase establishment of nuclear transfer-generated embryonic stem cell lines significantly without leading to obvious abnormalities. However, another inhibitor, valproic acid, could not improve cloning efficiency. Suberoylanilide hydroxamic acid, oxamflatin, trichostatin A, and scriptaid are inhibitors for classes I and IIa/b histone deacetylase, whereas valproic acid is an inhibitor for classes I and IIa, suggesting that inhibiting class IIb histone deacetylase is an important step for reprogramming mouse cloning efficiency.

  3. Emergence and spread of O16-ST131 and O25b-ST131 clones among faecal CTX-M-producing Escherichia coli in healthy individuals in Hunan Province, China

    PubMed Central

    Zhong, Yi-Ming; Liu, Wen-En; Liang, Xiang-Hui; Li, Yan-Ming; Jian, Zi-Juan; Hawkey, Peter M.

    2015-01-01

    Objectives The objectives of this study were to determine CTX-M-producing Escherichia coli ST131 strain prevalence in stool specimens from healthy subjects in central China and to molecularly characterize clonal groups. Methods From November 2013 to January 2014, stool specimens from healthy individuals in Hunan Province were screened for ESBL-producing E. coli using chromogenic medium and CTX-M genotypes and phylogenetic groups were determined. ST131 clonal groups were detected by PCR and characterized for antibiotic resistance, fimH, gyrA and parC alleles, plasmid-mediated quinolone resistance determinants, virulence genotypes and PFGE patterns. Results Among 563 subjects, 287 (51.0%) exhibited the presence of faecal ESBL-producing E. coli, all of which produced CTX-M enzymes. The most common CTX-M genotypes were CTX-M-14 (48.4%), CTX-M-15 (27.5%) and CTX-M-27 (15.0%). Of the 287 CTX-M-producing isolates, 32 (11.1%) belonged to the ST131 clone. O16-ST131 isolates were dominant (75%) and contained the fimH41 allele. The remaining eight (25%) ST131 isolates were of the O25b subgroup and contained fimH30 or fimH41. Ciprofloxacin resistance was found in 100% of the O25b-ST131 isolates, whereas only 8% of the O16-ST131 isolates were resistant. All of the O25b-ST131 isolates except one showed gyrA1AB and parC1aAB mutations; most of the O16-ST131 isolates had gyrA1A and parC1b mutations. The virulence genotypes of O16-ST131 resembled those of the O25b-ST131 isolates. The 32 ST131 isolates formed one large group at the 64% similarity level. They comprised 15 PFGE groups (defined at ≥85% similarity). Conclusions O16-ST131 isolates have emerged as the predominant type of ST131 isolate in faecal CTX-M-producing E. coli in healthy individuals in China. PMID:25957581

  4. Multipartite asymmetric quantum cloning

    SciTech Connect

    Iblisdir, S.; Gisin, N.; Acin, A.; Cerf, N.J.; Filip, R.; Fiurasek, J.

    2005-10-15

    We investigate the optimal distribution of quantum information over multipartite systems in asymmetric settings. We introduce cloning transformations that take N identical replicas of a pure state in any dimension as input and yield a collection of clones with nonidentical fidelities. As an example, if the clones are partitioned into a set of M{sub A} clones with fidelity F{sup A} and another set of M{sub B} clones with fidelity F{sup B}, the trade-off between these fidelities is analyzed, and particular cases of optimal N{yields}M{sub A}+M{sub B} cloning machines are exhibited. We also present an optimal 1{yields}1+1+1 cloning machine, which is an example of a tripartite fully asymmetric cloner. Finally, it is shown how these cloning machines can be optically realized.

  5. Ethical issues in cloning.

    PubMed

    Satris, S

    2000-01-01

    There is great public concern with the ethics of human cloning. This paper briefly examines some of what I identify as pseudo-problems or myths associated with cloning, and some of the more substantial ethical concerns.

  6. Aristotle and headless clones.

    PubMed

    Mosteller, Timothy

    2005-01-01

    Cloned organisms can be genetically altered so that they do not exhibit higher brain functioning. This form of therapeutic cloning allows for genetically identical organs and tissues to be harvested from the clone for the use of the organism that is cloned. "Spare parts" cloning promises many opportunities for future medical advances. What is the ontological and ethical status of spare parts, headless clones? This paper attempts to answer this question from the perspective of Aristotle's view of the soul. Aristotle's metaphysics as applied to his view of biological essences generates an ethic that can contribute to moral reasoning regarding the use of headless spare parts clones. The task of this paper is to show the implications that Aristotle's view of the soul, if it is true, would have on the ethics of headless, spare parts cloning. PMID:16180113

  7. Aristotle and headless clones.

    PubMed

    Mosteller, Timothy

    2005-01-01

    Cloned organisms can be genetically altered so that they do not exhibit higher brain functioning. This form of therapeutic cloning allows for genetically identical organs and tissues to be harvested from the clone for the use of the organism that is cloned. "Spare parts" cloning promises many opportunities for future medical advances. What is the ontological and ethical status of spare parts, headless clones? This paper attempts to answer this question from the perspective of Aristotle's view of the soul. Aristotle's metaphysics as applied to his view of biological essences generates an ethic that can contribute to moral reasoning regarding the use of headless spare parts clones. The task of this paper is to show the implications that Aristotle's view of the soul, if it is true, would have on the ethics of headless, spare parts cloning.

  8. 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.

  9. Comparing quantum cloning: A Fisher-information perspective

    NASA Astrophysics Data System (ADS)

    Song, Hongting; Luo, Shunlong; Li, Nan; Chang, Lina

    2013-10-01

    Perfect cloning of an unknown quantum state is impossible. Approximate cloning, which is optimal in various senses, has been found in many cases. Paradigmatic examples are Wootters-Zurek cloning and universal cloning. These cloning machines aim at optimal cloning of the full quantum states. However, in practice, what is important and relevant may only involve partial information in quantum states, rather than quantum states themselves. For example, signals are often encoded as parameters in quantum states, whose information content is well synthesized by quantum Fisher information. This raises the basic issue of evaluating the information transferring capability (e.g., distributing quantum Fisher information) of quantum cloning. We assess and compare Wootters-Zurek cloning and universal cloning from this perspective and show that, on average, Wootters-Zurek cloning performs better than universal cloning for the phase (as well as amplitude) parameter, although they are incomparable individually, and universal cloning has many advantages over Wootters-Zurek cloning in other contexts. Physical insights and related issues are further discussed.

  10. Cloning. Pigs is pigs.

    PubMed

    Prather, R S

    2000-09-15

    Since the first report of a cloned animal (Dolly the sheep) 3 years ago, cloning mammals has become something of a cottage industry. As Prather discusses in his Perspective, pigs can now be added to the august list of cloned animals, which includes cows, goats, and mice. This is a particularly spectacular achievement because pig cloning has turned out to be notoriously difficult. The pig is also a valuable domestic animal to have cloned because, being physiologically close to humans, its organs can be used in xenotransplantation.

  11. Cloning. Pigs is pigs.

    PubMed

    Prather, R S

    2000-09-15

    Since the first report of a cloned animal (Dolly the sheep) 3 years ago, cloning mammals has become something of a cottage industry. As Prather discusses in his Perspective, pigs can now be added to the august list of cloned animals, which includes cows, goats, and mice. This is a particularly spectacular achievement because pig cloning has turned out to be notoriously difficult. The pig is also a valuable domestic animal to have cloned because, being physiologically close to humans, its organs can be used in xenotransplantation. PMID:11012362

  12. Structural organization and transcription of the mouse gastric H+, K(+)-ATPase beta subunit gene.

    PubMed Central

    Canfield, V A; Levenson, R

    1991-01-01

    We have cloned and characterized the mouse gene encoding the beta subunit of H+, K(+)-ATPase (EC 3.6.1.36). The entire 10.5-kilobase transcription unit of the H+,K(+)-ATPase beta subunit gene was cloned in three overlapping cosmids encompassing approximately 46 kilobases of genomic DNA. A tight cluster of transcription initiation sites has been localized 24-25 nucleotides upstream of the translation start site and 28-29 nucleotides downstream of a TATA-like sequence. The H+, K(+)-ATPase beta subunit gene is split into seven exons encoding predicted structural domains of the beta subunit protein. The intracellular amino-terminal and putative transmembrane domains are encoded by individual exons, and the extracellular carboxyl-terminal domain is encoded by five exons. The exon/intron organization of the mouse H+,K(+)-ATPase beta subunit gene is identical to that of the mouse Na+,K(+)-ATPase beta 2 subunit gene. The conservation of genomic organization, together with the high sequence homology, indicates that the mouse H+,K(+)-ATPase beta and Na+,K(+)-ATPase beta 2 subunit genes originated from a common ancestral gene. Images PMID:1654563

  13. Cloning of ES cells and mice by nuclear transfer.

    PubMed

    Wakayama, Sayaka; Kishigami, Satoshi; Wakayama, Teruhiko

    2009-01-01

    We have been able to develop a stable nuclear transfer (NT) method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although the piezo unit is a complex tool, once mastered it is of great help not only in NT experiments, but also in almost all other forms of micromanipulation. Using this technique, embryonic stem (ntES) cell lines established from somatic cell nuclei can be generated relatively easily from a variety of mouse genotypes and cell types. Such ntES cells can be used not only for experimental models of human therapeutic cloning but also as a means of preserving mouse genomes instead of preserving germ cells. Here, we describe our most recent protocols for mouse cloning.

  14. When larger brains do not have more neurons: increased numbers of cells are compensated by decreased average cell size across mouse individuals

    PubMed Central

    Herculano-Houzel, Suzana; Messeder, Débora J.; Fonseca-Azevedo, Karina; Pantoja, Nilma A.

    2015-01-01

    There is a strong trend toward increased brain size in mammalian evolution, with larger brains composed of more and larger neurons than smaller brains across species within each mammalian order. Does the evolution of increased numbers of brain neurons, and thus larger brain size, occur simply through the selection of individuals with more and larger neurons, and thus larger brains, within a population? That is, do individuals with larger brains also have more, and larger, neurons than individuals with smaller brains, such that allometric relationships across species are simply an extension of intraspecific scaling? Here we show that this is not the case across adult male mice of a similar age. Rather, increased numbers of neurons across individuals are accompanied by increased numbers of other cells and smaller average cell size of both types, in a trade-off that explains how increased brain mass does not necessarily ensue. Fundamental regulatory mechanisms thus must exist that tie numbers of neurons to numbers of other cells and to average cell size within individual brains. Finally, our results indicate that changes in brain size in evolution are not an extension of individual variation in numbers of neurons, but rather occur through step changes that must simultaneously increase numbers of neurons and cause cell size to increase, rather than decrease. PMID:26082686

  15. Cloning pigs: advances and applications.

    PubMed

    Polejaeva, I A

    2001-01-01

    Although mouse embryonic stem cells have been used widely for over a decade as an important tool for introducing precise genetic modification into the genome, demonstrating the great value of this technology in a range of biomedical applications, similar technology does not exist for domestic animals. However, the development of somatic cell nuclear transfer has bypassed the need for embryonic stem cells from livestock. The production of offspring from differentiated cell nuclei provides information and opportunities in a number of areas including cellular differentiation, early development and ageing. However, the primary significance of cloning is probably in the opportunities that this technology brings to genetic manipulation. Potential applications of gene targeting in livestock species are described with particular emphasis on the generation of pigs that can be used for xenotransplantation, and the production of improved models for human physiology and disease. The development of techniques for somatic cell nuclear transfer in pigs and the challenges associated with this technology are also reviewed.

  16. The mouse genome informatics and the mouse genome database

    SciTech Connect

    Maltais, L.J.; Blackburn, R.E.; Bradt, D.W.

    1994-09-01

    The Mouse Genome Database (MGD) is a centralized, comprehensive database of the mouse genome that includes genetic mapping data, comparative mapping data, gene descriptions, mutant phenotype descriptions, strains and allelic polymorphism data, inbred strain characteristics, physical mapping data, and molecular probes and clones data. Data in MGD are obtained from the published literature and by electronic transfer from laboratories working on large backcross panels of mice. MGD provides tools that enable the user to search the database, retrieve data, generate reports, analyze data, annotate records, and build genetic maps. The Encyclopedia of the Mouse Genome provides a graphic user interface to mouse genome data. It consists of software tools including: LinkMap, a graphic display of genetic linkage maps with the ability to magnify regions of high locus density: CytoMap, a graphic display of cytogenetic maps showing banded chromosomes with cytogenetic locations of genes and chromosomal aberrations; CATS, a catalog searching tool for text retrieval of mouse locus descriptions. These software tools provide access to the following data sets: Chromosome Committee Reports, MIT Genome Center data, GBASE reports, Mouse Locus Catalog (MLC), and Mouse Cytogenetic Mapping Data. The MGD is available to the scientific community through the World Wide Web (WWW) and Gopher. In addition GBASE can be accessed via the Internet.

  17. The Synergistic Enhancement of Cloning Efficiency in Individualized Human Pluripotent Stem Cells by Peroxisome Proliferative-activated Receptor-γ (PPARγ) Activation and Rho-associated Kinase (ROCK) Inhibition.

    PubMed

    Kajabadi, Nasim-Sadat; Ghoochani, Ali; Peymani, Maryam; Ghaedi, Kamran; Kiani-Esfahani, Abbas; Hashemi, Motahareh-Sadat; Nasr-Esfahani, Mohammad Hossein; Baharvand, Hossein

    2015-10-23

    Although human pluripotent stem cells (hPSCs) provide valuable sources for regenerative medicine, their applicability is dependent on obtaining both suitable up-scaled and cost effective cultures. The Rho-associated kinase (ROCK) inhibitor Y-27632 permits hPSC survival upon dissociation; however, cloning efficiency is often still low. Here we have shown that pioglitazone, a selective peroxisome proliferative-activated receptor-γ agonist, along with Y-27632 synergistically diminished dissociation-induced apoptosis and increased cloning efficiency (2-3-fold versus Y-27632) without affecting pluripotency of hPSCs. Pioglitazone exerted its positive effect by inhibition of glycogen synthase kinase (GSK3) activity and enhancement of membranous β-catenin and E-cadherin proteins. These effects were reversed by GW-9662, an irreversible peroxisome proliferative-activated receptor-γ antagonist. This novel setting provided a step toward hPSC manipulation and its biomedical applications.

  18. Cloning adult animals - what is the genetic age of the clones?

    PubMed

    Yang, X; Tian, X C

    2000-01-01

    The rapid progress in cloning research along with its many ramifications will soon have a significant beneficial impact on basic research, agriculture, and biomedicine. However, for the nuclear transfer technology to reach its fullest potential, it is important to understand whether the cloning procedure can reverse cellular aging and generate clones with normal genetic and physiological age, similar to those produced from natural reproduction. Telomere shortening is believed to correlate with cellular aging both in vitro and in vivo. Telomere lengths in cells of cloned individuals thus may reflect their genetic age. However, controversies have developed over whether the eroded telomere in somatic cells used for nuclear transfer can be restored during the cloning process.

  19. Genome Clone Libraries and Data from the Integrated Molecular Analysis of Genomes and their Expression (I.M.A.G.E.) Consortium

    DOE Data Explorer

    The I.M.A.G.E. Consortium was initiated in 1993 by four academic groups on a collaborative basis after informal discussions led to a common vision of how to achieve an important goal in the study of the human genome: the Integrated Molecular Analysis of Genomes and their Expression Consortium's primary goal is to create arrayed cDNA libraries and associated bioinformatics tools, and make them publicly available to the research community. The primary organisms of interest include intensively studied mammalian species, including human, mouse, rat and non-human primate species. The Consortium has also focused on several commonly studied model organisms; as part of this effort it has arrayed cDNAs from zebrafish, and Fugu (pufferfish) as well as Xenopus laevis and X. tropicalis (frog). Utilizing high speed robotics, over nine million individual cDNA clones have been arrayed into 384-well microtiter plates, and sufficient replicas have been created to distribute copies both to sequencing centers and to a network of five distributors located worldwide. The I.M.A.G.E. Consortium represents the world's largest public cDNA collection, and works closely with the National Institutes of Health's Mammalian Gene Collection(MGC) to help it achieve its goal of creating a full-length cDNA clone for every human and mouse gene. I.M.A.G.E. is also a member of the ORFeome Collaboration, working to generate a complete set of expression-ready open reading frame clones representing each human gene. Custom informatics tools have been developed in support of these projects to better allow the research community to select clones of interest and track and collect all data deposited into public databases about those clones and their related sequences. I.M.A.G.E. clones are publicly available, free of any royalties, and may be used by anyone agreeing with the Consortium's guidelines.

  20. Vitamin D for combination photodynamic therapy of skin cancer in individuals with vitamin D deficiency: Insights from a preclinical study in a mouse model of squamous cell carcinoma

    NASA Astrophysics Data System (ADS)

    Anand, Sanjay; Thomas, Erik; Hasan, Tayyaba; Maytin, Edward V.

    2016-03-01

    Combination photodynamic therapy (cPDT) in which vitamin D (VD) is given prior to aminolevulinate, a precursor (pro-drug) for protoporphyrin IX (PpIX), is an approach developed in our laboratory. We previously showed that 1α,25- dihydroxyvitamin D3 (calcitriol), given prior to PDT, enhances accumulation of PpIX and improves cell death post-PDT in a mouse skin cancer model. However, since calcitriol poses a risk for hypercalcemia, we replaced systemic calcitriol with oral cholecalciferol (D3), administered as a high (tenfold, "10K") diet over a ten-day period. Here, we ask whether VD deficiency might alter the response to cPDT. Nude mice were fed a VD-deficient diet for at least 4 weeks ("deficient"); controls were fed a normal 1,000 IU/kg diet ("1K"). Human A431 cells were implanted subcutaneously and mice were switched to the 10K diet or continued on their baseline diets (controls). In other experiments, mice received a human equivalent dose of 50,000 IU D3 by oral gavage, to simulate administration of a single, high-dose VD pill. At various times, tumors were harvested and serum was collected to measure levels of VD metabolic intermediates. A significant increase in PpIX levels and in the expression of differentiation and proliferation markers in tumor tissue was observed after VD supplementation of both the deficient and 1K mice. Further results describing mechanistic details of PpIX enhancement through alteration of heme- and VD-metabolic enzyme levels will be presented. Based on these results, a clinical study using oral vitamin D prior to PDT for human skin cancer should be performed.

  1. Efficacy of PARP inhibition in Pde6a mutant mouse models for retinitis pigmentosa depends on the quality and composition of individual human mutations

    PubMed Central

    Jiao, K; Sahaboglu, A; Zrenner, E; Ueffing, M; Ekström, P A R; Paquet-Durand, F

    2016-01-01

    Retinitis pigmentosa (RP), an inherited blinding disease, is caused by a variety of different mutations that affect retinal photoreceptor function and survival. So far there is neither effective treatment nor cure. We have previously shown that poly(ADP-ribose)polymerase (PARP) acts as a common and critical denominator of cell death in photoreceptors, qualifying it as a potential target for future therapeutic intervention. A significant fraction of RP-causing mutations affect the genes for the rod photoreceptor phosphodiesterase 6A (PDE6A) subunit, but it is not known whether they all engage the same death pathway. Analysing three homozygous point mutations (Pde6a R562W, D670G, and V685M) and one compound heterozygous Pde6aV685M/R562W mutation in mouse models that match human RP patients, we demonstrate excessive activation of PARP, which correlated in time with the progression of photoreceptor degeneration. The causal involvement of PARP activity in the neurodegenerative process was confirmed in organotypic retinal explant cultures treated with the PARP-selective inhibitor PJ34, using different treatment time-points and durations. Remarkably, the neuroprotective efficacy of PARP inhibition correlated inversely with the strength of the genetically induced insult, with the D670G mutant showing the best treatment effects. Our results highlight PARP as a target for neuroprotective interventions in RP caused by PDE6A mutations and are a first attempt towards personalized, genotype-matched therapy development for RP. In addition, for each of the different mutant situations, our work identifies windows of opportunity for an optimal treatment regimen for further in vivo experimentation and possibly clinical studies. PMID:27551530

  2. Cloning of cDNA and genomic DNA encoding human type XVIII collagen and localization of the [alpha]1 (XVIII) collagen gene to mouse chromosome 10 and human chromosome 21

    SciTech Connect

    Oh, S.P.; Warman, M.L.; Timmons, S.; Olsen, B.R.; Knoll, J.H.M. ); Seldin, M.F. ); Cheng, Sou-De )

    1994-02-01

    Types XV and XVIII collagen belong to a unique and novel subclass of the collagen superfamily for which the authors have proposed the name the MULTIPLEXIN family. Members of this class contain polypeptides with multiple triple-helical domains separated and flanked by non-triple-helical regions. In this paper, they report the isolation of human cDNAs and genomic DNAs encoding the [alpha]1 (XVIII) collagen chain. Utilizing a genomic clone as probe, they have mapped the COL18A1 gene to chromosome 21q22.3 by fluorescence in situ hybridization. In addition, using an interspecific backcross panel, they have shown that the murine Col18a1 locus is on chromosome 10, close to the loci for Col6a1 and Col6a2. 16 refs., 5 figs.

  3. Networks of protein kinases and phosphatases in the individual phases of contextual fear conditioning in the C57BL/6J mouse.

    PubMed

    Mucic, Goran; Sase, Sunetra; Stork, Oliver; Lubec, Gert; Li, Lin

    2015-03-01

    Although protein kinases and phosphatases have been reported to be involved in fear memory, information about these signalling molecules in the individual phases of contextual fear conditioning (cFC) is limited. C57BL/6J mice were tested in cFC, sacrificed and hippocampi were used for screening of approximately 800 protein kinases and phosphatases by protein microarrays with subsequent Western blot confirmation of threefold higher or lower hippocampal levels as compared to foot shock controls. Immunoblotting of the protein kinases and phosphatases screened out was carried out by Western blotting. A network of protein kinases and phosphatases was generated (STRING 9.1). Animals learned the task in the paradigm and protein kinase and phosphatase levels were determined in the individual phases acquisition, consolidation and retrieval and compared to foot shock controls. Protein kinases discoidin containing receptor 2 (DDR2), mitogen activated protein kinase kinase kinase 7 (TAK1), protein phosphatases dual specificity protein phosphatase (PTEN) and protein phosphatase 2a (PP2A) were modulated in the individual phases of cFC. Phosphatidyl-inositol-3,4,5-triphosphate 3-phosphatase and phosphatidylinositol-3 kinase (PI3K) that is interacting with PTEN were modulated as well. Freezing time was correlating with PP2A levels in the retrieval phase of cFC. The abovementioned protein kinases, phosphatases and inositol-signalling enzymes were not reported so far in cFC and the results are relevant for interpretation of previous and design of future studies in cFC or fear memory. Protein phosphatase PP2A was, however, the only signalling compound tested that was directly linked to retrieval in the cFC. PMID:25461266

  4. Method for cloning genes

    SciTech Connect

    Weissman, S.M.; Pereira, D.; Sood, A.

    1988-04-19

    This patent describes a recombinant cloning vehicle comprising an inserted human gene, the improvement wherein the cloning vehicle is isolated from a recombinant clone which is isolated and identified by a process comprising the steps of: (a) effecting cDNA synthesis on a mixture of mRNAs containing a target mRNA coding for a major hisitocompatibility antigen, and isolating the resultant cDNA mixture; (b) inserting the resultant cDNA into recombinant cloning vehicles, and transforming hosts with the vehicles; and (c) separating the transformants and isolating and identifying a recombinant clone containing a DNA segment which is homologous over at least a portion thereof to at least one oligonucleotide probe specific for the DNA segment.

  5. Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning.

    PubMed

    Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan; Wu, Zhenfang

    2013-02-01

    Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 ± 0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 ± 0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 ± 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a

  6. On cloning human beings.

    PubMed

    de Melo-Martin, Inmaculada

    2002-06-01

    The purpose of this paper is to show that arguments for and against cloning fail to make their case because of one or both of the following reasons: 1) they take for granted customary beliefs and assumptions that are far from being unquestionable; 2) they tend to ignore the context in which human cloning is developed. I will analyze some of the assumptions underlying the main arguments that have been offered for and against cloning. Once these assumptions are critically analyzed, arguments both rejecting and supporting human cloning seem to lose weight. I will first briefly present the main arguments that have been proposed against cloning and I will argue that they fail to establish their case. In the next section I will evaluate some of the positive arguments that have been offered supporting such technology. This analysis will show that the case for cloning also fails. Finally, I will maintain that because critics and especially supporters of this technology neglect the context in which human cloning is developed and might be implemented, their arguments are far from compelling.

  7. Mink-mouse interspecific hybridomas.

    PubMed

    Ufimtseva, E G; Galakhar, N L; Matjakhina, L D; Khlebodarova, T M; Djatchenko, S N

    1991-08-01

    Mink-mouse interspecific hybridomas were produced by fusion of the american mink spleen cells with the NSO cells. Seven cloned lines of the mink-mouse hybridoma were isolated, and their functional mink Ig secretion and karyological characteristics are given. During cytogenetic analysis of mink-mouse hybridoma cell lines, we observed the elimination of mink chromosomes, and inter- and intralineral variability of the numbers of the cells with particular quantities of mink DNA. We did not find that the characteristic peculiarities of mink DNA distribution in the hybridoma cell lines had any bearing upon the secretion or non-secretion of mink Ig. There was no synthesis of lambda-L-chains of mink Ig in line 7 cells because the line lost the lambda-gene. With the aid of in situ hybridization with 3H-labeled total mink DNA, a considerable transformation of hybridoma cell karyotype was observed. Multiple integration of the mink DNA into mouse chromosomes and the appearance of chromosomes not characteristic for either the mink or mouse parent cells were noted. Increasing numbers of cells with translocations of mink chromosomes fragments into mouse chromosomes were found in the hybridoma lines cultivated for lengthy periods. PMID:1937502

  8. Robert Koch: the grandfather of cloning?

    PubMed

    Weiss, Robin A

    2005-11-18

    This year marks the centenary of Robert Koch's Nobel Prize for discovering the cause of tuberculosis. Koch was also the first scientist to isolate the anthrax and cholera microbes. Yet perhaps one of his greatest contributions to biology is the least appreciated: his method for propagating individual colonies of bacteria on plates, a technique that came to be called cloning.

  9. Mink-mouse hybridomas that secrete mink immunoglobulin G.

    PubMed

    Galakhar, N L; Djatchenko, S N; Fomicheva, I I; Mechetina, L V; Taranin, A V; Belousov, E S; Nayakshin, A M; Baranov, O K

    1988-11-25

    Optimum conditions were established to obtain mink-mouse interspecific hybridomas secreting mink IgG in fusions of mouse myelomas with mink immune spleen cells. Minks were immunized with allogeneic IgG, and the spleen cells were fused with three mouse myeloma lines P3-X63-Ag8.653, NSO and Sp2/0-Ag14. Of these, P3-X63-Ag8.653 and NSO were found to be the best fusion partners giving the highest yield of hybrid clones and number of IgG secreting clones. Cloning of mink-mouse hybridomas was efficient when BALB/c nu/nu peritoneal and spleen cells were used as feeders. The ten clonal lines produced secreted intact mink IgG molecules as shown by SDS-PAGE and subsequent immunoblotting. The secretion level of IgG ranged from 5 to 200 ng/ml in the clonal lines.

  10. 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

  11. 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.

  12. Do Managers Clone Themselves?

    ERIC Educational Resources Information Center

    Baron, Alma S.

    1981-01-01

    A recent questionnaire survey provides statistics on male managers' views of female managers. The author recommends that male managers break out of their cloning behavior and that the goal ought to be a plurality in management. (Author/WD)

  13. Statement on Human Cloning

    MedlinePlus

    ... form Search American Association for the Advancement of Science Statement on Human Cloning Print Email Tweet The American Association for the Advancement of Science (AAAS) recognizes the intense debates within our society ...

  14. The relative cytotoxicity of personal care preservative systems in Balb/C 3T3 clone A31 embryonic mouse cells and the effect of selected preservative systems upon the toxicity of a standard rinse-off formulation.

    PubMed

    Smith, C N; Alexander, B R

    2005-10-01

    Biocide chemicals are commonly used as preservatives for cosmetic and personal care products and the conditions for their use are stipulated in Annex VI of the Cosmetics Directive. In these studies the cytotoxicity (EC50 and EC90) of a range of preservatives including the isothiazolinone family, formaldehyde donors, parabens mixtures and organic acids have been established in the Balb/C 3T3 clone A31 fibroblast cell-line following a 1h exposure. Cell viability was established using the neutral red uptake assay 24h after exposure. The potency of the preservatives spanned several orders of magnitude from the isothiazolinones (EC50<10ppm) to the organic acids (EC50>10,000ppm). Although these values are directly proportional to the anti-microbial efficacy of the actives, they do not reflect the addition levels commonly used to preserve formulations, which are intended to provide prolonged protection against a wide spectrum of spoilage organisms. In a further study, the cytotoxic profile of an unpreserved standard rinse-off body wash formulation was assessed. Two concentrations of the formulation were selected: 0.1% v/v (EC98) and 0.15% v/v (EC82) to study the effects of selected preservative chemicals at recommended addition levels upon the cytotoxicity of the formulation. At 0.1%, only preservation with benzoate/sorbate at the highest addition level increased the toxicity, whereas at 0.15%, preservation with 2-bromo-2-nitro-propane-1,3-diol increased the cytotoxicity of the formulation. No other preservatives, including isothiazolinones and formaldehyde donors affected the basal cytotoxicity of the formulation. Theses studies have provided a standardised assessment of the cytotoxicity of cosmetic preservatives and demonstrated that preservation of a rinse-off formulation at recommended addition levels is unlikely to affect the cytotoxic profile.

  15. cDNA cloning of a mouse mammary epithelial cell surface protein reveals the existence of epidermal growth factor-like domains linked to factor VIII-like sequences

    SciTech Connect

    Stubbs, J.D.; Bui, A. San Francisco State Univ., CA ); Lekutis, C.; Singer, K.L.; Srinivasan, U.; Parry, G. ); Yuzuki, D. )

    1990-11-01

    A 2.1-kilobase cDNA coding for a surface protein of mammary epithelial cells has been isolated from a mouse mammary gland {lambda}gt11 cDNA library. Sequence analysis of this cDNA reveals an open reading frame of 1,389 base pairs that defines a protein with a molecular mass of 51.5 dKa. Structural analysis of the predicted sequence identifies two putative functional domains of the protein: (i) an N-terminal cysteine-rich region that is similar to epidermal growth factor-like domains of Drosophila Notch-1 protein and (ii) a large segment of the sequence that exhibited 54.5% identify with C-terminal domains of human coagulation factors VIII and V. These similarities in structure are used to predict the possible functions of the protein and its means of interaction with the cell surface. mRNA expression was detectable in mammary tissue from nonpregnant animals but was maximal in the lactating gland. In cultured cells, mRNA levels also correlated with the degree of cellular differentiation.

  16. [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.

  17. Stability of the JP2 clone of Aggregatibacter actinomycetemcomitans.

    PubMed

    Haubek, D; Ennibi, O-K; Vaeth, M; Poulsen, S; Poulsen, K

    2009-09-01

    The JP2 clone of Aggregatibacter actinomycetemcomitans is strongly associated with aggressive periodontitis. To obtain information about colonization dynamics of the JP2 clone, we used PCR to examine its presence in 365 Moroccan juveniles from whom periodontal plaque samples were collected at baseline and after one and two years. Periodontal attachment loss was measured at baseline and at the two-year follow-up. At baseline, 43 (12%) carriers of the JP2 clone were found. Nearly half (44 %) of these were persistently colonized with the clone. The relative risk for the development of aggressive periodontitis, adjusted for the concomitant presence of other genotypes of A. actinomycetemcomitans, was highest for individuals continuously infected by the JP2 clone (RR = 13.9; 95% CI, 9.0 to 21.4), indicating a relationship between infectious dose and disease, which further substantiates the evidence for the JP2 clone as a causal factor in aggressive periodontitis.

  18. Consumers' attitudes toward consumption of cloned beef. The impact of exposure to technological information about animal cloning.

    PubMed

    Aizaki, Hideo; Sawada, Manabu; Sato, Kazuo

    2011-10-01

    Novel food technologies, such as cloning, have been introduced into the meat production sector; however, their use is not widely supported by many consumers. This study was designed to assess whether Japanese consumers' attitudes toward consumption of cloned beef (specifically, beef derived from bovine embryo and somatic cell-cloned cattle) would change after they were provided with technological information on animal cloning through a web-based survey. The results revealed that most respondents did not discriminate between their attitudes toward the consumption of the two types of cloned beef, and that most respondents did not change their attitudes toward cloned beef after receiving the technological information. The respondents' individual characteristics, including their knowledge about the food safety of cloned beef and their basic knowledge about animal cloning, influenced the likelihood of a change in their attitudes after they received the information. In conclusion, some consumers might become less uncomfortable about the consumption of cloned beef by the straightforward provision of technological information about animal cloning; however, most consumers are likely to maintain their attitudes.

  19. Consumers' attitudes toward consumption of cloned beef. The impact of exposure to technological information about animal cloning.

    PubMed

    Aizaki, Hideo; Sawada, Manabu; Sato, Kazuo

    2011-10-01

    Novel food technologies, such as cloning, have been introduced into the meat production sector; however, their use is not widely supported by many consumers. This study was designed to assess whether Japanese consumers' attitudes toward consumption of cloned beef (specifically, beef derived from bovine embryo and somatic cell-cloned cattle) would change after they were provided with technological information on animal cloning through a web-based survey. The results revealed that most respondents did not discriminate between their attitudes toward the consumption of the two types of cloned beef, and that most respondents did not change their attitudes toward cloned beef after receiving the technological information. The respondents' individual characteristics, including their knowledge about the food safety of cloned beef and their basic knowledge about animal cloning, influenced the likelihood of a change in their attitudes after they received the information. In conclusion, some consumers might become less uncomfortable about the consumption of cloned beef by the straightforward provision of technological information about animal cloning; however, most consumers are likely to maintain their attitudes. PMID:21723894

  20. Virulence Potential and Genome-Wide Characterization of Drug Resistant Streptococcus pneumoniae Clones Selected In Vivo by the 7-Valent Pneumococcal Conjugate Vaccine

    PubMed Central

    Frazão, Nelson; Hiller, N. Luisa; Powell, Evan; Earl, Josh; Ahmed, Azad; Sá-Leão, Raquel; de Lencastre, Hermínia; Ehrlich, Garth D.; Tomasz, Alexander

    2013-01-01

    We used mouse models of pneumococcal colonization and disease combined with full genome sequencing to characterize three major drug resistant clones of S. pneumoniae that were recovered from the nasopharynx of PCV7-immunized children in Portugal. The three clones – serotype 6A (ST2191), serotype 15A (ST63) and serotype 19A (ST276) carried some of the same drug resistance determinants already identified in nasopharyngeal isolates from the pre-PCV7 era. The three clones were able to colonize efficiently the mouse nasopharyngeal mucosa where populations of these pneumococci were retained for as long as 21 days. During this period, the three clones were able to asymptomatically invade the olfactory bulbs, brain, lungs and the middle ear mucosa and established populations in these tissues. The virulence potential of the three clones was poor even at high inoculum (105 CFU per mouse) concentrations in the mouse septicemia model and was undetectable in the pneumonia model. Capsular type 3 transformants of clones 6A and 19A prepared in the laboratory produced lethal infection at low cell concentration (103 CFU per mouse) but the same transformants became impaired in their potential to colonize, indicating the importance of the capsular polysaccharide in both disease and colonization. The three clones were compared to the genomes of 56 S. pneumoniae strains for which sequence information was available in the public databank. Clone 15A (ST63) only differed from the serotype 19F clone G54 in a very few genes including serotype so that this clone may be considered the product of a capsular switch. While no strain with comparable degree of similarity to clone 19A (ST276) was found among the sequenced isolates, by MLST this clone is a single locust variant (SLV) of Denmark14-ST230 international clone. Clone 6A (ST2191) was most similar to the penicillin resistant Hungarian serotype 19A clone. PMID:24069360

  1. Cloning of the complete Mycoplasma pneumoniae genome.

    PubMed Central

    Wenzel, R; Herrmann, R

    1989-01-01

    The complete genome of Mycoplasma pneumoniae was cloned in an ordered library consisting of 34 overlapping or adjacent cosmids, one plasmid and two lambda phages. The genome size was determined by adding up the sizes of either the individual unique EcoRI restriction fragments of the gene bank or of the XhoI fragments of genomic M. pneumoniae DNA. The values from these calculations, 835 and 849 kbp, are in good agreement. An XhoI restriction map was constructed by identifying adjacent DNA fragments by probing with selected cosmid clones. Images PMID:2506532

  2. Evidence for a human-specific Escherichia coli clone.

    PubMed

    Clermont, Olivier; Lescat, Mathilde; O'Brien, Claire L; Gordon, David M; Tenaillon, Olivier; Denamur, Erick

    2008-04-01

    Escherichia coli is a widespread commensal of the vertebrate intestinal tract. Until recently, no strong association between a particular clone and a given host species has been found. However, members of the B2 subgroup VIII clone with an O81 serotype appear to be human host specific. To determine the degree of host specificity exhibited by this clone, a PCR-based assay was used to screen 723 faecal and clinical isolates from humans, and 904 faecal isolates from animals. This clone was not detected among the animal isolates, but was discovered in people living in Africa, Europe and South America. The clone is rarely isolated from people suffering from intestinal or extraintestinal disease and is avirulent in a mouse model of extraintestinal infection. Fine-scale epidemiological analysis suggests that this clone is competitively dominant relative to other members of the B2 phylogenetic group and that it has increased in frequency over the past 20 years. This clone appears to be a good candidate for use as a probiotic, and may be suitable as an indicator of human faecal contamination in microbial source tracking studies.

  3. Production of Cloned Mice by Nuclear Transfer of Cumulus Cells

    PubMed Central

    Kurd, Soleiman; Zarei, Mohammad Ali; Fathi, Fardin; Ghadimi, Tayyeb; Hakhamaneshi, Mohammad Saeed; Jalili, Ali

    2013-01-01

    Background Over the past several years, mammals have been successfully cloned by either the splitting of an early stage embryo or nuclear transfer of adult somatic cells (NT) into oocytes. Although it has been 15 years since the generation of the first cloned mammals from somatic cells by NT, the success rate for producing live offspring by this technique is low regardless of the cell type and animal species used. However, these techniques have the potential to be important tools for future research in basic biology. In the present study, we described our experiences in producing successfully cloned mouse using NT method and piezo-actuated micromanipulator. Methods B6D2F1 mice, 8-12 weeks old, were superovulated with injections of 5 IU of pregnant mare serum gonadotropin and 5 IU of human chorionic gonadotropin administered 48 hr apart. Enucleation and donor nuclei cumulus cell injection were performed with a piezo-actuated micromanipulator after which activation and trichostatin A treatment were used for reconstructed oocytes. Two-cell stage cloned embryos that developed in the mWM medium were transferred into the oviducts of pseudopregnant NMRI mice. Results Of 367 oocytes collected, 131 (69%) developed into 2-cell stage embryos. Of these, 5 (1%) live pups were successfully delivered. We used NMRI foster mother to raise the pups by lactation. One adult cloned mouse was mated, after which she delivered and raised normal offspring. Conclusion For mouse cloning, the present study also successfully tested the capability of somatic cell nuclear transfer SCNT using a piezo unit. PMID:23919122

  4. Extremal quantum cloning machines

    SciTech Connect

    Chiribella, G.; D'Ariano, G. M.; Perinotti, P.; Cerf, N.J.

    2005-10-15

    We investigate the problem of cloning a set of states that is invariant under the action of an irreducible group representation. We then characterize the cloners that are extremal in the convex set of group covariant cloning machines, among which one can restrict the search for optimal cloners. For a set of states that is invariant under the discrete Weyl-Heisenberg group, we show that all extremal cloners can be unitarily realized using the so-called double-Bell states, whence providing a general proof of the popular ansatz used in the literature for finding optimal cloners in a variety of settings. Our result can also be generalized to continuous-variable optimal cloning in infinite dimensions, where the covariance group is the customary Weyl-Heisenberg group of displacement000.

  5. Phenotypic abnormalities observed in aged cloned mice from embryonic stem cells after long-term maintenance.

    PubMed

    Shimozawa, Nobuhiro; Sotomaru, Yusuke; Eguchi, Natsuko; Suzuki, Shuzo; Hioki, Kyoji; Usui, Toshimi; Kono, Tomohiro; Ito, Mamoru

    2006-09-01

    Somatic/embryonic stem cell cloning has made it possible to produce an individual genomically identical to another individual. However, the cloned animals have a variety of abnormalities caused by the aberrant gene modification, with insufficient reprogramming in cloning. We previously reported abnormalities in cloned mice at birth. In this study, we examined what abnormalities could be seen in cloned mice after long-term maintenance. The aged cloned mice showed multiple abnormalities: increase of body weight, some phenotypic abnormalities in the kidneys, testes and thymus, and lower urea nitrogen in their serum biochemical values. The kidneys of all cloned mice were hypertrophied, with a metamorphic or whitish appearance. The multiple lesions, including the enlarged renal pelvis and distension of the renal veins in histology, might be the result of urine accumulation by urinary tract obstruction. The testes of the cloned mice were atrophied, and showed no sperm formation in histology. In contrast, the thymus was rather hypertrophied, and a comparably increased number of lymphocytes were observed in the medulla, consisting mainly of T cells. By conducting a progeny test between the cloned mice, it was confirmed that these abnormalities in the aged cloned mice were not transmitted to their offspring, indicating that the incomplete reprogramming in clones might be in part responsible for the abnormalities detected in aged clones. These results indicate that the postnatal abnormalities observed in aged cloned mice are varied and can be restored through the germ line. PMID:16940284

  6. Efficient high-resolution genetic mapping of mouse interspersed repetitive sequence PCR products, toward integrated genetic and physical mapping of the mouse genome.

    PubMed Central

    McCarthy, L; Hunter, K; Schalkwyk, L; Riba, L; Anson, S; Mott, R; Newell, W; Bruley, C; Bar, I; Ramu, E

    1995-01-01

    The ability to carry out high-resolution genetic mapping at high throughput in the mouse is a critical rate-limiting step in the generation of genetically anchored contigs in physical mapping projects and the mapping of genetic loci for complex traits. To address this need, we have developed an efficient, high-resolution, large-scale genome mapping system. This system is based on the identification of polymorphic DNA sites between mouse strains by using interspersed repetitive sequence (IRS) PCR. Individual cloned IRS PCR products are hybridized to a DNA array of IRS PCR products derived from the DNA of individual mice segregating DNA sequences from the two parent strains. Since gel electrophoresis is not required, large numbers of samples can be genotyped in parallel. By using this approach, we have mapped > 450 polymorphic probes with filters containing the DNA of up to 517 backcross mice, potentially allowing resolution of 0.14 centimorgan. This approach also carries the potential for a high degree of efficiency in the integration of physical and genetic maps, since pooled DNAs representing libraries of yeast artificial chromosomes or other physical representations of the mouse genome can be addressed by hybridization of filter representations of the IRS PCR products of such libraries. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:7777502

  7. 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.

  8. Applications of quantum cloning

    NASA Astrophysics Data System (ADS)

    Pomarico, E.; Sanguinetti, B.; Sekatski, P.; Zbinden, H.; Gisin, N.

    2011-10-01

    Quantum Cloning Machines (QCMs) allow for the copying of information, within the limits imposed by quantum mechanics. These devices are particularly interesting in the high-gain regime, i.e., when one input qubit generates a state of many output qubits. In this regime, they allow for the study of certain aspects of the quantum to classical transition. The understanding of these aspects is the root of the two recent applications that we will review in this paper: the first one is the Quantum Cloning Radiometer, a device which is able to produce an absolute measure of spectral radiance. This device exploits the fact that in the quantum regime information can be copied with only finite fidelity, whereas when a state becomes macroscopic, this fidelity gradually increases to 1. Measuring the fidelity of the cloning operation then allows to precisely determine the absolute spectral radiance of the input optical source. We will then discuss whether a Quantum Cloning Machine could be used to produce a state visible by the naked human eye, and the possibility of a Bell Experiment with humans playing the role of detectors.

  9. The Cloning of America.

    ERIC Educational Resources Information Center

    Dobson, Judith E.; Dobson, Russell L.

    1981-01-01

    Proposes that the U.S. school system purports to prize human variability, but many educators are engaged in activities that seek to homogenize students. Describes these activities, including diagnosis, labeling, ability grouping, and positive reinforcement. Presents suggestions for counselors to combat sources of cloning and self-validation. (RC)

  10. Human therapeutic cloning.

    PubMed

    Lanza, R P; Cibelli, J B; West, M D

    1999-09-01

    Somatic cell nuclear 'reprogramming' in livestock species is now routine in many laboratories. Here, Robert Lanza, Jose Cibelli and Michael West discuss how these techniques may soon be used to clone genetically matched cells and tissues for transplantation into patients suffering from a wide range of disorders that result from tissue loss or dysfunction.

  11. [Nuclear transfer and therapeutic cloning].

    PubMed

    Xu, Xiao-Ming; Lei, An-Min; Hua, Jin-Lian; Dou, Zhong-Ying

    2005-03-01

    Nuclear transfer and therapeutic cloning have widespread and attractive prospects in animal agriculture and biomedical applications. We reviewed that the quality of oocytes and nuclear reprogramming of somatic donor cells were the main reasons of the common abnormalities in cloned animals and the low efficiency of cloning and showed the problems and outlets in therapeutic cloning, such as some basic problems in nuclear transfer affected clinical applications of therapeutic cloning. Study on isolation and culture of nuclear transfer embryonic stem (ntES) cells and specific differentiation of ntES cells into important functional cells should be emphasized and could enhance the efficiency. Adult stem cells could help to cure some great diseases, but could not replace therapeutic cloning. Ethics also impeded the development of therapeutic cloning. It is necessary to improve many techniques and reinforce the research of some basic theories, then somatic nuclear transfer and therapeutic cloning may apply to agriculture reproduction and benefit to human life better.

  12. The First Human Cloned Embryo.

    ERIC Educational Resources Information Center

    Cibelli, Jose B.; Lanza, Robert P.; West, Michael D.; Ezzell, Carol

    2002-01-01

    Describes a process known as parthenogenesis which produces cloned, early-stage embryos and human embryos generated only from eggs. Speculates that this technology puts therapeutic cloning within reach. (DDR)

  13. Sequential cloning of chromosomes

    SciTech Connect

    Lacks, S.A.

    1991-12-31

    A method for sequential cloning of chromosomal DNA and chromosomal DNA cloned by this method are disclosed. The method includes the selection of a target organism having a segment of chromosomal DNA to be sequentially cloned. A first DNA segment, having a first restriction enzyme site on either side. homologous to the chromosomal DNA to be sequentially cloned is isolated. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes.

  14. Cloning of Plasmodium falciparum by single-cell sorting.

    PubMed

    Miao, Jun; Li, Xiaolian; Cui, Liwang

    2010-10-01

    Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples.

  15. Probabilistic Cloning and Quantum Computation

    NASA Astrophysics Data System (ADS)

    Gao, Ting; Yan, Feng-Li; Wang, Zhi-Xi

    2004-06-01

    We discuss the usefulness of quantum cloning and present examples of quantum computation tasks for which the cloning offers an advantage which cannot be matched by any approach that does not resort to quantum cloning. In these quantum computations, we need to distribute quantum information contained in the states about which we have some partial information. To perform quantum computations, we use a state-dependent probabilistic quantum cloning procedure to distribute quantum information in the middle of a quantum computation.

  16. Tests of the mouse visual system.

    PubMed

    Pinto, L H; Enroth-Cugell, C

    2000-07-01

    To apply the approach of forward genetics (e.g., gene identification with mutagenesis and screening, followed by positional cloning) to the mouse, it is necessary to have available screening tests that can be applied rapidly to individual mice and that give a reliable assessment of visual function. This paper reviews the strengths and limitations of two anatomical tests related to visual function, fundus examination and retinal histological examination. Two tests that do not depend on behavior of a conscious animal are reviewed: the electroretinogram and the visual evoked potentials of the cortex. Eight behavioral tests are also summarized: maze-based tests, cued fear conditioning, tests based on conditioned suppression, visual placing, optokinetic nystagmus, pupillary reflex, and light-induced shifts in circadian phase. It is recommended that retinal histology, the electroretinogram, and visual-evoked potentials be used at the present time for screening because they assess the function and structure of the visual system rapidly and reliably. In fact, the electroretinogram (or visually evoked potentials) can be recorded from several animals simultaneously in response to the same stimulus. It is also recommended that efforts be made to develop more appropriate, automated, behavioral tests of visual perception than are now available, particularly tests that rely solely on rewarding visually evoked behavior. Two other promising behavioral tests are cued fear conditioning and variants of maze tests.

  17. Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes

    PubMed Central

    Throop, Andrea L.; LaBaer, Joshua

    2015-01-01

    The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular recombinational cloning technologies, including the commercially available Gateway® (Life Technologies) and In-Fusion® (Clontech) cloning technologies. PMID:25827088

  18. Mouse models of myelodysplastic syndromes

    PubMed Central

    Beachy, Sarah H.; Aplan, Peter D.

    2010-01-01

    Synopsis Three general approaches have been used in an attempt to model myelodysplastic syndrome (MDS) in mice, including treatment with mutagens or carcinogens, xenotransplantation of human MDS cells, and genetic engineering of mouse hematopoietic cells. Xenotransplantation of cells from MDS patients has proved difficult, possibly due to the innate characteristics of the MDS clone and microenvironmental influences, including adverse effects of a host immune response. Genetic engineering of hematopoietic cells or mice has been accomplished by in vitro transfer of genes to mouse hematopoietic cells with subsequent transplantation into an irradiated host, or by modification of the mouse germline to generate mice with altered expression of genes of interest. A number of genes have been studied using these approaches, including RUNX1, Evi1, Npm1, SALL4B, NUP98-HOXD13, BCL2/NRAS, Arid4a, Polg and Dido. This review discusses the phenotypes observed in available mouse models for MDS with a concentration on a model that leads to aberrant expression of conserved homeobox (HOX) genes that are important regulators of normal hematopoiesis. Utilizing these models of MDS should allow a more complete understanding of the disease process and provide a platform for pre-clinical testing of therapeutic approaches. PMID:20359631

  19. Keith's MAGIC: Cloning and the Cell Cycle.

    PubMed

    Wells, D N

    2013-10-01

    Abstract Professor Keith Campbell's critical contribution to the discovery that a somatic cell from an adult animal can be fully reprogrammed by oocyte factors to form a cloned individual following nuclear transfer (NT)(Wilmut et al., 1997 ) overturned a dogma concerning the reversibility of cell fate that many scientists had considered to be biologically impossible. This seminal experiment proved the totipotency of adult somatic nuclei and finally confirmed that adult cells could differentiate without irreversible changes to the genetic material.

  20. Therapeutic and reproductive cloning--implications and recommendations.

    PubMed

    Ng, S C; Ng, Y; Liow, S L; Chen, N

    2003-03-01

    The announcement of Dolly's birth took the world by storm, mainly because what was thought impossible has become possible. Optimism that new approaches in agriculture and medicine abound, as much as fear and imagination leading to Frankenstein-like scenarios. Scientifically, cloning refers to replicating an animal with the same nuclear genetic material; whilst it may refer to embryos as the source material, the current storm refers to differentiated ("somatic") cells. Cloning technology is useful in the following areas: agriculture, to produce animals of superior or specific qualities; endangered animals, to increase genetic diversity through widening the gene pool; understanding fundamental questions in developmental embryology, through the use of laboratory animals; and in human therapy, to produce cells and possibly tissues for repair and regeneration. In the first 2 instances, it is reproductive cloning. In the last instance, it is therapeutic cloning, as no individuals are "produced". Human reproductive cloning is not allowed by all governments that have deliberated on it, and therapeutic cloning is allowed by some under certain circumstances. As therapeutic cloning has great potential in cures of many diseases, it should be allowed but with safeguards to prevent abuse and reproductive cloning in the human.

  1. Cloning of Homo sapiens? No!

    PubMed

    McKinnell, Robert G

    2002-01-01

    Animal cloning by nuclear transplantation was first developed in the northern leopard frog, Rana pipiens. It was soon extended to other amphibian species and within time, to various mammalian species. The production of a cloned sheep (Dolly) from an adult nuclear donor reawakened interest in human cloning. Nuclear transfer for the production of animal clones has served experimental biology well. Nonetheless, the potential burden of developmental hazards, scientists and funds diverted from more needy causes, as well as the potential assault on the concept of family has led the author to oppose human cloning.

  2. Cloning of Homo sapiens? No!

    PubMed

    McKinnell, Robert G

    2002-01-01

    Animal cloning by nuclear transplantation was first developed in the northern leopard frog, Rana pipiens. It was soon extended to other amphibian species and within time, to various mammalian species. The production of a cloned sheep (Dolly) from an adult nuclear donor reawakened interest in human cloning. Nuclear transfer for the production of animal clones has served experimental biology well. Nonetheless, the potential burden of developmental hazards, scientists and funds diverted from more needy causes, as well as the potential assault on the concept of family has led the author to oppose human cloning. PMID:11841468

  3. Clonal diversity and clone formation in the parthenogenetic Caucasian rock Lizard Darevskia dahli [corrected].

    PubMed

    Vergun, Andrey A; Martirosyan, Irena A; Semyenova, Seraphima K; Omelchenko, Andrey V; Petrosyan, Varos G; Lazebny, Oleg E; Tokarskaya, Olga N; Korchagin, Vitaly I; Ryskov, Alexey P

    2014-01-01

    The all-female Caucasian rock lizard species Darevskia dahli and other parthenogenetic species of this genus reproduce normally via true parthenogenesis. Previously, the genetic diversity of this species was analyzed using allozymes, mitochondrial DNA, and DNA fingerprint markers. In the present study, variation at three microsatellite loci was studied in 111 specimens of D. dahli from five populations from Armenia, and new information regarding clonal diversity and clone formation in D. dahli was obtained that suggests a multiple hybridization origin. All individuals but one were heterozygous at the loci studied. Based on specific allele combinations, 11 genotypes were identified among the individuals studied. Individuals with the same genotypes formed distinct clonal lineages: one major clone was represented by 72 individuals, an intermediate clone was represented by 21 individuals, and nine other clones were rare and represented by one or several individuals. A new approach based on the detection and comparison of genotype-specific markers formed by combinations of parental-specific markers was developed and used to identify at least three hybridization founder events that resulted in the initial formation of one major and two rare clones. All other clones, including the intermediate and seven rare clones, probably arose through postformation microsatellite mutations of the major clone. This approach can be used to identify hybridization founder events and to study clone formation in other unisexual taxa.

  4. Clonal diversity and clone formation in the parthenogenetic Caucasian rock lizard Darevskia dahlia.

    PubMed

    Vergun, Andrey A; Martirosyan, Irena A; Semyenova, Seraphima K; Omelchenko, Andrey V; Petrosyan, Varos G; Lazebny, Oleg E; Tokarskaya, Olga N; Korchagin, Vitaly I; Ryskov, Alexey P

    2014-01-01

    The all-female Caucasian rock lizard species Darevskia dahli and other parthenogenetic species of this genus reproduce normally via true parthenogenesis. Previously, the genetic diversity of this species was analyzed using allozymes, mitochondrial DNA, and DNA fingerprint markers. In the present study, variation at three microsatellite loci was studied in 111 specimens of D. dahli from five populations from Armenia, and new information regarding clonal diversity and clone formation in D. dahli was obtained that suggests a multiple hybridization origin. All individuals but one were heterozygous at the loci studied. Based on specific allele combinations, 11 genotypes were identified among the individuals studied. Individuals with the same genotypes formed distinct clonal lineages: one major clone was represented by 72 individuals, an intermediate clone was represented by 21 individuals, and nine other clones were rare and represented by one or several individuals. A new approach based on the detection and comparison of genotype-specific markers formed by combinations of parental-specific markers was developed and used to identify at least three hybridization founder events that resulted in the initial formation of one major and two rare clones. All other clones, including the intermediate and seven rare clones, probably arose through postformation microsatellite mutations of the major clone. This approach can be used to identify hybridization founder events and to study clone formation in other unisexual taxa. PMID:24618670

  5. Emergence of clones in sexual populations

    NASA Astrophysics Data System (ADS)

    Neher, Richard A.; Vucelja, Marija; Mezard, Mark; Shraiman, Boris I.

    2013-01-01

    In sexual population, recombination reshuffles genetic variation and produces novel combinations of existing alleles, while selection amplifies the fittest genotypes in the population. If recombination is more rapid than selection, populations consist of a diverse mixture of many genotypes, as is observed in many populations. In the opposite regime, which is realized for example in the facultatively sexual populations that outcross in only a fraction of reproductive cycles, selection can amplify individual genotypes into large clones. Such clones emerge when the fitness advantage of some of the genotypes is large enough that they grow to a significant fraction of the population despite being broken down by recombination. The occurrence of this ‘clonal condensation’ depends, in addition to the outcrossing rate, on the heritability of fitness. Clonal condensation leads to a strong genetic heterogeneity of the population which is not adequately described by traditional population genetics measures, such as linkage disequilibrium. Here we point out the similarity between clonal condensation and the freezing transition in the random energy model of spin glasses. Guided by this analogy we explicitly calculate the probability, Y, that two individuals are genetically identical as a function of the key parameters of the model. While Y is the analog of the spin-glass order parameter, it is also closely related to rate of coalescence in population genetics: two individuals that are part of the same clone have a recent common ancestor.

  6. [Media, cloning, and bioethics].

    PubMed

    Costa, S I; Diniz, D

    2000-01-01

    This article was based on an analysis of three hundred articles from mainstream Brazilian periodicals over a period of eighteen months, beginning with the announcement of the Dolly case in February 1997. There were two main objectives: to outline the moral constants in the press associated with the possibility of cloning human beings and to identify some of the moral assumptions concerning scientific research with non-human animals that were published carelessly by the media. The authors conclude that there was a haphazard spread of fear concerning the cloning of human beings rather than an ethical debate on the issue, and that there is a serious gap between bioethical reflections and the Brazilian media.

  7. Overlap extension PCR cloning.

    PubMed

    Bryksin, Anton; Matsumura, Ichiro

    2013-01-01

    Rising demand for recombinant proteins has motivated the development of efficient and reliable cloning methods. Here we show how a beginner can clone virtually any DNA insert into a plasmid of choice without the use of restriction endonucleases or T4 DNA ligase. Chimeric primers encoding plasmid sequence at the 5' ends and insert sequence at the 3' ends are designed and synthesized. Phusion(®) DNA polymerase is utilized to amplify the desired insert by PCR. The double-stranded product is subsequently employed as a pair of mega-primers in a PCR-like reaction with circular plasmids. The original plasmids are then destroyed in restriction digests with Dpn I. The product of the overlap extension PCR is used to transform competent Escherichia coli cells. Phusion(®) DNA polymerase is used for both the amplification and fusion reactions, so both steps can be monitored and optimized in the same way. PMID:23996437

  8. Probabilistic cloning of equidistant states

    SciTech Connect

    Jimenez, O.; Roa, Luis; Delgado, A.

    2010-08-15

    We study the probabilistic cloning of equidistant states. These states are such that the inner product between them is a complex constant or its conjugate. Thereby, it is possible to study their cloning in a simple way. In particular, we are interested in the behavior of the cloning probability as a function of the phase of the overlap among the involved states. We show that for certain families of equidistant states Duan and Guo's cloning machine leads to cloning probabilities lower than the optimal unambiguous discrimination probability of equidistant states. We propose an alternative cloning machine whose cloning probability is higher than or equal to the optimal unambiguous discrimination probability for any family of equidistant states. Both machines achieve the same probability for equidistant states whose inner product is a positive real number.

  9. Platelet-mediated transformation of mtDNA-less human cells: Analysis of phenotypic variability among clones from normal individuals-and complementation behavior of the tRNA[sup Lys] mutation causing myoclonic epilepsy and ragged red fibers

    SciTech Connect

    Chomyn, A.; Lai, S.T.; Shakeley, R.; Attardi, G. ); Bresolin, N.; Scarlato, G. )

    1994-06-01

    In the present work, the authors demonstrate the possibility of using human blood platelets as mitochondrial donors for the repopulation of mtDNA-less ([rho][sup o]) cells. The noninvasive nature of platelet isolation, combined with the prolonged viability of platelet mitochondria and the simplicity and efficiency of the mitochondria-transfer procedure, has substantially increased the applicability of the [rho][sup o] cell transformation approach for mitochondrial genetic analysis and for the study of mtDNA-linked diseases. This approach has been applied to platelets from several normal human individuals and one individual affected by the myoclonic-epilepsy-and-ragged-red-fibers (MERRF) encephalomyopathy. A certain variability in respiratory capacity was observed among the platelet-derived [rho][sup o] cell transformants from a given normal subject, and it was shown to be unrelated to their mtDNA content. The results of sequential transfer of mitochondria from selected transformants into a [rho][sup o] cell line different from the first [rho][sup o] acceptor strongly suggest that this variability reflected, at least in part, differences in nuclear gene content and/or activity among the original recipient cells. A much greater variability in respiratory capacity was observed among the transformants derived from the MERRF patient and was found to be related to the presence and amount of the mitochondrial tRNA[sup Lys] mutation associated with the MERRF syndrome. An analysis of the relationship between proportion of mtDNA carrying the MERRF mutation and degree of respiratory activity in various transformations derived from the MERRF patient revealed an unusual complementation behavior of the tRNA[sup Lys] mutation, possibly reflecting the distribution of mutant mtDNA among the platelet mitochondria. 29 refs., 4 figs., 1 tab.

  10. Sequential cloning of chromosomes

    DOEpatents

    Lacks, Sanford A.

    1995-07-18

    A method for sequential cloning of chromosomal DNA of a target organism is disclosed. A first DNA segment homologous to the chromosomal DNA to be sequentially cloned is isolated. The first segment has a first restriction enzyme site on either side. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism's chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction (class IIS) enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes.

  11. Sequential cloning of chromosomes

    DOEpatents

    Lacks, S.A.

    1995-07-18

    A method for sequential cloning of chromosomal DNA of a target organism is disclosed. A first DNA segment homologous to the chromosomal DNA to be sequentially cloned is isolated. The first segment has a first restriction enzyme site on either side. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction (class IIS) enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes. 9 figs.

  12. Experimental eavesdropping based on optimal quantum cloning.

    PubMed

    Bartkiewicz, Karol; Lemr, Karel; Cernoch, Antonín; Soubusta, Jan; Miranowicz, Adam

    2013-04-26

    The security of quantum cryptography is guaranteed by the no-cloning theorem, which implies that an eavesdropper copying transmitted qubits in unknown states causes their disturbance. Nevertheless, in real cryptographic systems some level of disturbance has to be allowed to cover, e.g., transmission losses. An eavesdropper can attack such systems by replacing a noisy channel by a better one and by performing approximate cloning of transmitted qubits which disturb them but below the noise level assumed by legitimate users. We experimentally demonstrate such symmetric individual eavesdropping on the quantum key distribution protocols of Bennett and Brassard (BB84) and the trine-state spherical code of Renes (R04) with two-level probes prepared using a recently developed photonic multifunctional quantum cloner [Lemr et al., Phys. Rev. A 85, 050307(R) (2012)]. We demonstrated that our optimal cloning device with high-success rate makes the eavesdropping possible by hiding it in usual transmission losses. We believe that this experiment can stimulate the quest for other operational applications of quantum cloning.

  13. Experimental eavesdropping based on optimal quantum cloning.

    PubMed

    Bartkiewicz, Karol; Lemr, Karel; Cernoch, Antonín; Soubusta, Jan; Miranowicz, Adam

    2013-04-26

    The security of quantum cryptography is guaranteed by the no-cloning theorem, which implies that an eavesdropper copying transmitted qubits in unknown states causes their disturbance. Nevertheless, in real cryptographic systems some level of disturbance has to be allowed to cover, e.g., transmission losses. An eavesdropper can attack such systems by replacing a noisy channel by a better one and by performing approximate cloning of transmitted qubits which disturb them but below the noise level assumed by legitimate users. We experimentally demonstrate such symmetric individual eavesdropping on the quantum key distribution protocols of Bennett and Brassard (BB84) and the trine-state spherical code of Renes (R04) with two-level probes prepared using a recently developed photonic multifunctional quantum cloner [Lemr et al., Phys. Rev. A 85, 050307(R) (2012)]. We demonstrated that our optimal cloning device with high-success rate makes the eavesdropping possible by hiding it in usual transmission losses. We believe that this experiment can stimulate the quest for other operational applications of quantum cloning. PMID:23679725

  14. End Sequencing and Finger Printing of Human & Mouse BAC Libraries

    SciTech Connect

    Fraser, C

    2005-09-27

    This project provided for continued end sequencing of existing and new BAC libraries constructed to support human sequencing as well as to initiate BAC end sequencing from the mouse BAC libraries constructed to support mouse sequencing. The clones, the sequences, and the fingerprints are now an available resource for the community at large. Research and development of new metaodologies for BAC end sequencing have reduced costs and increase throughput.

  15. Induction of autophagy improves embryo viability in cloned mouse embryos

    PubMed Central

    Shen, XingHui; Zhang, Na; Wang, ZhenDong; Bai, GuangYu; Zheng, Zhong; Gu, YanLi; Wu, YanShuang; Liu, Hui; Zhou, DongJie; Lei, Lei

    2015-01-01

    Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components. Moreover, autophagy is essential for preimplantation development in mammals. Here we show that autophagy is also important for reprogramming in somatic cell nuclear transfer (SCNT). Our data indicate that unlike fertilized oocytes, autophagy is not triggered in SCNT embryos during 6 hours of activation. Mechanistically, the inhibited autophagic induction during SCNT activation is due to the cytochalasin B (CB) caused depolymerization of actin filaments. In this study, we induced autophagy during SCNT activation by rapamycin and pp242, which could restore the expected level of autophagy and significantly enhance the development of SCNT embryos to the blastocyst stage when compared with the control (68.5% and 68.7% vs. 41.5%, P < 0.05). Furthermore, the treatment of rapamycin and pp242 accelerates active DNA demethylation indicated by the conversion of 5 mC to 5 hmC, and treatment of rapamycin improves degradation of maternal mRNA as well. Thus, our findings reveal that autophagy is important for development of SCNT embryos and inhibited autophagic induction during SCNT activation might be one of the serious causes of low efficiency of SCNT. PMID:26643778

  16. To clone or not to clone--a Jewish perspective.

    PubMed Central

    Lipschutz, J H

    1999-01-01

    Many new reproductive methods such as artificial insemination, in vitro fertilisation, freezing of human embryos, and surrogate motherhood were at first widely condemned but are now seen in Western society as not just ethically and morally acceptable, but beneficial in that they allow otherwise infertile couples to have children. The idea of human cloning was also quickly condemned but debate is now emerging. This article examines cloning from a Jewish perspective and finds evidence to support the view that there is nothing inherently wrong with the idea of human cloning. A hypothesis is also advanced suggesting that even if a body was cloned, the brain, which is the essence of humanity, would remain unique. This author suggests that the debate should be changed from "Is cloning wrong?" to "When is cloning wrong?". PMID:10226913

  17. Ethical issues in animal cloning.

    PubMed

    Fiester, Autumn

    2005-01-01

    The issue of human reproductive cloning has recently received a great deal attention in public discourse. Bioethicists, policy makers, and the media have been quick to identify the key ethical issues involved in human reproductive cloning and to argue, almost unanimously, for an international ban on such attempts. Meanwhile, scientists have proceeded with extensive research agendas in the cloning of animals. Despite this research, there has been little public discussion of the ethical issues raised by animal cloning projects. Polling data show that the public is decidedly against the cloning of animals. To understand the public's reaction and fill the void of reasoned debate about the issue, we need to review the possible objections to animal cloning and assess the merits of the anti-animal cloning stance. Some objections to animal cloning (e.g., the impact of cloning on the population of unwanted animals) can be easily addressed, while others (e.g., the health of cloned animals) require more serious attention by the public and policy makers.

  18. Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

    PubMed

    Lam, Kathy N; Hall, Michael W; Engel, Katja; Vey, Gregory; Cheng, Jiujun; Neufeld, Josh D; Charles, Trevor C

    2014-01-01

    High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones. PMID:24911009

  19. To clone or not to clone--whither the law?

    PubMed

    Lupton, M L

    1999-01-01

    The cloning of Dolly the lamb from adult cells by scientists at the Roslin Laboratories near Edinburgh in February 1997 has startled the world because it now opens the way to clone adult human beings. The reaction to Ian Wilmut's breakthrough has been instant and largely negative. Bills were rushed into both the US Senate and House of Representatives aimed at banning the cloning of human beings. Human cloning is premature at this stage, but there are many positive spin-offs of cloning in the field of genetic engineering, such as the production of human proteins such as blood clotting factors which aid in healing wounds. Progress by means of cloning can also be made into devising a cure for Parkinson's Disease amongst others. No lesser ethicist than John C. Fletcher of the University of Virginia foresees circumstances in which human cloning is acceptable e.g. to enable a couple to replace a dying child, to enable a couple, one of whom is infertile, to clone a child from either partner. Extensive regulation of cloning by the law is inevitable but, in doing so, the legislation should be careful not to outlaw research in this area which could be beneficial to mankind. PMID:10436743

  20. To clone or not to clone--whither the law?

    PubMed

    Lupton, M L

    1999-01-01

    The cloning of Dolly the lamb from adult cells by scientists at the Roslin Laboratories near Edinburgh in February 1997 has startled the world because it now opens the way to clone adult human beings. The reaction to Ian Wilmut's breakthrough has been instant and largely negative. Bills were rushed into both the US Senate and House of Representatives aimed at banning the cloning of human beings. Human cloning is premature at this stage, but there are many positive spin-offs of cloning in the field of genetic engineering, such as the production of human proteins such as blood clotting factors which aid in healing wounds. Progress by means of cloning can also be made into devising a cure for Parkinson's Disease amongst others. No lesser ethicist than John C. Fletcher of the University of Virginia foresees circumstances in which human cloning is acceptable e.g. to enable a couple to replace a dying child, to enable a couple, one of whom is infertile, to clone a child from either partner. Extensive regulation of cloning by the law is inevitable but, in doing so, the legislation should be careful not to outlaw research in this area which could be beneficial to mankind.

  1. Cloning from stem cells: different lineages, different species, same story.

    PubMed

    Oback, Björn

    2009-01-01

    Following nuclear transfer (NT), the most stringent measure of extensive donor cell reprogramming is development into viable offspring. This is referred to as cloning efficiency and quantified as the proportion of cloned embryos transferred into surrogate mothers that survive into adulthood. Cloning efficiency depends on the ability of the enucleated recipient cell to carry out the reprogramming reactions ('reprogramming ability') and the ability of the nuclear donor cell to be reprogrammed ('reprogrammability'). It has been postulated that reprogrammability of the somatic donor cell epigenome is inversely proportional to its differentiation status. In order to test this hypothesis, reprogrammability was compared between undifferentiated stem cells and their differentiated isogenic progeny. In the mouse, cells of divergent differentiation status from the neuronal, haematopoietic and skin epithelial lineage were tested. In cattle and deer, skeletal muscle and antler cells, respectively, were used as donors. No conclusive correlation between differentiation status and cloning efficiency was found, indicating that somatic donor cell type may not be the limiting factor for cloning success. This may reflect technical limitations of the NT-induced reprogramming assay. Alternatively, differentiation status and reprogrammability may be unrelated, making all cells equally difficult to reprogramme once they have left the ground state of pluripotency.

  2. Dairy cattle exploratory and social behaviors: is there an effect of cloning?

    PubMed

    Coulon, M; Baudoin, C; Depaulis-Carre, M; Heyman, Y; Renard, J P; Richard, C; Deputte, B L

    2007-11-01

    While an increasing number of animals are produced by means of somatic cloning, behavioral studies on cloned animals are still rare. The aim of this study was to investigate whether the somatic cloning procedure has an influence on locomotion, exploratory, vocal and social behaviors of heifers. Ten heifers were used in the present study. Five of them were cloned heifers derived from somatic cells of three different Prim'Holstein cows and five others were same-age control heifers produced by artificial insemination. In addition to observations of social behaviors in the stable group, each animal was placed individually for a short time in an unfamiliar environment. Our results failed to show any statistical differences between clones and their controls both in frequencies of agonistic and non-agonistic behaviors. However, cloned heifers showed significantly more non-agonistic and less agonistic behaviors towards other cloned partners than towards control ones. This result also stood for control heifers. As far as their Hierarchical Index was concerned, three cloned heifers were highest ranking and two others lowest ranking. In this herd, social dominance appeared to be linked to body weight and age rather than to a cloning effect. In an unfamiliar environment, cloned and control subjects exhibited the same level of locomotion and vocalization. However, cloned heifers showed more exploratory behaviors than did control ones. This difference could be due to environmental factors during the postnatal period rather than to cloning.

  3. Cloning humans? Biological, ethical, and social considerations

    PubMed Central

    Ayala, Francisco J.

    2015-01-01

    There are, in mankind, two kinds of heredity: biological and cultural. Cultural inheritance makes possible for humans what no other organism can accomplish: the cumulative transmission of experience from generation to generation. In turn, cultural inheritance leads to cultural evolution, the prevailing mode of human adaptation. For the last few millennia, humans have been adapting the environments to their genes more often than their genes to the environments. Nevertheless, natural selection persists in modern humans, both as differential mortality and as differential fertility, although its intensity may decrease in the future. More than 2,000 human diseases and abnormalities have a genetic causation. Health care and the increasing feasibility of genetic therapy will, although slowly, augment the future incidence of hereditary ailments. Germ-line gene therapy could halt this increase, but at present, it is not technically feasible. The proposal to enhance the human genetic endowment by genetic cloning of eminent individuals is not warranted. Genomes can be cloned; individuals cannot. In the future, therapeutic cloning will bring enhanced possibilities for organ transplantation, nerve cells and tissue healing, and other health benefits. PMID:26195738

  4. Cloning humans? Biological, ethical, and social considerations.

    PubMed

    Ayala, Francisco J

    2015-07-21

    There are, in mankind, two kinds of heredity: biological and cultural. Cultural inheritance makes possible for humans what no other organism can accomplish: the cumulative transmission of experience from generation to generation. In turn, cultural inheritance leads to cultural evolution, the prevailing mode of human adaptation. For the last few millennia, humans have been adapting the environments to their genes more often than their genes to the environments. Nevertheless, natural selection persists in modern humans, both as differential mortality and as differential fertility, although its intensity may decrease in the future. More than 2,000 human diseases and abnormalities have a genetic causation. Health care and the increasing feasibility of genetic therapy will, although slowly, augment the future incidence of hereditary ailments. Germ-line gene therapy could halt this increase, but at present, it is not technically feasible. The proposal to enhance the human genetic endowment by genetic cloning of eminent individuals is not warranted. Genomes can be cloned; individuals cannot. In the future, therapeutic cloning will bring enhanced possibilities for organ transplantation, nerve cells and tissue healing, and other health benefits.

  5. Cloning humans? Biological, ethical, and social considerations.

    PubMed

    Ayala, Francisco J

    2015-07-21

    There are, in mankind, two kinds of heredity: biological and cultural. Cultural inheritance makes possible for humans what no other organism can accomplish: the cumulative transmission of experience from generation to generation. In turn, cultural inheritance leads to cultural evolution, the prevailing mode of human adaptation. For the last few millennia, humans have been adapting the environments to their genes more often than their genes to the environments. Nevertheless, natural selection persists in modern humans, both as differential mortality and as differential fertility, although its intensity may decrease in the future. More than 2,000 human diseases and abnormalities have a genetic causation. Health care and the increasing feasibility of genetic therapy will, although slowly, augment the future incidence of hereditary ailments. Germ-line gene therapy could halt this increase, but at present, it is not technically feasible. The proposal to enhance the human genetic endowment by genetic cloning of eminent individuals is not warranted. Genomes can be cloned; individuals cannot. In the future, therapeutic cloning will bring enhanced possibilities for organ transplantation, nerve cells and tissue healing, and other health benefits. PMID:26195738

  6. Therapeutic cloning and reproductive liberty.

    PubMed

    Sparrow, Robert

    2009-04-01

    Concern for "reproductive liberty" suggests that decisions about embryos should normally be made by the persons who would be the genetic parents of the child that would be brought into existence if the embryo were brought to term. Therapeutic cloning would involve creating and destroying an embryo, which, if brought to term, would be the offspring of the genetic parents of the person undergoing therapy. I argue that central arguments in debates about parenthood and genetics therefore suggest that therapeutic cloning would be prima facie unethical unless it occurred with the consent of the parents of the person being cloned. Alternatively, if therapeutic cloning is thought to be legitimate, this undermines the case for some uses of reproductive cloning by implying that the genetic relation it establishes between clones and DNA donors does not carry the same moral weight as it does in cases of normal reproduction.

  7. Frequent occurrence of highly expanded but unrelated B-cell clones in patients with multiple myeloma.

    PubMed

    Kriangkum, Jitra; Motz, Sarah N; Debes Marun, Carina S; Lafarge, Sandrine T; Gibson, Spencer B; Venner, Christopher P; Johnston, James B; Belch, Andrew R; Pilarski, Linda M

    2013-01-01

    Clonal diversity in multiple myeloma (MM) includes both MM-related and MM-unrelated clonal expansions which are subject to dominance exerted by the MM clone. Here we show evidence for the existence of minor but highly expanded unrelated B-cell clones in patients with MM defined by their complementary determining region 3 (CDR3) peak. We further characterize these clones over the disease and subsequent treatment. Second clones were identified by their specific IgH-VDJ sequences that are distinct from those of dominant MM clones. Clonal frequencies were determined through semi-quantitative PCR, quantitative PCR and single-cell polymerase chain reaction of the clone-specific sequence. In 13/74 MM patients, more than one dominant CDR3 peak was identified with 12 patients (16%) being truly biclonal. Second clones had different frequencies, were found in different locations and were found in different cell types from the dominant MM clone. Where analysis was possible, they were shown to have chromosomal characteristic distinct from those of the MM clone. The frequency of the second clone also changed over the course of the disease and often persisted despite treatment. Molecularly-defined second clones are infrequent in monoclonal gammopathy of undetermined significance (MGUS, 1/43 individuals or 2%), suggesting that they may arise at relatively late stages of myelomagenesis. In further support of our findings, biclonal gammopathy and concomitant MM and CLL (chronic lymphocytic leukemia) were confirmed to originate from two unrelated clones. Our data supports the idea that the clone giving rise to symptomatic myeloma exerts clonal dominance to prevent expansion of other clones. MM and second clones may arise from an underlying niche permissive of clonal expansion. The clinical significance of these highly expanded but unrelated clones remains to be confirmed. Overall, our findings add new dimensions to evaluating related and unrelated clonal expansions in MM and the

  8. Ultraviolet survival and sensitizing effect of caffeine in mouse hybrid cells

    SciTech Connect

    Zampetti-Bosseler, F.; Delhaise, P.; Limbosch, S.

    1980-10-01

    In a previous paper it was reported that three hybrid cell lines between mouse lymphoma cells (L5178YS) and mouse fibroblasts (A9) were more resistant to x rays than either of the parental cells. In this work, these hybrids displayed a degree of resistance to uv light either higher than (hybrid clone 3) or similar to (hybrid clones 1 and 2) that of the more resistant parent (A9). The enhanced resistance of hybrid clone 3 to uv was related neither to changes in cell shape, ploidy, and growth rate nor to an increase in a caffeine-sensitive recovery process after uv irradiation.

  9. What is Cloning?

    MedlinePlus

    ... two. Twinning happens in the first days after egg and sperm join, while the embryo is made ... individuals. Since they developed from the same fertilized egg, the resulting individuals are genetically identical. Artificial embryo ...

  10. Recent progress and problems in animal cloning.

    PubMed

    Tsunoda, Y; Kato, Y

    2002-01-01

    It is remarkable that mammalian somatic cell nuclei can form whole individuals if they are transferred to enucleated oocytes. Advancements in nuclear transfer technology can now be applied for genetic improvement and increase of farm animals, rescue of endangered species, and assisted reproduction and tissue engineering in humans. Since July 1998, more than 200 calves have been produced by nuclear transfer of somatic cell nuclei in Japan, but half of them were stillborn or died within several months of parturition. Morphologic abnormalities have also been observed in cloned calves and embryonic stem cell-derived mice. In this review, we discuss the present situation and problems with animal cloning and the possibility for its application to human medicine.

  11. Neisseria meningitidis; clones, carriage, and disease.

    PubMed

    Read, R C

    2014-05-01

    Neisseria meningitidis, the cause of meningococcal disease, has been the subject of sophisticated molecular epidemiological investigation as a consequence of the significant public health threat posed by this organism. The use of multilocus sequence typing and whole genome sequencing classifies the organism into clonal complexes. Extensive phenotypic, genotypic and epidemiological information is available on the PubMLST website. The human nasopharynx is the sole ecological niche of this species, and carrier isolates show extensive genetic diversity as compared with hyperinvasive lineages. Horizontal gene exchange and recombinant events within the meningococcal genome during residence in the human nasopharynx result in antigenic diversity even within clonal complexes, so that individual clones may express, for example, more than one capsular polysaccharide (serogroup). Successful clones are capable of wide global dissemination, and may be associated with explosive epidemics of invasive disease. PMID:24766477

  12. ISOLATION OF MOUSE NEUTROPHILS

    PubMed Central

    Swamydas, Muthulekha; Luo, Yi; Dorf, Martin E.; Lionakis, Michail S.

    2015-01-01

    Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcome of infected individuals. This unit describes a reproducible density gradient centrifugation-based protocol that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice both at the steady state and following infection with Candida albicans as described in UNIT 19.6. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or Fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver or the spleen. Finally, methods for isolating neutrophils from mouse peritoneal fluid and peripheral blood are included. Mouse neutrophils isolated by these protocols can be used for examining several aspects of cellular function ex vivo including pathogen binding, phagocytosis and killing, neutrophil chemotaxis, oxidative burst, degranulation and cytokine production, and for performing neutrophil adoptive transfer experiments. PMID:26237011

  13. Cloning to reproduce desired genotypes.

    PubMed

    Westhusin, M E; Long, C R; Shin, T; Hill, J R; Looney, C R; Pryor, J H; Piedrahita, J A

    2001-01-01

    Cloned sheep, cattle, goats, pigs and mice have now been produced using somatic cells for nuclear transplantation. Animal cloning is still very inefficient with on average less than 10% of the cloned embryos transferred resulting in a live offspring. However successful cloning of a variety of different species and by a number of different laboratory groups has generated tremendous interest in reproducing desired genotypes. Some of these specific genotypes represent animal cell lines that have been genetically modified. In other cases there is a significant demand for cloning animals characterized by their inherent genetic value, for example prize livestock, household pets and rare or endangered species. A number of different variables may influence the ability to reproduce a specific genotype by cloning. These include species, source of recipient ova, cell type of nuclei donor, treatment of donor cells prior to nuclear transfer, and the techniques employed for nuclear transfer. At present, there is no solid evidence that suggests cloning will be limited to only a few specific animals, and in fact, most data collected to date suggests cloning will be applicable to a wide variety of different animals. The ability to reproduce any desired genotype by cloning will ultimately depend on the amount of time and resources invested in research.

  14. Human cloning and child welfare.

    PubMed Central

    Burley, J; Harris, J

    1999-01-01

    In this paper we discuss an objection to human cloning which appeals to the welfare of the child. This objection varies according to the sort of harm it is expected the clone will suffer. The three formulations of it that we will consider are: 1. Clones will be harmed by the fearful or prejudicial attitudes people may have about or towards them (H1); 2. Clones will be harmed by the demands and expectations of parents or genotype donors (H2); 3. Clones will be harmed by their own awareness of their origins, for example the knowledge that the genetic donor is a stranger (H3). We will show why these three versions of the child welfare objection do not necessarily supply compelling reasons to ban human reproductive cloning. The claim that we will develop and defend in the course of our discussion is that even if it is the case that a cloned child will suffer harms of the type H1-H3, it is none the less permissible to conceive by cloning so long as these cloning-induced welfare deficits are not such as to blight the existence of the resultant child, whoever this may be. PMID:10226914

  15. Therapeutic cloning: The ethical limits

    SciTech Connect

    Whittaker, Peter A. . E-mail: p.whittaker@lancaster.ac.uk

    2005-09-01

    A brief outline of stem cells, stem cell therapy and therapeutic cloning is given. The position of therapeutic cloning with regard to other embryonic manipulations - IVF-based reproduction, embryonic stem formation from IVF embryos and reproductive cloning - is indicated. The main ethically challenging stages in therapeutic cloning are considered to be the nuclear transfer process including the source of eggs for this and the destruction of an embryo to provide stem cells for therapeutic use. The extremely polarised nature of the debate regarding the status of an early human embryo is noted, and some potential alternative strategies for preparing immunocompatible pluripotent stem cells are indicated.

  16. Genotoxic effects of 1 GeV/amu Fe ions in mouse kidney epithelial cells

    NASA Astrophysics Data System (ADS)

    Kronenberg, A.; Gauny, S. S.; Connolly, L.; Turker, M.

    Human exploration of space places individuals in environments where they are exposed to charged particle radiation. The goal of our studies is to assess the genotoxic and mutagenic effects of high energy Fe ions (1 GeV/amu) in kidney epithelial cells of the mouse irradiated either in vitro or in vivo. The initial study focused on establishing the toxicity of these heavy ions (LET=159 keV/micron) in two Aprt heterozygous kidney epithelial cell lines: K06 cells derived from a C57BL6/129Sv animal, and clone 4a cells derived from a C57BL6/DBA2 animal. Cells were exposed in vitro to graded doses of Fe ions (0-300 cGy) and the toxicity of the treatment was established using colony forming assays. Experiments were performed in triplicate at the NASA Space Radiation Laboratory at Brookhaven National Laboratory. The results indicate that Fe ions are toxic to mouse kidney epithelial cells and that no shoulder is observed on the survival curve for cells from either genetic background. The clone 4a cells were more sensitive to Fe ion exposures than the K06 cells. The D(37) for clone 4a cells was 92 cGy and the D(10) was 212 cGy. The more resistant K06 cells had a D(37) of 192 cGy and an estimated D(10) of 388 cGy. Parallel experiments are underway to establish the RBE's for cell killing for these two cell lines. Supported by NASA grant T-403X to A. Kronenberg

  17. Clone history shapes Populus drought responses.

    PubMed

    Raj, Sherosha; Bräutigam, Katharina; Hamanishi, Erin T; Wilkins, Olivia; Thomas, Barb R; Schroeder, William; Mansfield, Shawn D; Plant, Aine L; Campbell, Malcolm M

    2011-07-26

    Just as animal monozygotic twins can experience different environmental conditions by being reared apart, individual genetically identical trees of the genus Populus can also be exposed to contrasting environmental conditions by being grown in different locations. As such, clonally propagated Populus trees provide an opportunity to interrogate the impact of individual environmental history on current response to environmental stimuli. To test the hypothesis that current responses to an environmental stimulus, drought, are contingent on environmental history, the transcriptome- level drought responses of three economically important hybrid genotypes-DN34 (Populus deltoides × Populus nigra), Walker [P. deltoides var. occidentalis × (Populus laurifolia × P. nigra)], and Okanese [Walker × (P. laurifolia × P. nigra)]-derived from two different locations were compared. Strikingly, differences in transcript abundance patterns in response to drought were based on differences in geographic origin of clones for two of the three genotypes. This observation was most pronounced for the genotypes with the longest time since establishment and last common propagation. Differences in genome-wide DNA methylation paralleled the transcriptome level trends, whereby the clones with the most divergent transcriptomes and clone history had the most marked differences in the extent of total DNA methylation, suggesting an epigenomic basis for the clone history-dependent transcriptome divergence. The data provide insights into the interplay between genotype and environment in the ecologically and economically important Populus genus, with implications for the industrial application of Populus trees and the evolution and persistence of these important tree species and their associated hybrids. PMID:21746919

  18. Isolation of the mouse homologue of BRCA1 and genetic mapping to mouse chromosome 11

    SciTech Connect

    Bennett, L.M.; Haugen-Strano, A.; Cochran, C.

    1995-10-10

    The BRCA1 gene is in large part responsible for hereditary human breast and ovarian cancer. Here we report the isolation of the murine Brca1 homologue cDNA clones. In addition, we identified genomic P1 clones that contain most, if not all, of the mouse Brca1 locus. DNA sequence analysis revealed that the mouse and human coding regions are 75% identical at the nucleotide level while the predicted amino acid identity is only 58%. A DNA sequence variant in the Brcal locus was identified and used to map this gene on a (Mus m. musculus Czech II x C57BL/KsJ)F1 x C57BL/KsJ intersubspecific backcross to distal mouse chromosome 11. The mapping of this gene to a region highly syntenic with human chromosome 17, coupled with Southern and Northern analyses, confirms that we isolated the murine Brcal homologue rather than a related RING finger gene. The isolation of the mouse Brca1 homologue will facilitate the creation of mouse models for germline BRCA1 defects. 12 refs., 3 figs.

  19. [The discrete horror of cloning].

    PubMed

    Guibourg, Ricardo A

    2009-01-01

    The author raises the topic of cloning after the decision of the Argentine government, which concerned for the "dignity of the human person", passed a decree of need and urgency, No. 200/97 (Annex), prohibiting cloning experiments with human beings. Therefore, considering that the topic is so terribly urgent and necessary, the author feels it is timely to consider it.

  20. CATO: The Clone Alignment Tool.

    PubMed

    Henstock, Peter V; LaPan, Peter

    2016-01-01

    High-throughput cloning efforts produce large numbers of sequences that need to be aligned, edited, compared with reference sequences, and organized as files and selected clones. Different pieces of software are typically required to perform each of these tasks. We have designed a single piece of software, CATO, the Clone Alignment Tool, that allows a user to align, evaluate, edit, and select clone sequences based on comparisons to reference sequences. The input and output are designed to be compatible with standard data formats, and thus suitable for integration into a clone processing pipeline. CATO provides both sequence alignment and visualizations to facilitate the analysis of cloning experiments. The alignment algorithm matches each of the relevant candidate sequences against each reference sequence. The visualization portion displays three levels of matching: 1) a top-level summary of the top candidate sequences aligned to each reference sequence, 2) a focused alignment view with the nucleotides of matched sequences displayed against one reference sequence, and 3) a pair-wise alignment of a single reference and candidate sequence pair. Users can select the minimum matching criteria for valid clones, edit or swap reference sequences, and export the results to a summary file as part of the high-throughput cloning workflow.

  1. Animal Cloning and Food Safety

    MedlinePlus

    ... from clones and their offspring out of the food chain until CVM could further evaluate the issue. back to top FDA Studies Cloning For more than five years, CVM ... evaluate the safety of food from these animals. The resulting report, called a ...

  2. CATO: The Clone Alignment Tool.

    PubMed

    Henstock, Peter V; LaPan, Peter

    2016-01-01

    High-throughput cloning efforts produce large numbers of sequences that need to be aligned, edited, compared with reference sequences, and organized as files and selected clones. Different pieces of software are typically required to perform each of these tasks. We have designed a single piece of software, CATO, the Clone Alignment Tool, that allows a user to align, evaluate, edit, and select clone sequences based on comparisons to reference sequences. The input and output are designed to be compatible with standard data formats, and thus suitable for integration into a clone processing pipeline. CATO provides both sequence alignment and visualizations to facilitate the analysis of cloning experiments. The alignment algorithm matches each of the relevant candidate sequences against each reference sequence. The visualization portion displays three levels of matching: 1) a top-level summary of the top candidate sequences aligned to each reference sequence, 2) a focused alignment view with the nucleotides of matched sequences displayed against one reference sequence, and 3) a pair-wise alignment of a single reference and candidate sequence pair. Users can select the minimum matching criteria for valid clones, edit or swap reference sequences, and export the results to a summary file as part of the high-throughput cloning workflow. PMID:27459605

  3. CATO: The Clone Alignment Tool

    PubMed Central

    Henstock, Peter V.; LaPan, Peter

    2016-01-01

    High-throughput cloning efforts produce large numbers of sequences that need to be aligned, edited, compared with reference sequences, and organized as files and selected clones. Different pieces of software are typically required to perform each of these tasks. We have designed a single piece of software, CATO, the Clone Alignment Tool, that allows a user to align, evaluate, edit, and select clone sequences based on comparisons to reference sequences. The input and output are designed to be compatible with standard data formats, and thus suitable for integration into a clone processing pipeline. CATO provides both sequence alignment and visualizations to facilitate the analysis of cloning experiments. The alignment algorithm matches each of the relevant candidate sequences against each reference sequence. The visualization portion displays three levels of matching: 1) a top-level summary of the top candidate sequences aligned to each reference sequence, 2) a focused alignment view with the nucleotides of matched sequences displayed against one reference sequence, and 3) a pair-wise alignment of a single reference and candidate sequence pair. Users can select the minimum matching criteria for valid clones, edit or swap reference sequences, and export the results to a summary file as part of the high-throughput cloning workflow. PMID:27459605

  4. [The discrete horror of cloning].

    PubMed

    Guibourg, Ricardo A

    2009-01-01

    The author raises the topic of cloning after the decision of the Argentine government, which concerned for the "dignity of the human person", passed a decree of need and urgency, No. 200/97 (Annex), prohibiting cloning experiments with human beings. Therefore, considering that the topic is so terribly urgent and necessary, the author feels it is timely to consider it. PMID:19860340

  5. [Scientific ethics of human cloning].

    PubMed

    Valenzuela, Carlos Y

    2005-01-01

    True cloning is fission, budding or other types of asexual reproduction. In humans it occurs in monozygote twinning. This type of cloning is ethically and religiously good. Human cloning can be performed by twinning (TWClo) or nuclear transfer (NTClo). Both methods need a zygote or a nuclear transferred cell, obtained in vitro (IVTec). They are under the IVTec ethics. IVTecs use humans (zygotes, embryos) as drugs or things; increase the risk of malformations; increase development and size of abnormalities and may cause long-term changes. Cloning for preserving extinct (or almost extinct) animals or humans when sexual reproduction is not possible is ethically valid. The previous selection of a phenotype in human cloning violates some ethical principles. NTClo for reproductive or therapeutic purposes is dangerous since it increases the risk for nucleotide or chromosome mutations, de-programming or re-programming errors, aging or malignancy of the embryo cells thus obtained.

  6. Therapeutic cloning: promises and issues

    PubMed Central

    Kfoury, Charlotte

    2007-01-01

    Advances in biotechnology necessitate both an understanding of scientific principles and ethical implications to be clinically applicable in medicine. In this regard, therapeutic cloning offers significant potential in regenerative medicine by circumventing immunorejection, and in the cure of genetic disorders when used in conjunction with gene therapy. Therapeutic cloning in the context of cell replacement therapy holds a huge potential for de novo organogenesis and the permanent treatment of Parkinson’s disease, Duchenne muscular dystrophy, and diabetes mellitus as shown by in vivo studies. Scientific roadblocks impeding advancement in therapeutic cloning are tumorigenicity, epigenetic reprogramming, mitochondrial heteroplasmy, interspecies pathogen transfer, low oocyte availability. Therapeutic cloning is also often tied to ethical considerations concerning the source, destruction and moral status of IVF embryos based on the argument of potential. Legislative and funding issues are also addressed. Future considerations would include a distinction between therapeutic and reproductive cloning in legislative formulations. PMID:18523539

  7. Denaturing gradient gel electrophoresis can rapidly display the bacterial diversity contained in 16S rDNA clone libraries.

    PubMed

    Burr, M D; Clark, S J; Spear, C R; Camper, A K

    2006-05-01

    Two different strategies for molecular analysis of bacterial diversity, 16S rDNA cloning and denaturing gradient gel electrophoresis (DGGE), were combined into a single protocol that took advantage of the best attributes of each: the ability of cloning to package DNA sequence information and the ability of DGGE to display a community profile. In this combined protocol, polymerase chain reaction products from environmental DNA were cloned, and then DGGE was used to screen the clone libraries. Both individual clones and pools of randomly selected clones were analyzed by DGGE, and these migration patterns were compared to the conventional DGGE profile produced directly from environmental DNA. For two simple bacterial communities (biofilm from a humics-fed laboratory reactor and planktonic bacteria filtered from an urban freshwater pond), pools of 35-50 clones produced DGGE profiles that contained most of the bands visible in the conventional DGGE profiles, indicating that the clone pools were adequate for identifying the dominant genotypes. However, DGGE profiles of two different pools of 50 clones from a lawn soil clone library were distinctly different from each other and from the conventional DGGE profile, indicating that this small number of clones poorly represented the bacterial diversity in soil. Individual clones with the same apparent DGGE mobility as prominent bands in the humics reactor community profiles were sequenced from the clone plasmid DNA rather than from bands excised from the gel. Because a longer fragment was cloned (approximately 1500 bp) than was actually analyzed in DGGE (approximately 350 bp), far more sequence information was available using this approach that could have been recovered from an excised gel band. This clone/DGGE protocol permitted rapid analysis of the microbial diversity in the two moderately complex systems, but was limited in its ability to represent the diversity in the soil microbial community. Nonetheless, clone/DGGE is

  8. Following the very initial growth of biological RNA viral clones.

    PubMed

    Cuevas, José M; Moya, Andrés; Sanjuán, Rafael

    2005-02-01

    Due to their extremely high genetic diversity, which is a direct consequence of high mutation rates, RNA viruses are often described as molecular quasispecies. According to this theory, RNA virus populations cannot be understood in terms of individual viral clones, as they are clouds of interconnected mutants, but this prediction has not yet been demonstrated experimentally. The goal of this study was to determine the fitness of individual clones sampled from a given RNA virus population, a necessary previous step to test the above prediction. To do so, limiting dilutions of a vesicular stomatitis virus population were employed to isolate single viral clones and their initial growth dynamics were followed, corresponding to the release of the first few hundred viral particles. This technique is useful for estimating basic fitness parameters, such as intracellular growth rate, viral yield per cell, rate at which cells are infected and time spent in cell-to-cell transmission. A combination of these parameters allows estimation of the fitness of individual clones, which seems to be determined mainly by their ability to complete infection cycles more quickly. Interestingly, fitness was systematically higher for initial clones than for their derived populations. In addition to environmental changes, such as cellular defence mechanisms, these differences are attributable to high RNA virus mutation rates.

  9. Animal cloning: problems and prospects.

    PubMed

    Wells, D N

    2005-04-01

    An efficient animal cloning technology would provide many new opportunities for livestock agriculture, human medicine, and animal conservation. Nuclear cloning involves the production of animals that are genetically identical to the donor cells used in a technique known as nuclear transfer (NT). However, at present it is an inefficient process: in cattle, only around 6% of the embryos transferred to the reproductive tracts of recipient cows result in healthy, longterm surviving clones. Of concern are the high losses throughout gestation, during birth and in the post-natal period through to adulthood. Many of the pregnancy losses relate to failure of the placenta to develop and function correctly. Placental dysfunction may also have an adverse influence on postnatal health. These anomalies are probably due to incorrect epigenetic reprogramming of the donor genome following NT, leading to inappropriate patterns of gene expression during the development of clones. Whilst some physiological tests on surviving clones suggest normality, other reports indicate a variety of post-natal clone-associated abnormalities. This variability in outcome may reflect species-specific and/or cloning methodological differences. Importantly, to date it appears that these clone-associated phenotypes are not transmitted to offspring following sexual reproduction. This indicates that they represent epigenetic errors, rather than genetic errors, which are corrected during gametogenesis. Whilst this needs confirmation at the molecular level, it provides initial confidence in the first application of NT in agriculture, namely, the production of small numbers of cloned sires from genetically elite bulls, for natural mating, to effectively disseminate genetic gain. In addition to the animal welfare concerns with the technology, the underlying health of the animals and the consequential effect on food safety are critical aspects that require investigation to gain regulatory and consumer

  10. Human cloning: can it be made safe?

    PubMed

    Rhind, Susan M; Taylor, Jane E; De Sousa, Paul A; King, Tim J; McGarry, Michelle; Wilmut, Ian

    2003-11-01

    There are continued claims of attempts to clone humans using nuclear transfer, despite the serious problems that have been encountered in cloning other mammals. It is known that epigenetic and genetic mechanisms are involved in clone failure, but we still do not know exactly how. Human reproductive cloning is unethical, but the production of cells from cloned embryos could offer many potential benefits. So, can human cloning be made safe?

  11. Cloning and characterization of a mammalian pseudouridine synthase.

    PubMed Central

    Chen, J; Patton, J R

    1999-01-01

    This report describes the cloning and characterization of a pseudouridine (psi) synthase from mouse that we have named mouse pseudouridine synthase 1 (mpus1p). The cDNA is approximately 1.5 kb and when used as a probe on a Northern blot of mouse RNA from tissues and cultured cells, several bands were detected. The open reading frame is 393 amino acids and has 35% identity over its length with yeast psi synthase 1 (pus1p). The recombinant protein was expressed in Escherichia coli and the purified protein converted specific uridines to psi in a number of tRNA substrates. The positions modified in stoichiometric amounts in vitro were 27/28 in the anticodon stem and also positions 34 and 36 in the anticodon of an intron containing tRNA. A human cDNA was also cloned and the smaller open reading frame (348 amino acids) was 92% identical over its length with mpus1p but is shorter by 45 amino acids at the amino terminus. The expressed recombinant human protein has no activity on any of the tRNA substrates, most probably the result of the truncated open reading frame. PMID:10094309

  12. Cloning and pharmacological characterization of a rat kappa opioid receptor.

    PubMed Central

    Meng, F; Xie, G X; Thompson, R C; Mansour, A; Goldstein, A; Watson, S J; Akil, H

    1993-01-01

    A full-length cDNA was isolated from a rat striatal library by using low-stringency screening with two PCR fragments, one spanning transmembrane domains 3-6 of the mouse delta opioid receptor and the other unidentified but homologous to the mouse delta receptor from rat brain. The novel cDNA had a long open reading frame encoding a protein of 380 residues with 59% identity to the mouse delta receptor and topography consistent with a seven-helix guanine nucleotide-binding protein-coupled receptor. COS-1 cells transfected with the coding region of this clone showed high-affinity binding to kappa opioid receptor-selective ligands such as dynorphin A and U-50,488 and also nonselective opioid ligands such as bremazocine, ethylketocyclazocine, and naloxone. Not bound at all (or bound with low affinity) were dynorphin A-(2-13), enantiomers of naloxone and levophanol [i.e., (+)-naloxone and dextrorphan], and selective mu and delta opioid receptor ligands. Activation of the expressed receptor by kappa receptor agonists led to inhibition of cAMP. Finally, in situ hybridization revealed a mRNA distribution in rat brain that corresponded well to the distribution of binding sites labeled with kappa-selective ligands. These observations indicate that we have cloned a cDNA encoding a rat kappa receptor of the kappa 1 subtype. Images Fig. 3 PMID:8234341

  13. DNA cloning: A personal view after 40 years

    PubMed Central

    Cohen, Stanley N.

    2013-01-01

    In November 1973, my colleagues A. C. Y. Chang, H. W. Boyer, R. B. Helling, and I reported in PNAS that individual genes can be cloned and isolated by enzymatically cleaving DNA molecules into fragments, linking the fragments to an autonomously replicating plasmid, and introducing the resulting recombinant DNA molecules into bacteria. A few months later, Chang and I reported that genes from unrelated bacterial species can be combined and propagated using the same approach and that interspecies recombinant DNA molecules can produce a biologically functional protein in a foreign host. Soon afterward, Boyer’s laboratory and mine published our collaborative discovery that even genes from animal cells can be cloned in bacteria. These three PNAS papers quickly led to the use of DNA cloning methods in multiple areas of the biological and chemical sciences. They also resulted in a highly public controversy about the potential hazards of laboratory manipulation of genetic material, a decision by Stanford University and the University of California to seek patents on the technology that Boyer and I had invented, and the application of DNA cloning methods for commercial purposes. In the 40 years that have passed since publication of our findings, use of DNA cloning has produced insights about the workings of genes and cells in health and disease and has altered the nature of the biotechnology and biopharmaceutical industries. Here, I provide a personal perspective of the events that led to, and followed, our report of DNA cloning. PMID:24043817

  14. DNA cloning: a personal view after 40 years.

    PubMed

    Cohen, Stanley N

    2013-09-24

    In November 1973, my colleagues A. C. Y. Chang, H. W. Boyer, R. B. Helling, and I reported in PNAS that individual genes can be cloned and isolated by enzymatically cleaving DNA molecules into fragments, linking the fragments to an autonomously replicating plasmid, and introducing the resulting recombinant DNA molecules into bacteria. A few months later, Chang and I reported that genes from unrelated bacterial species can be combined and propagated using the same approach and that interspecies recombinant DNA molecules can produce a biologically functional protein in a foreign host. Soon afterward, Boyer's laboratory and mine published our collaborative discovery that even genes from animal cells can be cloned in bacteria. These three PNAS papers quickly led to the use of DNA cloning methods in multiple areas of the biological and chemical sciences. They also resulted in a highly public controversy about the potential hazards of laboratory manipulation of genetic material, a decision by Stanford University and the University of California to seek patents on the technology that Boyer and I had invented, and the application of DNA cloning methods for commercial purposes. In the 40 years that have passed since publication of our findings, use of DNA cloning has produced insights about the workings of genes and cells in health and disease and has altered the nature of the biotechnology and biopharmaceutical industries. Here, I provide a personal perspective of the events that led to, and followed, our report of DNA cloning.

  15. Pathophysiology of gene-targeted mouse models for cystic fibrosis.

    PubMed

    Grubb, B R; Boucher, R C

    1999-01-01

    Pathophysiology of Gene-Targeted Mouse Models for Cystic Fibrosis. Physiol. Rev. 79, Suppl.: S193-S214, 1999. - Mutations in the gene causing the fatal disease cystic fibrosis (CF) result in abnormal transport of several ions across a number of epithelial tissues. In just 3 years after this gene was cloned, the first CF mouse models were generated. The CF mouse models generated to date have provided a wealth of information on the pathophysiology of the disease in a variety of organs. Heterogeneity of disease in the mouse models is due to the variety of gene-targeting strategies used in the generation of the CF mouse models as well as the diversity of the murine genetic background. This paper reviews the pathophysiology in the tissues and organs (gastrointestinal, airway, hepatobiliary, pancreas, reproductive, and salivary tissue) involved in the disease in the various CF mouse models. Marked similarities to and differences from the human disease have been observed in the various murine models. Some of the CF mouse models accurately reflect the ion-transport abnormalities and disease phenotype seen in human CF patients, especially in gastrointestinal tissue. However, alterations in airway ion transport, which lead to the devastating lung disease in CF patients, appear to be largely absent in the CF mouse models. Reasons for these unexpected findings are discussed. This paper also reviews pharmacotherapeutic and gene therapeutic studies in the various mouse models. PMID:9922382

  16. Cloning: revisiting an old debate.

    PubMed

    Verhey, Allen D

    1994-09-01

    The debate about cloning that took place 25 years ago, although directed toward a different sort of cloning, elucidates fundamental issues currently at stake in reproductive technologies and research. Paul Ramsey and Joseph Fletcher were participants in this early debate. The differences between Ramsey and Fletcher about the meaning and sufficiency of freedom, the understanding and weighing of good and evil, the connection between embodiment and personhood, the relationship of humans with nature, and the meaning of parenthood suggest both a broader agenda for the debate about cloning and a cautious move forward in the development of embryo-splitting.

  17. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    NASA Astrophysics Data System (ADS)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  18. Cloning changes the response to obesity of innate immune factors in blood, liver, and adipose tissues in domestic pigs.

    PubMed

    Rødgaard, Tina; Skovgaard, Kerstin; Stagsted, Jan; Heegaard, Peter M H

    2013-06-01

    The objective of this study was to evaluate the usefulness of cloned pigs as porcine obesity models reflecting obesity-associated changes in innate immune factor gene expression profiles. Liver and adipose tissue expression of 43 innate immune genes as well as serum concentrations of six immune factors were analyzed in lean and diet-induced obese cloned domestic pigs and compared to normal domestic pigs (obese and lean). The number of genes affected by obesity was lower in cloned animals than in control animals. All genes affected by obesity in adipose tissues of clones were downregulated; both upregulation and downregulation were observed in the controls. Cloning resulted in a less differentiated adipose tissue expression pattern. Finally, the serum concentrations of two acute-phase proteins (APPs), haptoglobin (HP) and orosomucoid (ORM), were increased in obese clones as compared to obese controls as well as lean clones and controls. Generally, the variation in phenotype between individual pigs was not reduced in cloned siblings as compared to normal siblings. Therefore, we conclude that cloning limits both the number of genes responding to obesity as well as the degree of tissue-differentiated gene expression, concomitantly with an increase in APP serum concentrations only seen in cloned, obese pigs. This may suggest that the APP response seen in obese, cloned pigs is a consequence of the characteristic skewed gene response to obesity in cloned pigs, as described in this work. This should be taken into consideration when using cloned animals as models for innate responses to obesity.

  19. Human therapeutic cloning (NTSC): applying research from mammalian reproductive cloning.

    PubMed

    French, Andrew J; Wood, Samuel H; Trounson, Alan O

    2006-01-01

    Human therapeutic cloning or nuclear transfer stem cells (NTSC) to produce patient-specific stem cells, holds considerable promise in the field of regenerative medicine. The recent withdrawal of the only scientific publications claiming the successful generation of NTSC lines afford an opportunity to review the available research in mammalian reproductive somatic cell nuclear transfer (SCNT) with the goal of progressing human NTSC. The process of SCNT is prone to epigenetic abnormalities that contribute to very low success rates. Although there are high mortality rates in some species of cloned animals, most surviving clones have been shown to have normal phenotypic and physiological characteristics and to produce healthy offspring. This technology has been applied to an increasing number of mammals for utility in research, agriculture, conservation, and biomedicine. In contrast, attempts at SCNT to produce human embryonic stem cells (hESCs) have been disappointing. Only one group has published reliable evidence of success in deriving a cloned human blastocyst, using an undifferentiated hESC donor cell, and it failed to develop into a hESC line. When optimal conditions are present, it appears that in vitro development of cloned and parthenogenetic embryos, both of which may be utilized to produce hESCs, may be similar to in vitro fertilized embryos. The derivation of ESC lines from cloned embryos is substantially more efficient than the production of viable offspring. This review summarizes developments in mammalian reproductive cloning, cell-to-cell fusion alternatives, and strategies for oocyte procurement that may provide important clues facilitating progress in human therapeutic cloning leading to the successful application of cell-based therapies utilizing autologous hESC lines.

  20. Methylotroph cloning vehicle

    DOEpatents

    Hanson, Richard S.; Allen, Larry N.

    1989-04-25

    A cloning vehicle comprising: a replication determinant effective for replicating the vehicle in a non-C.sub.1 -utilizing host and in a C.sub.1 -utilizing host; DNA effective to allow the vehicle to be mobilized from the non-C.sub.1 -utilizing host to the C.sub.1 -utilizing host; DNA providing resistance to two antibiotics to which the wild-type C.sub.1 -utilizing host is susceptible, each of the antibiotic resistance markers having a recognition site for a restriction endonuclease; a cos site; and a means for preventing replication in the C.sub.1 -utilizing host. The vehicle is used for complementation mapping as follows. DNA comprising a gene from the C.sub.1 -utilizing organism is inserted at the restriction nuclease recognition site, inactivating the antibiotic resistance marker at that site. The vehicle can then be used to form a cosmid structure to infect the non-C.sub.1 -utilizing (e.g., E. coli) host, and then conjugated with a selected C.sub.1 -utilizing mutant. Resistance to the other antibiotic by the mutant is a marker of the conjugation. Other phenotypical changes in the mutant, e.g., loss of an auxotrophic trait, is attributed to the C.sub.1 gene. The vector is also used to inactivate genes whose protein products catalyze side reactions that divert compounds from a biosynthetic pathway to a desired product, thereby producing an organism that makes the desired product in higher yields.

  1. Impermanence of bacterial clones

    PubMed Central

    Bobay, Louis-Marie; Traverse, Charles C.; Ochman, Howard

    2015-01-01

    Bacteria reproduce asexually and pass on a single genome copied from the parent, a reproductive mode that assures the clonal descent of progeny; however, a truly clonal bacterial species is extremely rare. The signal of clonality can be interrupted by gene uptake and exchange, initiating homologous recombination that results in the unique sequence of one clone being incorporated into another. Because recombination occurs sporadically and on local scales, these events are often difficult to recognize, even when considering large samples of completely sequenced genomes. Moreover, several processes can produce the appearance of clonality in populations that undergo frequent recombination. The rates and consequences of recombination have been studied in Escherichia coli for over 40 y, and, during this time, there have been several shifting views of its clonal status, population structure, and rates of gene exchange. We reexamine the studies and retrace the evolution of the methods that have assessed the extent of DNA flux, largely focusing on its impact on the E. coli genome. PMID:26195749

  2. Social Individualism.

    ERIC Educational Resources Information Center

    Cornille, Thomas A.; Harrigan, John

    Relationships between individuals and society have often been presented from the perspective of the social institution. Social psychology has addressed the variables that affect the individual in relationships with larger groups. Social individualism is a conceptual framework that explores the relationship of the individual and society from the…

  3. A mouse embryonic stem cell bank for inducible overexpression of human chromosome 21 genes

    PubMed Central

    2010-01-01

    Background Dosage imbalance is responsible for several genetic diseases, among which Down syndrome is caused by the trisomy of human chromosome 21. Results To elucidate the extent to which the dosage imbalance of specific human chromosome 21 genes perturb distinct molecular pathways, we developed the first mouse embryonic stem (ES) cell bank of human chromosome 21 genes. The human chromosome 21-mouse ES cell bank includes, in triplicate clones, 32 human chromosome 21 genes, which can be overexpressed in an inducible manner. Each clone was transcriptionally profiled in inducing versus non-inducing conditions. Analysis of the transcriptional response yielded results that were consistent with the perturbed gene's known function. Comparison between mouse ES cells containing the whole human chromosome 21 (trisomic mouse ES cells) and mouse ES cells overexpressing single human chromosome 21 genes allowed us to evaluate the contribution of single genes to the trisomic mouse ES cell transcriptome. In addition, for the clones overexpressing the Runx1 gene, we compared the transcriptome changes with the corresponding protein changes by mass spectroscopy analysis. Conclusions We determined that only a subset of genes produces a strong transcriptional response when overexpressed in mouse ES cells and that this effect can be predicted taking into account the basal gene expression level and the protein secondary structure. We showed that the human chromosome 21-mouse ES cell bank is an important resource, which may be instrumental towards a better understanding of Down syndrome and other human aneuploidy disorders. PMID:20569505

  4. Individual differences and evidence-based psychopharmacology.

    PubMed

    Belmaker, Rh; Bersudsky, Yuly; Agam, Galila

    2012-09-27

    Individual differences in response to pharmacologic treatment limits the usefulness of mean data obtained from randomized controlled trials. These individual differences exist even in genetically uniform inbred mouse strains. While stratification can be of value in large studies, the individual patient history is the most effective currently available guide for personalized medicine in psychopharmacology.

  5. A Clone of Your Own.

    ERIC Educational Resources Information Center

    Bilodeau, Kirsten

    1997-01-01

    Describes an activity used at the Washington Park Arboretum that helps students understand cloning through plant propagation. Students also learn how to make a pot from recycled newspapers and how to make soil that is appropriate for the plants. (DDR)

  6. Cloning of a quantum measurement

    SciTech Connect

    Bisio, Alessandro; D'Ariano, Giacomo Mauro; Perinotti, Paolo; Sedlak, Michal

    2011-10-15

    We analyze quantum algorithms for cloning of a quantum measurement. Our aim is to mimic two uses of a device performing an unknown von Neumann measurement with a single use of the device. When the unknown device has to be used before the bipartite state to be measured is available we talk about 1{yields}2 learning of the measurement, otherwise the task is called 1{yields}2 cloning of a measurement. We perform the optimization for both learning and cloning for arbitrary dimension d of the Hilbert space. For 1{yields}2 cloning we also propose a simple quantum network that achieves the optimal fidelity. The optimal fidelity for 1{yields}2 learning just slightly outperforms the estimate and prepare strategy in which one first estimates the unknown measurement and depending on the result suitably prepares the duplicate.

  7. Human Cloning: Let's Discuss It.

    ERIC Educational Resources Information Center

    Taras, Loretta; Stavroulakis, Anthea M.; Ortiz, Mary T.

    1999-01-01

    Describes experiences with holding discussions on cloning at a variety of levels in undergraduate biology courses. Discusses teaching methods used and student reactions to the discussions. Contains 12 references. (WRM)

  8. Human cloning and 'posthuman' society.

    PubMed

    Blackford, Russell

    2005-01-01

    Since early 1997, when the creation of Dolly the sheep by somatic cell nuclear transfer was announced in Nature, numerous government reports, essays, articles and books have considered the ethical problems and policy issues surrounding human reproductive cloning. In this article, I consider what response a modern liberal society should give to the prospect of human cloning, if it became safe and practical. Some opponents of human cloning have argued that permitting it would place us on a slippery slope to a repugnant future society, comparable to that portrayed in Aldous Huxley's novel, Brave New World. I conclude that, leaving aside concerns about safety, none of the psychological or social considerations discussed in this article provides an adequate policy justification for invoking the state's coercive powers to prevent human cloning.

  9. Are cloned quantum states macroscopic?

    PubMed

    Fröwis, F; Dür, W

    2012-10-26

    We study quantum states produced by optimal phase covariant quantum cloners. We argue that cloned quantum superpositions are not macroscopic superpositions in the spirit of Schrödinger's cat, despite their large particle number. This is indicated by calculating several measures for macroscopic superpositions from the literature, as well as by investigating the distinguishability of the two superposed cloned states. The latter rapidly diminishes when considering imperfect detectors or noisy states and does not increase with the system size. In contrast, we find that cloned quantum states themselves are macroscopic, in the sense of both proposed measures and their usefulness in quantum metrology with an optimal scaling in system size. We investigate the applicability of cloned states for parameter estimation in the presence of different kinds of noise.

  10. Human cloning and 'posthuman' society.

    PubMed

    Blackford, Russell

    2005-01-01

    Since early 1997, when the creation of Dolly the sheep by somatic cell nuclear transfer was announced in Nature, numerous government reports, essays, articles and books have considered the ethical problems and policy issues surrounding human reproductive cloning. In this article, I consider what response a modern liberal society should give to the prospect of human cloning, if it became safe and practical. Some opponents of human cloning have argued that permitting it would place us on a slippery slope to a repugnant future society, comparable to that portrayed in Aldous Huxley's novel, Brave New World. I conclude that, leaving aside concerns about safety, none of the psychological or social considerations discussed in this article provides an adequate policy justification for invoking the state's coercive powers to prevent human cloning. PMID:16007753

  11. Psychological aspects of human cloning and genetic manipulation: the identity and uniqueness of human beings.

    PubMed

    Morales, N M

    2009-01-01

    Human cloning has become one of the most controversial debates about reproduction in Western civilization. Human cloning represents asexual reproduction, but the critics of human cloning argue that the result of cloning is not a new individual who is genetically unique. There is also awareness in the scientific community, including the medical community, that human cloning and the creation of clones are inevitable. Psychology and other social sciences, together with the natural sciences, will need to find ways to help the healthcare system, to be prepared to face the new challenges introduced by the techniques of human cloning. One of those challenges is to help the healthcare system to find specific standards of behaviour that could be used to help potential parents to interact properly with cloned babies or children created through genetic manipulation. In this paper, the concepts of personality, identity and uniqueness are discussed in relationship to the contribution of twin studies in these areas. The author argues that an individual created by human cloning techniques or any other type of genetic manipulation will not show the donor's characteristics to the extent of compromising uniqueness. Therefore, claims to such an effect are needlessly alarmist.

  12. Images of cloning and stem cell research in editorial cartoons in the United States.

    PubMed

    Giarelli, Ellen

    2006-01-01

    Through semiotic analysis of manifest and latent meanings in editorial cartoons, the author uncovers how cloning and stem cell research are represented in a popular mass medium. She identified 86 editorial cartoons published in the United States between 2001 and 2004 that referred to cloning and 20 that referred to stem cell research. Cartoonists portrayed people individually 224 times and 4 times in groups of more than 10. Men were portrayed in 64% of cartoons. Stem cell research was depicted as having a potential positive value, and cloning was depicted negatively. Some major messages are that cloning will lead to the mass production of evil, cloning creates monsters, and politics will influence who or what will be cloned. Analyzing popular images can allow access to public understanding about genetic technology and evaluation of public beliefs, preconceptions, and expectations as the public is educated on the use and value of services.

  13. Local cloning of entangled qubits

    SciTech Connect

    Choudhary, Sujit K.; Kunkri, Samir; Rahaman, Ramij; Roy, Anirban

    2007-11-15

    We discuss the exact cloning of orthogonal but entangled qubits under local operations and classical communication. The amount of entanglement necessary in a blank copy is obtained for various cases. Surprisingly, this amount is more than 1 ebit for certain sets of two nonmaximal but equally entangled states of two qubits. To clone any three Bell states, at least log{sub 2} 3 ebit is necessary.

  14. Cloning: questions answered and unsolved.

    PubMed

    Latham, Keith E

    2004-02-01

    Cloning by the transfer of adult somatic cell nuclei to oocytes has produced viable offspring in a variety of mammalian species. The technology is still in its initial stages of development. Studies to date have answered several basic questions related to such issues as genome potency, life expectancy of clones, mitochondrial fates, and feasibility of inter-species nuclear transfer. They have also raised new questions related to the control of nuclear reprogramming and function. These questions are reviewed here.

  15. Progesterone promotes propagation and viability of mouse embryonic stem cells.

    PubMed

    Shen, Shan-Wei; Song, Hou-Yan

    2009-10-25

    It has been known that estrogen-17beta stimulates proliferation of mouse embryonic stem (mES) cells. To explore the function of another steroid hormone progesterone, we used MTT method and BrdU incorporation assay to obtain growth curves, clone forming assay to detect the propagation and viability of individual mES cells, Western blot to test the expression of ES cell marker gene Oct-4, fluorescence activated cell sorter (FACS) to test cell cycle, and real-time PCR to detect the expressions of cyclins, cyclin-dependent kinases and proto-oncogenes. The results showed that progesterone promoted proliferation of mES cells. The number of clones was more in progesterone-treated group than that in the control group. The expression of pluripotency-associated transcriptional factor Oct-4 changed little after progesterone treatment as shown by Western blot, indicating that most of mES cells were in undifferentiated state. The results of FACS proved that progesterone promoted DNA synthesis in mES cells. The proportion of mES cells in S+G(2)/M phase was higher in progesterone-treated group than that in the control group. Cyclins and cyclin-dependent kinases, as well as proto-oncogenes (c-myc, c-fos) were up-regulated when cells were treated with progesterone. The results obtained indicate that progesterone promotes propagation and viability of mES cells. The up-regulation of cell cycle-related factors might contribute to the function of progesterone.

  16. Cloning goes to the movies.

    PubMed

    Cormick, Craig

    2006-10-01

    Public attitude research conducted by Biotechnology Australia shows that one of the major sources of information on human reproductive cloning is movies. Traditionally, understanding of new and emerging technologies has come through the mass media but human cloning, being so widely addressed through the popular culture of movies, is more effectively defined by Hollywood than the news media or science media. But how well are the science and social issues of cloning portrayed in box office hits such as The Island, Multiplicity, Star Wars: Attack of the Clones and Jurassic Park? These movies have enormous reach and undoubted influence, and are therefore worth analyzing in some detail. This study looks at 33 movies made between 1971 and 2005 that address human reproductive cloning, and it categorizes the films based on their genre and potential influence. Yet rather than simply rating the quality of the science portrayed, the study compares the key messages in these movies with public attitudes towards cloning, to examine the correlations.

  17. Islamic perspectives on human cloning.

    PubMed

    Sadeghi, Mahmoud

    2007-01-01

    The present paper seeks to assess various views from Islamic jurists relating to human cloning, which is one of the controversial topics in the recent past. Taking Islamic jurisprudence principles, such as the rule of necessity for self preservation and respect for human beings, the rule of la darar wa la dirar ('the necessity to refrain from causing harm to oneself and others') and the rule of usr wa haraj, one may indicate that if human cloning could not be prohibited, as such, it could still be opposed because it gives way to various harmful consequences, which include family disorder, chaos in the clone's family relationships, physical and mental diseases for clones and suffering of egg donors and surrogate mothers. However with due attention to the fact that the reasons behind the prohibition of abortion only restrict the destruction of human embryos in their post-implantation stages, human cloning for biomedical research and exploitation of stem cells from cloned embryos at the blastocyst stage for therapeutic purposes would be acceptable.

  18. Cloning goes to the movies.

    PubMed

    Cormick, Craig

    2006-10-01

    Public attitude research conducted by Biotechnology Australia shows that one of the major sources of information on human reproductive cloning is movies. Traditionally, understanding of new and emerging technologies has come through the mass media but human cloning, being so widely addressed through the popular culture of movies, is more effectively defined by Hollywood than the news media or science media. But how well are the science and social issues of cloning portrayed in box office hits such as The Island, Multiplicity, Star Wars: Attack of the Clones and Jurassic Park? These movies have enormous reach and undoubted influence, and are therefore worth analyzing in some detail. This study looks at 33 movies made between 1971 and 2005 that address human reproductive cloning, and it categorizes the films based on their genre and potential influence. Yet rather than simply rating the quality of the science portrayed, the study compares the key messages in these movies with public attitudes towards cloning, to examine the correlations. PMID:17214211

  19. Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: Homology to Epstein-Barr virus open reading frame BCRFI

    SciTech Connect

    Vieira, P.; De Waal-Malefyt, R.; Dang, M.N.; Johnson, K.E.; Kastelein, R.; Fiorentino, D.F.; DeVries, J.E.; Roncarolo, M.G.; Mosmann, T.R.; Moore, K.W. )

    1991-02-15

    The authors demonstrated the existence of human cytokine synthesis inhibitory factor (DSIF) (interleukin 10 (IL-10)). cDNA clones encoding human IL-10 (hIL-10) were isolated from a tetanus toxin-specific human T-cell clone. Like mouse IL-10, hIL-10 exhibits strong DNA and amino acid sequence homology to an open reading frame in the Epstein-Barr virus, BDRFL. hIL-10 and the BCRFI product inhibit cytokine synthesis by activated human peripheral blood mononuclear cells and by a mouse Th1 clone. Both hIL-10 and mouse IL-10 sustain the viability of a mouse mast cell line in culture, but BCRFI lacks comparable activity in this way, suggesting that BCRFI may have conserved only a subset of hIL-10 activities.

  20. Methylotroph cloning vehicle

    DOEpatents

    Hanson, R.S.; Allen, L.N.

    1989-04-25

    A cloning vehicle comprising: a replication determinant effective for replicating the vehicle in a non-C[sub 1]-utilizing host and in a C[sub 1]-utilizing host; DNA effective to allow the vehicle to be mobilized from the non-C[sub 1]-utilizing host to the C[sub 1]-utilizing host; DNA providing resistance to two antibiotics to which the wild-type C[sub 1]-utilizing host is susceptible, each of the antibiotic resistance markers having a recognition site for a restriction endonuclease; a cos site; and a means for preventing replication in the C[sub 1]-utilizing host. The vehicle is used for complementation mapping as follows. DNA comprising a gene from the C[sub 1]-utilizing organism is inserted at the restriction nuclease recognition site, inactivating the antibiotic resistance marker at that site. The vehicle can then be used to form a cosmid structure to infect the non-C[sub 1]-utilizing (e.g., E. coli) host, and then conjugated with a selected C[sub 1]-utilizing mutant. Resistance to the other antibiotic by the mutant is a marker of the conjugation. Other phenotypical changes in the mutant, e.g., loss of an auxotrophic trait, is attributed to the C[sub 1] gene. The vector is also used to inactivate genes whose protein products catalyze side reactions that divert compounds from a biosynthetic pathway to a desired product, thereby producing an organism that makes the desired product in higher yields. 3 figs.

  1. The rise and fall of horror autotoxicus and forbidden clones.

    PubMed

    Jennette, J Charles; Falk, Ronald J

    2010-09-01

    Cui and associates show that healthy individuals have natural autoantibodies (NAAs) specific for myeloperoxidase, proteinase 3, and glomerular basement membrane (GBM) with the same specificity as anti-neutrophil cytoplasmic antibodies and anti-GBM antibodies that are pathogenic. Although Ehrlich proposed horror autotoxicus and Burnet envisioned elimination of forbidden clones, NAAs are present in all healthy individuals and play beneficial homeostatic roles. Pathogenic autoimmunity is dysregulation of natural homeostatic autoimmunity rather than onset of a previously absent self-recognition.

  2. The rise and fall of horror autotoxicus and forbidden clones.

    PubMed

    Jennette, J Charles; Falk, Ronald J

    2010-09-01

    Cui and associates show that healthy individuals have natural autoantibodies (NAAs) specific for myeloperoxidase, proteinase 3, and glomerular basement membrane (GBM) with the same specificity as anti-neutrophil cytoplasmic antibodies and anti-GBM antibodies that are pathogenic. Although Ehrlich proposed horror autotoxicus and Burnet envisioned elimination of forbidden clones, NAAs are present in all healthy individuals and play beneficial homeostatic roles. Pathogenic autoimmunity is dysregulation of natural homeostatic autoimmunity rather than onset of a previously absent self-recognition. PMID:20805814

  3. Isolation of mouse cell proteoglycan mutants

    SciTech Connect

    Keller, K.M.; Keller, J.M.

    1986-05-01

    The sulfated proteoglycans on the surface of cultured mammalian cells have been implicated in a variety of phenomena. To obtain more direct evidence for the role of these molecules in specific cellular functions, they are isolating mutants that produce altered sulfated proteoglycans from a cloned line of Swiss mouse 3T3 cells. This cell type was selected because it exhibits contact inhibition of growth and there is extensive information on its' cell surface and extracellular proteoglycans and other glycoproteins. Cells were chemically mutagenized and subjected to one or more cycles of radiation suicide in the presence of /sup 35/S-sulfate. By replica plating, 150 clones, which appear to incorporate abnormal amounts of /sup 35/S-sulfate, have been selected. After recloning three times via the replica plating technique, the proteoglycans of 29 clones have thus far been analyzed. They have identified four clones which appear to make altered amounts of either cell surface heparan sulfate or chondroitin sulfate. The biochemical bases for the altered levels of the proteoglycans are under study. Of particular interest, however, is the fact that in this limited collection of mutants the chemical alterations correlate with specific altered cellular morphologies.

  4. Positional cloning of the murine flavivirus resistance gene.

    PubMed

    Perelygin, Andrey A; Scherbik, Svetlana V; Zhulin, Igor B; Stockman, Bronislava M; Li, Yan; Brinton, Margo A

    2002-07-01

    Inbred mouse strains exhibit significant differences in their susceptibility to viruses in the genus Flavivirus, which includes human pathogens such as yellow fever, Dengue, and West Nile virus. A single gene, designated Flv, confers this differential susceptibility and was mapped previously to a region of mouse chromosome 5. A positional cloning strategy was used to identify 22 genes from the Flv gene interval including 10 members of the 2'-5'-oligoadenylate synthetase gene family. One 2'-5'-oligoadenylate synthetase gene, Oas1b, was identified as Flv by correlation between genotype and phenotype in nine mouse strains. Susceptible mouse strains produce a protein lacking 30% of the C-terminal sequence as compared with the resistant counterpart because of the presence of a premature stop codon. The Oas1b gene differs from all the other murine Oas genes by a unique four-amino acid deletion in the P-loop located within the conserved RNA binding domain. Expression of the resistant allele of Oas1b in susceptible embryo fibroblasts resulted in partial inhibition of the replication of a flavivirus but not of an alpha togavirus.

  5. Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells

    PubMed Central

    Mizutani, Eiji; Torikai, Kohei; Wakayama, Sayaka; Nagatomo, Hiroaki; Ohinata, Yasuhide; Kishigami, Satoshi; Wakayama, Teruhiko

    2016-01-01

    Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38–77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal. PMID:27033801

  6. Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells.

    PubMed

    Mizutani, Eiji; Torikai, Kohei; Wakayama, Sayaka; Nagatomo, Hiroaki; Ohinata, Yasuhide; Kishigami, Satoshi; Wakayama, Teruhiko

    2016-01-01

    Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38-77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal. PMID:27033801

  7. Local cloning of two product states

    SciTech Connect

    Ji Zhengfeng; Feng Yuan; Ying Mingsheng

    2005-09-15

    Local quantum operations and classical communication (LOCC) put considerable constraints on many quantum information processing tasks such as cloning and discrimination. Surprisingly, however, discrimination of any two pure states survives such constraints in some sense. We show that cloning is not that lucky; namely, probabilistic LOCC cloning of two product states is strictly less efficient than global cloning. We prove our result by giving explicitly the efficiency formula of local cloning of any two product states.

  8. The Molecular Basis of Muscular Dystrophy in the mdx Mouse: A Point Mutation

    NASA Astrophysics Data System (ADS)

    Sicinski, Piotr; Geng, Yan; Ryder-Cook, Allan S.; Barnard, Eric A.; Darlison, Mark G.; Barnard, Pene J.

    1989-06-01

    The mdx mouse is an X-linked myopathic mutant, an animal model for human Duchenne muscular dystrophy. In both mouse and man the mutations lie within the dystrophin gene, but the phenotypic differences of the disease in the two species confer much interest on the molecular basis of the mdx mutation. The complementary DNA for mouse dystrophin has been cloned, and the sequence has been used in the polymerase chain reaction to amplify normal and mdx dystrophin transcripts in the area of the mdx mutation. Sequence analysis of the amplification products showed that the mdx mouse has a single base substitution within an exon, which causes premature termination of the polypeptide chain.

  9. CELL SURFACE ANTIGENS OF A MOUSE TESTICULAR TERATOMA

    PubMed Central

    Gooding, Linda R.; Edidin, Michael

    1974-01-01

    Rabbit antisera to a mouse testicular teratoma, absorbed with normal mouse tissues, react by immunofluorescence with plasma membrane antigens of a variety of transplantable mouse tumor cells and transformed fibroblast cell lines including Clone 1D, SV-40-3T3, and 3T12. Trypsin treatment of cells of "normal" lines, 3T3 and FR-SV-3T3, uncovers reactivity on these as well. Early passage mouse embryo fibroblast cell cultures do not react even after trypsinization. By cross-absorbtion studies, the anti-teratoma serum appears to react with an antigen common to most tumor cells investigated thus far. When this antigen on Clone 1D cells is "capped," H-2 antigens collect with the teratoma antigens in the cap indicating a physical association between the molecules. Molecules specified by both the H-2D and H-2K regions are bound to the teratoma antigens in the Clone 1D plasma membrane. This antigen is also found in soluble tumor cell fractions where it is believed to be free of H-2. A second cell surface antigen defined by anti-teratoma serum is expressed only by hepatoma and teratoma itself. This second antigen is apparently a secretory product of teratoma cells. A third surface antigen defined by anti-teratoma serum appears to be specific for the teratoma. PMID:4365513

  10. Expression of Differentiated Functions in Hepatoma Cell Hybrids: High Frequency of Induction of Mouse Albumin Production in Rat Hepatoma-Mouse Lymphoblast Hybrids*

    PubMed Central

    Malawista, Stephen E.; Weiss, Mary C.

    1974-01-01

    We have studied the production of serum albumin by somatic hybrids between well-differentiated 2s and 1s rat hepatoma cells (Faza), which produce serum albumin, and sub-diploid mouse leukemic lymphoblasts (Lc), which do not produce albumin. We determined the rat or mouse origin of the albumin by double immunodiffusion, using immuno-adsorbed noncrossreacting antisera. Each of 12 karyologically identified 2s hybrid clones (Lc2F) produces both rat and mouse albumin. Moreover, unlike 1s hybrids reported previously, eight of nine 1s hybrids (LcF) also produce mouse albumin; six of them produce rat albumin as well. One clone from the 1s cross produces only rat albumin. Images PMID:4362639

  11. 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

  12. Local cloning of entangled states

    SciTech Connect

    Gheorghiu, Vlad; Yu Li; Cohen, Scott M.

    2010-08-15

    We investigate the conditions under which a set S of pure bipartite quantum states on a DxD system can be locally cloned deterministically by separable operations, when at least one of the states is full Schmidt rank. We allow for the possibility of cloning using a resource state that is less than maximally entangled. Our results include that: (i) all states in S must be full Schmidt rank and equally entangled under the G-concurrence measure, and (ii) the set S can be extended to a larger clonable set generated by a finite group G of order |G|=N, the number of states in the larger set. It is then shown that any local cloning apparatus is capable of cloning a number of states that divides D exactly. We provide a complete solution for two central problems in local cloning, giving necessary and sufficient conditions for (i) when a set of maximally entangled states can be locally cloned, valid for all D; and (ii) local cloning of entangled qubit states with nonvanishing entanglement. In both of these cases, we show that a maximally entangled resource is necessary and sufficient, and the states must be related to each other by local unitary 'shift' operations. These shifts are determined by the group structure, so need not be simple cyclic permutations. Assuming this shifted form and partially entangled states, then in D=3 we show that a maximally entangled resource is again necessary and sufficient, while for higher-dimensional systems, we find that the resource state must be strictly more entangled than the states in S. All of our necessary conditions for separable operations are also necessary conditions for local operations and classical communication (LOCC), since the latter is a proper subset of the former. In fact, all our results hold for LOCC, as our sufficient conditions are demonstrated for LOCC, directly.

  13. The Mouse Genome Database (MGD): mouse biology and model systems.

    PubMed

    Bult, Carol J; Eppig, Janan T; Kadin, James A; Richardson, Joel E; Blake, Judith A

    2008-01-01

    The Mouse Genome Database, (MGD, http://www.informatics.jax.org/), integrates genetic, genomic and phenotypic information about the laboratory mouse, a primary animal model for studying human biology and disease. MGD data content includes comprehensive characterization of genes and their functions, standardized descriptions of mouse phenotypes, extensive integration of DNA and protein sequence data, normalized representation of genome and genome variant information including comparative data on mammalian genes. Data within MGD are obtained from diverse sources including manual curation of the biomedical literature, direct contributions from individual investigator's laboratories and major informatics resource centers such as Ensembl, UniProt and NCBI. MGD collaborates with the bioinformatics community on the development of data and semantic standards such as the Gene Ontology (GO) and the Mammalian Phenotype (MP) Ontology. MGD provides a data-mining platform that enables the development of translational research hypotheses based on comparative genotype, phenotype and functional analyses. Both web-based querying and computational access to data are provided. Recent improvements in MGD described here include the association of gene trap data with mouse genes and a new batch query capability for customized data access and retrieval.

  14. 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.

  15. The science, fiction, and reality of embryo cloning.

    PubMed

    Cohen, Jacques; Tomkin, Giles

    1994-09-01

    Although many scientists view cloning as a useful procedure for scientific research into early embryo development -- one that cannot currently be used to produce multiple copies of humans -- the popular literature has led some individuals to view it as sinister. To address the concerns of the public, various conceptions of cloning are distinguished and their basis in fact analyzed. The possible uses, benefits, and detriments of both embryo splitting and nuclear transplantation are explained. Once the nature and purposes of cloning are understood, and the distinctive ethical dilemmas created by embryo splitting and nuclear transplantation are sorted out, these procedures should be clinically implemented to assist in vitro fertilization treatment for those who are infertile and to further other therapeutic and investigational efforts in medicine.

  16. 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

  17. [Mystery and problems of cloning].

    PubMed

    Nikitin, V A

    2010-01-01

    The attention of investigators is attracted to the fact that, in spite of great efforts in mammalian cloning, advances that have been made in this area of research are not great, and cloned animals have developmental pathologies often incompatible with life and/or reproduction ability. It is yet not clear what technical or biological factors underlie this, and how they are connected or interact with each other, which is more realistic strategically. There is a great number of articles dealing with the influence of cloning with the nuclear transfer on genetic and epigenetic reprogramming of donor cells. At the same time we can see the practical absence of analytical investigations concerning the technology of cloning as such, its weak points, and possible sources of cellular trauma in the course of microsurgery of nuclear transfer or twinning. This article discusses step by step several nuclear transfer techniques and the methods of dividing early preimplanted embryos for twinning with the aim to reveal possible sources of cell damage during micromanipulation that may have negative influence on the development of cloned organisms. Several new author's technologies based on the study of cell biophysical characteristics are described, which allow one to avoid cellular trauma during manipulation and minimize the possibility of cell damage at any rate.

  18. Cytochrome P450 1b1 in polycyclic aromatic hydrocarbon (PAH)-induced skin carcinogenesis: Tumorigenicity of individual PAHs and coal-tar extract, DNA adduction and expression of select genes in the Cyp1b1 knockout mouse

    SciTech Connect

    Siddens, Lisbeth K.; Bunde, Kristi L.; Harper, Tod A.; McQuistan, Tammie J.; Löhr, Christiane V.; Bramer, Lisa M.; Waters, Katrina M.; Tilton, Susan C.; Krueger, Sharon K.; and others

    2015-09-01

    FVB/N mice wild-type, heterozygous or null for Cyp 1b1 were used in a two-stage skin tumor study comparing PAH, benzo[a]pyrene (BaP), dibenzo[def,p]chrysene (DBC), and coal tar extract (CTE, SRM 1597a). Following 20 weeks of promotion with TPA the Cyp 1b1 null mice, initiated with DBC, exhibited reductions in incidence, multiplicity, and progression. None of these effects were observed with BaP or CTE. The mechanism of Cyp 1b1-dependent alteration of DBC skin carcinogenesis was further investigated by determining expression of select genes in skin from DBC-treated mice 2, 4 and 8 h post-initiation. A significant reduction in levels of Cyp 1a1, Nqo1 at 8 h and Akr 1c14 mRNA was observed in Cyp 1b1 null (but not wt or het) mice, whereas no impact was observed in Gst a1, Nqo 1 at 2 and 4 h or Akr 1c19 at any time point. Cyp 1b1 mRNA was not elevated by DBC. The major covalent DNA adducts, dibenzo[def,p]chrysene-(±)-11,12-dihydrodiol-cis and trans-13,14-epoxide-deoxyadenosine (DBCDE-dA) were quantified by UHPLC-MS/MS 8 h post-initiation. Loss of Cyp1 b1 expression reduced DBCDE-dA adducts in the skin but not to a statistically significant degree. The ratio of cis- to trans-DBCDE-dA adducts was higher in the skin than other target tissues such as the spleen, lung and liver (oral dosing). These results document that Cyp 1b1 plays a significant role in bioactivation and carcinogenesis of DBC in a two-stage mouse skin tumor model and that loss of Cyp 1b1 has little impact on tumor response with BaP or CTE as initiators. - Highlights: • Cyp1b1 null mice exhibit lower skin cancer sensitivity to DBC but not BaP or CTE. • Cyp1b1 expression impacts expression of other PAH metabolizing enzymes. • cis/trans-DBCDE-dA ratio significantly higher in the skin than the spleen, lung or liver • Potency of DBC and CTE in mouse skin is higher than predicted by RPFs.

  19. The topsy-turvy cloning law.

    PubMed

    Brassington, Iain; Oultram, Stuart

    2011-03-01

    In debates about human cloning, a distinction is frequently drawn between therapeutic and reproductive uses of the technology. Naturally enough, this distinction influences the way that the law is framed. The general consensus is that therapeutic cloning is less morally problematic than reproductive cloning--one can hold this position while holding that both are morally unacceptable--and the law frequently leaves the way open for some cloning for the sake of research into new therapeutic techniques while banning it for reproductive purposes. We claim that the position adopted by the law has things the wrong way around: if we accept a moral distinction between therapeutic and reproductive cloning, there are actually more reasons to be morally worried about therapeutic cloning than about reproductive cloning. If cloning is the proper object of legal scrutiny, then, we ought to make sure that we are scrutinising the right kind of clone.

  20. Building a Brainier Mouse.

    ERIC Educational Resources Information Center

    Tsien, Joe Z.

    2000-01-01

    Describes a genetic engineering project to build an intelligent mouse. Cites understanding the molecular basis of learning and memory as a very important step. Concludes that while science will never create a genius mouse that plays the stock market, it can turn a mouse into a quick learner with a better memory. (YDS)

  1. Isolation and characterization of bovine and mouse terminal deoxynucleotidyltransferase cDNAs expressible in mammalian cells.

    PubMed Central

    Koiwai, O; Yokota, T; Kageyama, T; Hirose, T; Yoshida, S; Arai, K

    1986-01-01

    We have isolated nearly full-length cDNA clones of terminal deoxynucleotidyltransferase (TdT) from calf thymus and mouse lymphoma cDNA libraries. The libraries were constructed using the pcD vector system which permits the expression of cDNA inserts in mammalian cells. The bovine TdT cDNA clone contains an open reading frame coding for 520 amino acids, Mr 59,678. The mouse TdT cDNA clone contains an open reading frame of 1,587 bp, whose translated cDNA encodes a 60,004 dalton protein. The mouse TdT cDNA clone contains 60 bp in the 3' end region of the coding sequence not found in the bovine TdT cDNA sequence, otherwise, the clones share about 80% homology. A possible nuclear-localization-sequence (Pro-Arg-Lys-Lys-Arg-Pro-Arg) was conserved in the N-terminal region in the mouse and bovine cDNA clones. Bovine and mouse cDNAs transfected into COS7 monkey fibroblasts directed the synthesis of enzymatically active protein of Mr 60,000 which was detected immunologically using polyclonal rabbit antibody against bovine TdT. Bovine TdT expressed in COS7 cells by nearly full-length cDNA clone was localized in the nucleus and the translational product of pOK103 lacking the nuclear-localization-sequence was localized in the cytoplasm. Images PMID:3755527

  2. An accurate clone-based haplotyping method by overlapping pool sequencing

    PubMed Central

    Li, Cheng; Cao, Changchang; Tu, Jing; Sun, Xiao

    2016-01-01

    Chromosome-long haplotyping of human genomes is important to identify genetic variants with differing gene expression, in human evolution studies, clinical diagnosis, and other biological and medical fields. Although several methods have realized haplotyping based on sequencing technologies or population statistics, accuracy and cost are factors that prohibit their wide use. Borrowing ideas from group testing theories, we proposed a clone-based haplotyping method by overlapping pool sequencing. The clones from a single individual were pooled combinatorially and then sequenced. According to the distinct pooling pattern for each clone in the overlapping pool sequencing, alleles for the recovered variants could be assigned to their original clones precisely. Subsequently, the clone sequences could be reconstructed by linking these alleles accordingly and assembling them into haplotypes with high accuracy. To verify the utility of our method, we constructed 130 110 clones in silico for the individual NA12878 and simulated the pooling and sequencing process. Ultimately, 99.9% of variants on chromosome 1 that were covered by clones from both parental chromosomes were recovered correctly, and 112 haplotype contigs were assembled with an N50 length of 3.4 Mb and no switch errors. A comparison with current clone-based haplotyping methods indicated our method was more accurate. PMID:27095193

  3. [Cloning: necessary reflections on the imaginary].

    PubMed

    Minahim, María Auxiliadora

    2009-01-01

    The article covers the innumerable reasons given for using cloning for therapeutic and reproductive purposes. The most commonly used argument in favour of the procedure has been that of preserving human dignity, which would include the wide exercising of personal autonomy without restrictions of an ethical nature. This view is countered by questions relating to the use of the technique, namely self-determination and the loss of the integrity of the species, which would include the transformation of a generation through the production of human beings and tissues. It must also be made clear that therapeutic cloning (which is carried out through the use of stem cells) is not yet a reality in the scientific world, with the result that the procedure that is supposedly necessary, which argues in favour of the destruction of the young embryo is misleading, as are also certain discourses used to refer to the theme and the science. Criminal law, on prohibiting this practice is anticipating it becoming a reality, protecting legal rights that affect supra-individual interests, such as the destruction of the young embryo, one of the issues of concern to ADIN (Acción Directa de Inconstitucionalidad en Brasil - Direct Action on Unconstitutionality in Brazil) 3510-0.

  4. Positional Gene Cloning in Experimental Populations.

    PubMed

    Jagodic, Maja; Stridh, Pernilla

    2016-01-01

    Positional cloning is a technique that identifies a trait-associated gene based on its location in the genome and involves methods such as linkage analysis, association mapping, and bioinformatics. This approach can be used for gene identification even when little is known about the molecular basis of the trait. Vast majority of traits are regulated by multiple genomic loci called quantitative trait loci (QTL). We describe experimental populations and designs that can be used for positional cloning, including backcrosses, intercrosses, and heterogeneous stocks, and advantages and disadvantages of different approaches. Once the phenotype and genotype of each individual in an experimental population have been determined, QTL identification can be accomplished. We describe the statistical tools used to identify the existence, location, and significance of QTLs. These different methods have advantages and disadvantages to consider when selecting the appropriate model to be used, which is briefly discussed.Although the objective of QTL mapping is to identify genomic regions associated with a trait, the ultimate goal is to identify the gene and the genetic variation (which is often quantitative trait nucleotide, QTN) or haplotype that is responsible for the phenotype. By discovering the function of causative variants or haplotypes we can understand the molecular changes that lead to the phenotype. We briefly describe how the genomic sequences can be exploited to identify QTNs and how these can be validated in congenic strains and functionally tested to understand their influence on phenotype expression. PMID:25103675

  5. The mouse homologue of the tuberin gene (TSC2) maps to a conserved synteny group between mouse chromosome 17 and human 16p13.3

    SciTech Connect

    Olsson, P.G.; Sutherland, H.F.; Nowicka, U.

    1995-01-01

    The tuberous sclerosis gene (TSC2) on human chromosome 16p13.3 has recently been identified. Several markers from this region have previously been shown to be members of a conserved synteny group, in the mouse located on chromosome 17. The mouse region includes markers D17Lon1, D17Lon2, D17Lon3, and D17Lon4, which are linked to the {alpha}-globin pseudogene Hba-ps4 on chromosome 17, while the corresponding human markers, NK12, NK92, sazD, and KM17, are linked to the functional {alpha}-globin locus near the tip of chromosome 16p. Since the human TSC2 maps in close proximity to NK12, we wanted to investigate whether a mouse gene, homologous to TSC2, was present on mouse chromosome 17 and thus included in the conserved synteny group. During the characterization of transcripts from the human PKD1 region on human chromosome 16p13.3, we isolated three short clones encoding fragments of TSC2 from a human fetal brain cDNA library enriched for transcripts from the PKD1 region. These TSC2 clones were used as probes to screen a mouse teratocarcinoma (PCC4) cDNA library (Stratagene), at a final stringency of 0.3 x SSC, 0.1% SDS at 65{degrees}C. One of the positive clones isolated, mTS-1, had a 2.8-kb insert. Two hundred bases from each end of the insert were sequenced, showing 88 and 83.5% identity to the human tuberin nucleotide sequence, with the 5{prime} end of the clone starting at position 2351 and the 3{prime} end ending at position 5265. The high degree of homology to the human tuberin sequence suggests that clone mTS-1 is indeed derived from the mouse homologue of TSC2. 11 refs., 1 fig.

  6. Individual Education.

    ERIC Educational Resources Information Center

    Corsini, Raymond

    1981-01-01

    Paper presented at the 66th Convention of the International Association of Pupil Personnel Workers, October 20, 1980, Baltimore, Maryland, describes individual education based on the principles of Alfred Adler. Defines six advantages of individual education, emphasizing student responsibility, mutual respect, and allowing students to progress at…

  7. Integration of Mouse Phenome Data Resources

    SciTech Connect

    Hancock, John M; Adams, Neils; Aidinis, Vassilis; Blake, Judith A; Bogue, Molly; Brown, Steve D M; Chesler, Elissa J; Davidson, Duncan; Duran, Christopher; Eppig, Janan T; Gailus-Durner, Valerie; Gkoutos, Georgios V; Greenaway, Simon; Angelis, Martin Hrabe de; Kollias, George; Leblanc, Sophie; Lee, Kirsty; Lengger, Christoph; Maier, Holger; Mallon, Ann-Marie; Masuya, Hiroshi; Melvin, David; Muller, Werner; Parkinson, Helen; Proctor, Glenn; Reuveni, Eli; Schofield, Paul; Shukla, Aadya; Smith, Cynthia; Toyoda, Tetsuro; Vasseur, Laurent; Wakana, Shigeharu; Walling, Alison; White, Jacqui; Wood, Joe; Zouberakis, Michalis

    2008-01-01

    Understanding the functions encoded in the mouse genome will be central to an understanding of the genetic basis of human disease. To achieve this it will be essential to be able to characterise the phenotypic consequences of variation and alterations in individual genes. Data on the phenotypes of mouse strains are currently held in a number of different forms (detailed descriptions of mouse lines, first line phenotyping data on novel mutations, data on the normal features of inbred lines, etc.) at many sites worldwide. For the most efficient use of these data sets, we have set in train a process to develop standards for the description of phenotypes (using ontologies), and file formats for the description of phenotyping protocols and phenotype data sets. This process is ongoing, and needs to be supported by the wider mouse genetics and phenotyping communities to succeed. We invite interested parties to contact us as we develop this process further.

  8. Molecular cloning, purification and immunogenicity of recombinant Brucella abortus 544 malate dehydrogenase protein.

    PubMed

    Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon; Kim, Suk

    2016-03-01

    The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis.

  9. Molecular cloning, purification and immunogenicity of recombinant Brucella abortus 544 malate dehydrogenase protein

    PubMed Central

    Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon

    2016-01-01

    The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis. PMID:27051349

  10. Mammalian cloning: possibilities and threats.

    PubMed

    Mitalipov, S M; Wolf, D P

    2000-10-01

    The cloning of mammals originated with the production of limited numbers of genetically identical offspring by blastomere separation or embryo splitting. In the past few years, remarkable progress has been reported in cloning by nuclear transfer (NT) with donor nuclei recovered from embryonic, fetal or adult cells. Factors that contribute to the successful reprogramming of the transferred nucleus and the normal term development of the newly reconstructed embryo include the cell cycle stage of both the donor nucleus and recipient cytoplast, the timing of fusion and cytoplast activation, and the source of donor nuclei. The possibility of producing live offspring by somatic cell NT carries potential applications in animal husbandry, biotechnology, transgenic and pharmaceutical production, biomedical research, and the preservation of endangered species. However, the low efficiencies of cloning by NT coupled with high embryonic, fetal and neonatal losses may restrict immediate commercial applications in agriculture. These limitations notwithstanding, the greatest benefits and practical implications of this new technology could be in transplantation medicine and therapeutic cloning.

  11. Clone Poems and the Microcomputer.

    ERIC Educational Resources Information Center

    Irizarry, Estelle

    1989-01-01

    Describes how students can use the computer to study and create clone poems (altering original Spanish-language poems by substituting words and expressions), and how students can gain a deeper appreciation of the original poem's poetic structure and semantics. (CB)

  12. Healthy ageing of cloned sheep

    PubMed Central

    Sinclair, K. D.; Corr, S. A.; Gutierrez, C. G.; Fisher, P. A.; Lee, J.-H.; Rathbone, A. J.; Choi, I.; Campbell, K. H. S.; Gardner, D. S.

    2016-01-01

    The health of cloned animals generated by somatic-cell nuclear transfer (SCNT) has been of concern since its inception; however, there are no detailed assessments of late-onset, non-communicable diseases. Here we report that SCNT has no obvious detrimental long-term health effects in a cohort of 13 cloned sheep. We perform musculoskeletal assessments, metabolic tests and blood pressure measurements in 13 aged (7–9 years old) cloned sheep, including four derived from the cell line that gave rise to Dolly. We also perform radiological examinations of all main joints, including the knees, the joint most affected by osteoarthritis in Dolly, and compare all health parameters to groups of 5-and 6-year-old sheep, and published reference ranges. Despite their advanced age, these clones are euglycaemic, insulin sensitive and normotensive. Importantly, we observe no clinical signs of degenerative joint disease apart from mild, or in one case moderate, osteoarthritis in some animals. Our study is the first to assess the long-term health outcomes of SCNT in large animals. PMID:27459299

  13. Healthy ageing of cloned sheep.

    PubMed

    Sinclair, K D; Corr, S A; Gutierrez, C G; Fisher, P A; Lee, J-H; Rathbone, A J; Choi, I; Campbell, K H S; Gardner, D S

    2016-01-01

    The health of cloned animals generated by somatic-cell nuclear transfer (SCNT) has been of concern since its inception; however, there are no detailed assessments of late-onset, non-communicable diseases. Here we report that SCNT has no obvious detrimental long-term health effects in a cohort of 13 cloned sheep. We perform musculoskeletal assessments, metabolic tests and blood pressure measurements in 13 aged (7-9 years old) cloned sheep, including four derived from the cell line that gave rise to Dolly. We also perform radiological examinations of all main joints, including the knees, the joint most affected by osteoarthritis in Dolly, and compare all health parameters to groups of 5-and 6-year-old sheep, and published reference ranges. Despite their advanced age, these clones are euglycaemic, insulin sensitive and normotensive. Importantly, we observe no clinical signs of degenerative joint disease apart from mild, or in one case moderate, osteoarthritis in some animals. Our study is the first to assess the long-term health outcomes of SCNT in large animals.

  14. Healthy ageing of cloned sheep.

    PubMed

    Sinclair, K D; Corr, S A; Gutierrez, C G; Fisher, P A; Lee, J-H; Rathbone, A J; Choi, I; Campbell, K H S; Gardner, D S

    2016-01-01

    The health of cloned animals generated by somatic-cell nuclear transfer (SCNT) has been of concern since its inception; however, there are no detailed assessments of late-onset, non-communicable diseases. Here we report that SCNT has no obvious detrimental long-term health effects in a cohort of 13 cloned sheep. We perform musculoskeletal assessments, metabolic tests and blood pressure measurements in 13 aged (7-9 years old) cloned sheep, including four derived from the cell line that gave rise to Dolly. We also perform radiological examinations of all main joints, including the knees, the joint most affected by osteoarthritis in Dolly, and compare all health parameters to groups of 5-and 6-year-old sheep, and published reference ranges. Despite their advanced age, these clones are euglycaemic, insulin sensitive and normotensive. Importantly, we observe no clinical signs of degenerative joint disease apart from mild, or in one case moderate, osteoarthritis in some animals. Our study is the first to assess the long-term health outcomes of SCNT in large animals. PMID:27459299

  15. Energy values of nine Populus clones

    SciTech Connect

    Strong, T.F.

    1980-01-01

    This paper compares calorific values for components of nine Populus clones. The components include stem wood, stem bark, and branches. Also compared are calorific values for clones of balsam poplar and black cottonwood parentages.

  16. Race quickens for the first human clone.

    PubMed

    Gross, M

    2001-04-01

    The dazzling creation of Dolly, the cloned sheep, led many states to legislate against the possibility of using similar technology to create human clones. But for some, this prize is proving too tempting to ignore. Michael Gross reports. PMID:11413008

  17. Computational analysis of small RNA cloning data.

    PubMed

    Berninger, Philipp; Gaidatzis, Dimos; van Nimwegen, Erik; Zavolan, Mihaela

    2008-01-01

    Cloning and sequencing is the method of choice for small regulatory RNA identification. Using deep sequencing technologies one can now obtain up to a billion nucleotides--and tens of millions of small RNAs--from a single library. Careful computational analyses of such libraries enabled the discovery of miRNAs, rasiRNAs, piRNAs, and 21U RNAs. Given the large number of sequences that can be obtained from each individual sample, deep sequencing may soon become an alternative to oligonucleotide microarray technology for mRNA expression profiling. In this report we present the methods that we developed for the annotation and expression profiling of small RNAs obtained through large-scale sequencing. These include a fast algorithm for finding nearly perfect matches of small RNAs in sequence databases, a web-accessible software system for the annotation of small RNA libraries, and a Bayesian method for comparing small RNA expression across samples.

  18. 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.

  19. An Infectious cDNA Clone of Zika Virus to Study Viral Virulence, Mosquito Transmission, and Antiviral Inhibitors.

    PubMed

    Shan, Chao; Xie, Xuping; Muruato, Antonio E; Rossi, Shannan L; Roundy, Christopher M; Azar, Sasha R; Yang, Yujiao; Tesh, Robert B; Bourne, Nigel; Barrett, Alan D; Vasilakis, Nikos; Weaver, Scott C; Shi, Pei-Yong

    2016-06-01

    The Asian lineage of Zika virus (ZIKV) has recently caused epidemics and severe disease. Unraveling the mechanisms causing increased viral transmissibility and disease severity requires experimental systems. We report an infectious cDNA clone of ZIKV that was generated using a clinical isolate of the Asian lineage. The cDNA clone-derived RNA is infectious in cells, generating recombinant ZIKV. The recombinant virus is virulent in established ZIKV mouse models, leading to neurological signs relevant to human disease. Additionally, recombinant ZIKV is infectious for Aedes aegypti and thus provides a means to examine virus transmission. The infectious cDNA clone was further used to generate a luciferase ZIKV that exhibited sensitivity to a panflavivirus inhibitor, highlighting its potential utility for antiviral screening. This ZIKV reverse genetic system, together with mouse and mosquito infection models, may help identify viral determinants of human virulence and mosquito transmission as well as inform vaccine and therapeutic strategies. PMID:27198478

  20. An Infectious cDNA Clone of Zika Virus to Study Viral Virulence, Mosquito Transmission, and Antiviral Inhibitors.

    PubMed

    Shan, Chao; Xie, Xuping; Muruato, Antonio E; Rossi, Shannan L; Roundy, Christopher M; Azar, Sasha R; Yang, Yujiao; Tesh, Robert B; Bourne, Nigel; Barrett, Alan D; Vasilakis, Nikos; Weaver, Scott C; Shi, Pei-Yong

    2016-06-01

    The Asian lineage of Zika virus (ZIKV) has recently caused epidemics and severe disease. Unraveling the mechanisms causing increased viral transmissibility and disease severity requires experimental systems. We report an infectious cDNA clone of ZIKV that was generated using a clinical isolate of the Asian lineage. The cDNA clone-derived RNA is infectious in cells, generating recombinant ZIKV. The recombinant virus is virulent in established ZIKV mouse models, leading to neurological signs relevant to human disease. Additionally, recombinant ZIKV is infectious for Aedes aegypti and thus provides a means to examine virus transmission. The infectious cDNA clone was further used to generate a luciferase ZIKV that exhibited sensitivity to a panflavivirus inhibitor, highlighting its potential utility for antiviral screening. This ZIKV reverse genetic system, together with mouse and mosquito infection models, may help identify viral determinants of human virulence and mosquito transmission as well as inform vaccine and therapeutic strategies.

  1. Cytochrome P450 1b1 in polycyclic aromatic hydrocarbon (PAH)-induced skin carcinogenesis: Tumorigenicity of individual PAHs and coal-tar extract, DNA adduction and expression of select genes in the Cyp1b1 knockout mouse.

    PubMed

    Siddens, Lisbeth K; Bunde, Kristi L; Harper, Tod A; McQuistan, Tammie J; Löhr, Christiane V; Bramer, Lisa M; Waters, Katrina M; Tilton, Susan C; Krueger, Sharon K; Williams, David E; Baird, William M

    2015-09-01

    FVB/N mice wild-type, heterozygous or null for Cyp 1b1 were used in a two-stage skin tumor study comparing PAH, benzo[a]pyrene (BaP), dibenzo[def,p]chrysene (DBC), and coal tar extract (CTE, SRM 1597a). Following 20 weeks of promotion with TPA the Cyp 1b1 null mice, initiated with DBC, exhibited reductions in incidence, multiplicity, and progression. None of these effects were observed with BaP or CTE. The mechanism of Cyp 1b1-dependent alteration of DBC skin carcinogenesis was further investigated by determining expression of select genes in skin from DBC-treated mice 2, 4 and 8h post-initiation. A significant reduction in levels of Cyp 1a1, Nqo1 at 8h and Akr 1c14 mRNA was observed in Cyp 1b1 null (but not wt or het) mice, whereas no impact was observed in Gst a1, Nqo 1 at 2 and 4h or Akr 1c19 at any time point. Cyp 1b1 mRNA was not elevated by DBC. The major covalent DNA adducts, dibenzo[def,p]chrysene-(±)-11,12-dihydrodiol-cis and trans-13,14-epoxide-deoxyadenosine (DBCDE-dA) were quantified by UHPLC-MS/MS 8h post-initiation. Loss of Cyp1 b1 expression reduced DBCDE-dA adducts in the skin but not to a statistically significant degree. The ratio of cis- to trans-DBCDE-dA adducts was higher in the skin than other target tissues such as the spleen, lung and liver (oral dosing). These results document that Cyp 1b1 plays a significant role in bioactivation and carcinogenesis of DBC in a two-stage mouse skin tumor model and that loss of Cyp 1b1 has little impact on tumor response with BaP or CTE as initiators. PMID:26049101

  2. Probabilistic cloning of three symmetric states

    SciTech Connect

    Jimenez, O.; Bergou, J.; Delgado, A.

    2010-12-15

    We study the probabilistic cloning of three symmetric states. These states are defined by a single complex quantity, the inner product among them. We show that three different probabilistic cloning machines are necessary to optimally clone all possible families of three symmetric states. We also show that the optimal cloning probability of generating M copies out of one original can be cast as the quotient between the success probability of unambiguously discriminating one and M copies of symmetric states.

  3. Phase-covariant quantum cloning of qudits

    SciTech Connect

    Fan Heng; Imai, Hiroshi; Matsumoto, Keiji; Wang, Xiang-Bin

    2003-02-01

    We study the phase-covariant quantum cloning machine for qudits, i.e., the input states in a d-level quantum system have complex coefficients with arbitrary phase but constant module. A cloning unitary transformation is proposed. After optimizing the fidelity between input state and single qudit reduced density operator of output state, we obtain the optimal fidelity for 1 to 2 phase-covariant quantum cloning of qudits and the corresponding cloning transformation.

  4. [Cloning: reproductive medicine or breeding program?].

    PubMed

    Zülicke, F

    1998-01-01

    The presentation of clone-sheep Dolly in February 1997 which was the result of a long and costly research process by Ian Wilmut's team at Roslin Institute near Edinburgh brought world-wide headlines and a continuous debate. But neither cloning and cloning experiments nor the debates about it and the possible application on humans are as new as it is shown in the media. The following article gives some facts and arguments to the field of cloning.

  5. Mouse BAC Ends Quality Assessment and Sequence Analyses

    PubMed Central

    Zhao, Shaying; Shatsman, Sofiya; Ayodeji, Bola; Geer, Keita; Tsegaye, Getahun; Krol, Margaret; Gebregeorgis, Elizabeth; Shvartsbeyn, Alla; Russell, Daniel; Overton, Larry; Jiang, Lingxia; Dimitrov, George; Tran, Kevin; Shetty, Jyoti; Malek, Joel A.; Feldblyum, Tamara; Nierman, William C.; Fraser, Claire M.

    2001-01-01

    A large-scale BAC end-sequencing project at The Institute for Genomic Research (TIGR) has generated one of the most extensive sets of sequence markers for the mouse genome to date. With a sequencing success rate of >80%, an average read length of 485 bp, and ABI3700 capillary sequencers, we have generated 449,234 nonredundant mouse BAC end sequences (mBESs) with 218 Mb total from 257,318 clones from libraries RPCI-23 and RPCI-24, representing 15× clone coverage, 7% sequence coverage, and a marker every 7 kb across the genome. A total of 191,916 BACs have sequences from both ends providing 12× genome coverage. The average Q20 length is 406 bp and 84% of the bases have phred quality scores ≥ 20. RPCI-24 mBESs have more Q20 bases and longer reads on average than RPCI-23 sequences. ABI3700 sequencers and the sample tracking system ensure that > 95% of mBESs are associated with the right clone identifiers. We have found that a significant fraction of mBESs contains L1 repeats and ∼48% of the clones have both ends with ≥ 100 bp contiguous unique Q20 bases. About 3% mBESs match ESTs and > 70% of matches were conserved between the mouse and the human or the rat. Approximately 0.1% mBESs contain STSs. About 0.2% mBESs match human finished sequences and > 70% of these sequences have EST hits. The analyses indicate that our high-quality mouse BAC end sequences will be a valuable resource to the community. PMID:11591651

  6. Economical phase-covariant cloning of qudits

    SciTech Connect

    Buscemi, Francesco; D'Ariano, Giacomo Mauro; Macchiavello, Chiara

    2005-04-01

    We derive the optimal N{yields}M phase-covariant quantum cloning for equatorial states in dimension d with M=kd+N, k integer. The cloning maps are optimal for both global and single-qudit fidelity. The map is achieved by an 'economical' cloning machine, which works without ancilla.

  7. Local cloning of arbitrarily entangled multipartite states

    SciTech Connect

    Kay, Alastair; Ericsson, Marie

    2006-01-15

    We examine the perfect cloning of nonlocal, orthogonal states using only local operations and classical communication. We provide a complete characterisation of the states that can be cloned under these restrictions, and their relation to distinguishability. We also consider the case of catalytic cloning, which we show provides no enhancement to the set of clonable states.

  8. Resolution of murine chlamydial genital infection by the adoptive transfer of a biovar-specific, Th1 lymphocyte clone.

    PubMed

    Igietseme, J U; Ramsey, K H; Magee, D M; Williams, D M; Kincy, T J; Rank, R G

    1993-01-01

    MoPn-specific T-cell clones were isolated from a T-cell line that was capable of curing chlamydial genital infection by the Chlamydia trachomatis agent of mouse pneumonitis (MoPn) after adoptive transfer. Two clones (designated as 2.14-0 and 2.14-3) were characterized by flow cytometry techniques to be homogenous for L3T4, CD3, and alpha/beta T cell receptor (TcR) T-helper cell markers. The two clones were biovar specific, because they reacted to MoPn but not the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis (GPIC) or C. trachomatis, serovar type E. Cytokine profile analysis, by a combination of bioassays, ELISA, and slot/Northern blotting for specific cytokine messenger RNAs, further revealed that cultures of antigen-stimulated clone 2.14-0 contained interleukin-2 (IL-2), tumor necrosis factor-alpha, and gamma interferon (a T helper 1 cell [Th1] profile). Clone 2.14-3 was also positive for gamma interferon, a level much lower than that of clone 2.14-0, and negative for IL-4 secretion, suggesting a Th1 profile as well. The ability of these clones to bring about the resolution of the chronic genital chlamydial infection of nude mice was tested by the adoptive transfer of 10(7) cells of each clone into the mice. By 4 weeks after cell transfer of clone 2.14-0, 81% of recipient nude mice (30 of 37) resolved the disease. In contrast, clone 2.14-3 or a control T-cell clone specific for a heterologous antigen were unable to resolve the infection in 20 recipients in each case, even after 100 days.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Subtractive cloning: new genes for studying inflammatory disorders.

    PubMed

    Rebrikov, Denis; Desai, Sejal; Kogan, Yakov N; Thornton, Angela M; Diatchenko, Luda

    2002-12-01

    Understanding of the biology of interaction between pathogens and host is the central question in studying inflammatory disorders. Subtractive DNA cloning is one of the most efficient and comprehensive methods available for identifying eukaryotic genes regulated under specific physiological conditions, including inflammation and host response. Here we explore the utility of subtractive DNA cloning and describe suppression subtractive hybridization (SSH), a polymerase chain reaction (PCR)-based DNA subtraction method that has been developed and evolved in our labs over several years. The SSH method possesses a number of advantages as compared to other subtractive cloning techniques, making it one of the most adventitious methods for cloning differentially expressed genes. Besides isolation of differentially expressed eukaryotic mRNAs, subtractive DNA cloning can be used to identify genes that are differentially expressed between diverse bacterial species. These genes can be of great interest, as some may encode strain-specific traits such as drug resistance, or bacterial surface proteins involved in determining the virulence of a particular strain. Other genes may be useful as markers for epidemiological or evolutionary studies. To demonstrate the potential of the SSH technique, we describe here the comprehensive characterization of 2 SSH subtracted libraries constructed in our laboratories. One library was created using eukaryotic cDNA subtraction and is specific for mRNAs up-regulated in CD25 positive cells from mouse lymph nodes as compared to CD25 negative cells. The second subtracted library is specific for a methicillin-resistant Staphylococcus aureus bacterial strain, but not in a methicillin-sensitive strain. The bacterial genomes of these 2 strains have been completely sequenced and this second library provides an excellent reference for testing the ability of SSH to recover all strain-specific gene content. The analysis of these 2 subtracted libraries

  10. Cloning humans, cloning literature: genetics and the imagination deficit.

    PubMed

    Van Dijck, J

    1999-01-01

    After the birth of Dolly, media stories on cloning were replete with references to well-known science fiction plots. This essay criticizes the 'imagination deficit' of scientists and journalists, first by problematizing the uncritical adoption of attentuated science fiction plots in the media coverage of Dolly, and second, by proposing to look at more expansive science fiction novels that carefully examine issues such as uniqueness and identity in relation to the new genetics.

  11. Whole-mount X-Gal staining of mouse tissues.

    PubMed

    Gierut, Jessica J; Jacks, Tyler E; Haigis, Kevin M

    2014-04-01

    Although the development of improved mouse models, including conditional deletions, marks an exciting time in mouse genetics, it is important to characterize and validate these models. Cre reporter strains allow researchers to assess the recombinase expression profile and function in individual Cre mouse lines. These strains are engineered to express a reporter gene (usually LacZ) following the removal of a floxed STOP cassette, thus marking cell lineages that can be targeted with a given Cre line. This protocol provides a detailed method for the histochemical detection of β-galactosidase activity in Cre mouse strains.

  12. Noncytotoxic T cell clones obtained from a human mixed leukocyte culture.

    PubMed

    Chu, M H; Wee, S L; Bach, F H

    1990-02-01

    Peripheral blood mononuclear cells from a DQW-1 homozygous individual were cocultured with irradiated lymphoblastoid cell line from a DQW-1 homozygous unrelated donor bearing BW35-DW1 haplotype. From T cell cloning of primary and twice-stimulated mixed leukocyte cultures (MLC), 7 and 11 T cell clones were obtained respectively. None of the 18 clones showed specific cytotoxic activity against the alloantigen of the stimulator cell as well as natural killer (NK)-like activity against K562 cells. However, most T cell clones from both primary and re-stimulated MLC demonstrated moderate cytotoxic activity in lectin-dependent cell-mediated cytolysis (LDCC) assay. Screening assay for cell-mediated lympholysis (CML) performed on growing microcultures obtained from restimulated MLC cloning confirmed the non-cytotoxic status of these T cell clones by showing that 41 out of 44 growing microcultures were not cytotoxic against the stimulator cell; the other 3 clones lyzed the target cell mildly. The cells from all 5 T cell clones detected for indirect fluorescence expressed CD3 and CD4 surface markers. Taken together, the results suggested that proliferation-regulating T cell subsets or factor(s) may be generated during the course of MLCs under the present responder-stimulator combination, and may suppress the development of alloreactive cytotoxic T cells and NK-like cells. PMID:2144231

  13. Predators induce cloning in echinoderm larvae.

    PubMed

    Vaughn, Dawn; Strathmann, Richard R

    2008-03-14

    Asexual propagation (cloning) is a widespread reproductive strategy of plants and animals. Although larval cloning is well documented in echinoderms, identified stimuli for cloning are limited to those associated with conditions favorable for growth and reproduction. Our research shows that larvae of the sand dollar Dendraster excentricus also clone in response to cues from predators. Predator-induced clones were smaller than uncloned larvae, suggesting an advantage against visual predators. Our results offer another ecological context for asexual reproduction: rapid size reduction as a defense.

  14. Probabilistic cloning of three nonorthogonal states

    NASA Astrophysics Data System (ADS)

    Zhang, Wen; Rui, Pinshu; Yang, Qun; Zhao, Yan; Zhang, Ziyun

    2015-04-01

    We study the probabilistic cloning of three nonorthogonal states with equal success probabilities. For simplicity, we assume that the three states belong to a special set. Analytical form of the maximal success probability for probabilistic cloning is calculated. With the maximal success probability, we deduce the explicit form of probabilistic quantum cloning machine. In the case of cloning, we get the unambiguous form of the unitary operation. It is demonstrated that the upper bound for probabilistic quantum cloning machine in (Qiu in J Phys A 35:6931, 2002) can be reached only if the three states are equidistant.

  15. Optimal quantum cloning via spin networks

    SciTech Connect

    Chen Qing; Cheng Jianhua; Wang Kelin; Du Jiangfeng

    2006-09-15

    In this paper we demonstrate that optimal 1{yields}M phase-covariant cloning quantum cloning is available via free dynamical evolution of spin networks. By properly designing the network and the couplings between spins, we show that optimal 1{yields}M phase-covariant cloning can be achieved if the initial state is prepared as a specific symmetric state. Especially, when M is an odd number, the optimal phase-covariant cloning can be achieved without ancillas. Moreover, we demonstrate that the same framework is capable for optimal 1{yields}2 universal cloning.

  16. No-cloning theorem on quantum logics

    SciTech Connect

    Miyadera, Takayuki; Imai, Hideki

    2009-10-15

    This paper discusses the no-cloning theorem in a logicoalgebraic approach. In this approach, an orthoalgebra is considered as a general structure for propositions in a physical theory. We proved that an orthoalgebra admits cloning operation if and only if it is a Boolean algebra. That is, only classical theory admits the cloning of states. If unsharp propositions are to be included in the theory, then a notion of effect algebra is considered. We proved that an atomic Archimedean effect algebra admitting cloning operation is a Boolean algebra. This paper also presents a partial result, indicating a relation between the cloning on effect algebras and hidden variables.

  17. Therapeutic and reproductive cloning: a critique.

    PubMed

    Bowring, Finn

    2004-01-01

    This article is a critical examination of the science and ethics of human cloning. It summarises the key scientific milestones in the development of nuclear transplantation, explains the importance of cloning to research into the medical potential of embryonic stem cells, and discusses the well-worn distinction between 'therapeutic' and 'reproductive' cloning. Suggesting that this distinction will be impossible to police, it goes on to consider the ethics of full human cloning. It is concluded that it represents an unacceptable form of parental despotism, and that the genetic engineering and cloning of future human beings will fracture the foundations of modern humanism.

  18. Individualizing Medicare.

    PubMed

    Chollet, D J

    1999-05-01

    Despite the enactment of significant changes to the Medicare program in 1997, Medicare's Hospital Insurance trust fund is projected to be exhausted just as the baby boom enters retirement. To address Medicare's financial difficulties, a number of reform proposals have been offered, including several to individualize Medicare financing and benefits. These proposals would attempt to increase Medicare revenues and reduce Medicare expenditures by having individuals bear risk--investment market risk before retirement and insurance market risk after retirement. Many fundamental aspects of these proposals have yet to be worked out, including how to guarantee a baseline level of saving for health insurance after retirement, how retirees might finance unanticipated health insurance price increases after retirement, the potential implications for Medicaid of inadequate individual saving, and whether the administrative cost of making the system fair and adequate ultimately would eliminate any rate-of-return advantages from allowing workers to invest their Medicare contributions in corporate stocks and bonds.

  19. Individualizing Medicare.

    PubMed

    Chollet, D J

    1999-05-01

    Despite the enactment of significant changes to the Medicare program in 1997, Medicare's Hospital Insurance trust fund is projected to be exhausted just as the baby boom enters retirement. To address Medicare's financial difficulties, a number of reform proposals have been offered, including several to individualize Medicare financing and benefits. These proposals would attempt to increase Medicare revenues and reduce Medicare expenditures by having individuals bear risk--investment market risk before retirement and insurance market risk after retirement. Many fundamental aspects of these proposals have yet to be worked out, including how to guarantee a baseline level of saving for health insurance after retirement, how retirees might finance unanticipated health insurance price increases after retirement, the potential implications for Medicaid of inadequate individual saving, and whether the administrative cost of making the system fair and adequate ultimately would eliminate any rate-of-return advantages from allowing workers to invest their Medicare contributions in corporate stocks and bonds. PMID:10915458

  20. Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

    SciTech Connect

    Deymier, Martin J.; Claiborne, Daniel T.; Ende, Zachary; Ratner, Hannah K.; Kilembe, William; Hunter, Eric

    2014-11-15

    The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens. - Highlights: • Our novel methodology demonstrates accurate amplification and cloning of full-length HIV-1 genomes. • A majority of plasma derived HIV variants from a chronically infected individual are infectious. • The transmitted/founder was more infectious than the majority of the variants from the chronically infected donor.

  1. Chromatin remodeling in nuclear cloning.

    PubMed

    Wade, Paul A; Kikyo, Nobuaki

    2002-05-01

    Nuclear cloning is a procedure to create new animals by injecting somatic nuclei into unfertilized oocytes. Recent successes in mammalian cloning with differentiated adult nuclei strongly indicate that oocyte cytoplasm contains unidentified remarkable reprogramming activities with the capacity to erase the previous memory of cell differentiation. At the heart of this nuclear reprogramming lies chromatin remodeling as chromatin structure and function define cell differentiation through regulation of the transcriptional activities of the cells. Studies involving the modification of chromatin elements such as selective uptake or release of binding proteins, covalent histone modifications including acetylation and methylation, and DNA methylation should provide significant insight into the molecular mechanisms of nuclear dedifferentiation and redifferentiation in oocyte cytoplasm.

  2. An encyclopedia of mouse DNA elements (Mouse ENCODE).

    PubMed

    Stamatoyannopoulos, John A; Snyder, Michael; Hardison, Ross; Ren, Bing; Gingeras, Thomas; Gilbert, David M; Groudine, Mark; Bender, Michael; Kaul, Rajinder; Canfield, Theresa; Giste, Erica; Johnson, Audra; Zhang, Mia; Balasundaram, Gayathri; Byron, Rachel; Roach, Vaughan; Sabo, Peter J; Sandstrom, Richard; Stehling, A Sandra; Thurman, Robert E; Weissman, Sherman M; Cayting, Philip; Hariharan, Manoj; Lian, Jin; Cheng, Yong; Landt, Stephen G; Ma, Zhihai; Wold, Barbara J; Dekker, Job; Crawford, Gregory E; Keller, Cheryl A; Wu, Weisheng; Morrissey, Christopher; Kumar, Swathi A; Mishra, Tejaswini; Jain, Deepti; Byrska-Bishop, Marta; Blankenberg, Daniel; Lajoie, Bryan R; Jain, Gaurav; Sanyal, Amartya; Chen, Kaun-Bei; Denas, Olgert; Taylor, James; Blobel, Gerd A; Weiss, Mitchell J; Pimkin, Max; Deng, Wulan; Marinov, Georgi K; Williams, Brian A; Fisher-Aylor, Katherine I; Desalvo, Gilberto; Kiralusha, Anthony; Trout, Diane; Amrhein, Henry; Mortazavi, Ali; Edsall, Lee; McCleary, David; Kuan, Samantha; Shen, Yin; Yue, Feng; Ye, Zhen; Davis, Carrie A; Zaleski, Chris; Jha, Sonali; Xue, Chenghai; Dobin, Alex; Lin, Wei; Fastuca, Meagan; Wang, Huaien; Guigo, Roderic; Djebali, Sarah; Lagarde, Julien; Ryba, Tyrone; Sasaki, Takayo; Malladi, Venkat S; Cline, Melissa S; Kirkup, Vanessa M; Learned, Katrina; Rosenbloom, Kate R; Kent, W James; Feingold, Elise A; Good, Peter J; Pazin, Michael; Lowdon, Rebecca F; Adams, Leslie B

    2012-08-13

    To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

  3. Cloning Expeditions: Risky but Rewarding

    PubMed Central

    2013-01-01

    In the 1980s, a good part of my laboratory was using the then-new recombinant DNA techniques to clone and characterize many important cell surface membrane proteins: GLUT1 (the red cell glucose transporter) and then GLUT2 and GLUT4, the red cell anion exchange protein (Band 3), asialoglycoprotein receptor subunits, sucrase-isomaltase, the erythropoietin receptor, and two of the subunits of the transforming growth factor β (TGF-β) receptor. These cloned genes opened many new fields of basic research, including membrane insertion and trafficking of transmembrane proteins, signal transduction by many members of the cytokine and TGF-β families of receptors, and the cellular physiology of glucose and anion transport. They also led to many insights into the molecular biology of several cancers, hematopoietic disorders, and diabetes. This work was done by an exceptional group of postdocs and students who took exceptionally large risks in developing and using novel cloning technologies. Unsurprisingly, all have gone on to become leaders in the fields of molecular cell biology and molecular medicine. PMID:24061478

  4. Cloning expeditions: risky but rewarding.

    PubMed

    Lodish, Harvey

    2013-12-01

    In the 1980s, a good part of my laboratory was using the then-new recombinant DNA techniques to clone and characterize many important cell surface membrane proteins: GLUT1 (the red cell glucose transporter) and then GLUT2 and GLUT4, the red cell anion exchange protein (Band 3), asialoglycoprotein receptor subunits, sucrase-isomaltase, the erythropoietin receptor, and two of the subunits of the transforming growth factor β (TGF-β) receptor. These cloned genes opened many new fields of basic research, including membrane insertion and trafficking of transmembrane proteins, signal transduction by many members of the cytokine and TGF-β families of receptors, and the cellular physiology of glucose and anion transport. They also led to many insights into the molecular biology of several cancers, hematopoietic disorders, and diabetes. This work was done by an exceptional group of postdocs and students who took exceptionally large risks in developing and using novel cloning technologies. Unsurprisingly, all have gone on to become leaders in the fields of molecular cell biology and molecular medicine.

  5. [Individualizing Education.

    ERIC Educational Resources Information Center

    Horrigan, William J.

    The individually guided education (IGE) program developed by the Kettering Foundation was implemented in September of 1973 at the John F. Kennedy Memorial Junior High School in Woburn, Massachusetts. The components of the program described in this speech include pupil and teacher scheduling, physical layout, pupil selection and adjustment,…

  6. Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus.

    PubMed Central

    Miller, R D; Hogg, J; Ozaki, J H; Gell, D; Jackson, S P; Riblet, R

    1995-01-01

    The gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) has been proposed recently as a candidate gene for the mouse severe combined immune deficiency (scid) locus. We have used a partial cDNA clone for human DNA-PKcs to map the mouse homologue using a large interspecific backcross panel. We found that the mouse gene for DNA-PKcs does not recombine with scid, consistent with the hypothesis that scid is a mutation in the mouse gene for DNA-PKcs. Images Fig. 3 PMID:7479885

  7. Sequence, molecular properties, and chromosomal mapping of mouse lumican

    NASA Technical Reports Server (NTRS)

    Funderburgh, J. L.; Funderburgh, M. L.; Hevelone, N. D.; Stech, M. E.; Justice, M. J.; Liu, C. Y.; Kao, W. W.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    PURPOSE. Lumican is a major proteoglycan of vertebrate cornea. This study characterizes mouse lumican, its molecular form, cDNA sequence, and chromosomal localization. METHODS. Lumican sequence was determined from cDNA clones selected from a mouse corneal cDNA expression library using a bovine lumican cDNA probe. Tissue expression and size of lumican mRNA were determined using Northern hybridization. Glycosidase digestion followed by Western blot analysis provided characterization of molecular properties of purified mouse corneal lumican. Chromosomal mapping of the lumican gene (Lcn) used Southern hybridization of a panel of genomic DNAs from an interspecific murine backcross. RESULTS. Mouse lumican is a 338-amino acid protein with high-sequence identity to bovine and chicken lumican proteins. The N-terminus of the lumican protein contains consensus sequences for tyrosine sulfation. A 1.9-kb lumican mRNA is present in cornea and several other tissues. Antibody against bovine lumican reacted with recombinant mouse lumican expressed in Escherichia coli and also detected high molecular weight proteoglycans in extracts of mouse cornea. Keratanase digestion of corneal proteoglycans released lumican protein, demonstrating the presence of sulfated keratan sulfate chains on mouse corneal lumican in vivo. The lumican gene (Lcn) was mapped to the distal region of mouse chromosome 10. The Lcn map site is in the region of a previously identified developmental mutant, eye blebs, affecting corneal morphology. CONCLUSIONS. This study demonstrates sulfated keratan sulfate proteoglycan in mouse cornea and describes the tools (antibodies and cDNA) necessary to investigate the functional role of this important corneal molecule using naturally occurring and induced mutants of the murine lumican gene.

  8. Molecular cloning and analysis of the Catsper1 gene promoter.

    PubMed

    Mata-Rocha, Minerva; Alvarado-Cuevas, Edith; Hernández-Sánchez, Javier; Cerecedo, Doris; Felix, Ricardo; Hernández-Reyes, Adriana; Tesoro-Cruz, Emiliano; Oviedo, Norma

    2013-05-01

    CatSper channels are essential for hyperactivity of sperm flagellum, progesterone-mediated chemotaxis and oocyte fertilization. Catsper genes are exclusively expressed in the testis during spermatogenesis, but the function and regulation of the corresponding promoter regions are unknown. Here, we report the cloning and characterization of the promoter regions in the human and murine Catsper1 genes. These promoter regions were identified and isolated from genomic DNA, and transcriptional activities were tested in vitro after transfection into human embryonic kidney 293, mouse Sertoli cells 1 and GC-1spg cell lines as well as by injecting plasmids directly into mouse testes. Although the human and murine Catsper1 promoters lacked a TATA box, a well-conserved CRE site was identified. Both sequences may be considered as TATAless promoters because their transcriptional activity was not affected after deletion of TATA box-like sites. Several transcription initiation sites were revealed by RNA ligase-mediated rapid amplification of the cDNA 5'-ends. We also found that the immediate upstream region and the first exon in the human CATSPER1 gene negatively regulate transcriptional activity. In the murine Catsper1 promoter, binding sites for transcription factors SRY, SOX9 and CREB were protected by the presence of nuclear testis proteins in DNAse degradation assays. Likewise, the mouse Catsper1 promoter exhibited transcriptional activity in both orientations and displayed significant expression levels in mouse testis in vivo, whereas the suppression of transcription signals in the promoter resulted in low expression levels. This study, thus, represents the first identification of the transcriptional control regions in the genes encoding the human and murine CatSper channels.

  9. Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease

    SciTech Connect

    Kane, S.E.; Troen, B.R.; Gal, S.; Ueda, K.; Pastan, I.; Gottesman, M.M.

    1988-08-01

    Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, the authors transfected cloned genes for mouse or human MEP into mouse MIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes were also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.

  10. Lgn1, a gene that determines susceptibility to Legionella pneumophila, maps to mouse chromosome 13

    SciTech Connect

    Dietrich, W.F.; Damron, D.M.; Lander, E.S.

    1995-04-10

    The intracellular pathogen Legionella pneumophila is unable to replicate in macrophages derived from most inbred mouse strains. Here, we report the mapping of a gene, called Lgn1, that determines whether mouse macrophages are permissive for the intracellular replication of L. pneumophila. Although Lgn1 has been previously reported to map to mouse chromosome 15, we show here that it actually maps to chromosome 13, between D13Mit128 and D13Mit70. In the absence of any regional candidates for Lgn1, this map position will facilitate positional cloning attempts directed at this gene. 22 refs., 2 figs., 2 tabs.

  11. The MOUSE Squad

    ERIC Educational Resources Information Center

    Borja, Rhea R.

    2004-01-01

    This article presents a New York city after-school program started by MOUSE (Making Opportunities for Upgrading Schools and Education), a national nonprofit group that teaches students how to fix computers, and equips them with the communication and problem-solving skills to help them in the working world. The MOUSE program is part of a trend…

  12. Individualized Communications

    NASA Technical Reports Server (NTRS)

    1997-01-01

    IntelliWeb and IntelliPrint, products from MicroMass Communications, utilize C Language Integrated Production System (CLIPS), a development and delivery expert systems tool developed at Johnson Space Center. IntelliWeb delivers personalized messages by dynamically creating single web pages or entire web sites based on information provided by each website visitor. IntelliPrint is a product designed to create tailored, individualized messages via printed media. The software uses proprietary technology to generate printed messages that are personally relevant and tailored to meet each individual's needs. Intelliprint is in use in many operations including Brystol-Myers Squibb's personalized newsletter, "Living at Your Best," geared to each recipient based on a health and lifestyle survey taken earlier; and SmithKline Beecham's "Nicorette Committed Quitters Program," in which customized motivational materials support participants in their attempt to quit smoking.

  13. Mouse genome database 2016.

    PubMed

    Bult, Carol J; Eppig, Janan T; Blake, Judith A; Kadin, James A; Richardson, Joel E

    2016-01-01

    The Mouse Genome Database (MGD; http://www.informatics.jax.org) is the primary community model organism database for the laboratory mouse and serves as the source for key biological reference data related to mouse genes, gene functions, phenotypes and disease models with a strong emphasis on the relationship of these data to human biology and disease. As the cost of genome-scale sequencing continues to decrease and new technologies for genome editing become widely adopted, the laboratory mouse is more important than ever as a model system for understanding the biological significance of human genetic variation and for advancing the basic research needed to support the emergence of genome-guided precision medicine. Recent enhancements to MGD include new graphical summaries of biological annotations for mouse genes, support for mobile access to the database, tools to support the annotation and analysis of sets of genes, and expanded support for comparative biology through the expansion of homology data.

  14. Mouse genome database 2016

    PubMed Central

    Bult, Carol J.; Eppig, Janan T.; Blake, Judith A.; Kadin, James A.; Richardson, Joel E.

    2016-01-01

    The Mouse Genome Database (MGD; http://www.informatics.jax.org) is the primary community model organism database for the laboratory mouse and serves as the source for key biological reference data related to mouse genes, gene functions, phenotypes and disease models with a strong emphasis on the relationship of these data to human biology and disease. As the cost of genome-scale sequencing continues to decrease and new technologies for genome editing become widely adopted, the laboratory mouse is more important than ever as a model system for understanding the biological significance of human genetic variation and for advancing the basic research needed to support the emergence of genome-guided precision medicine. Recent enhancements to MGD include new graphical summaries of biological annotations for mouse genes, support for mobile access to the database, tools to support the annotation and analysis of sets of genes, and expanded support for comparative biology through the expansion of homology data. PMID:26578600

  15. Mouse genome database 2016.

    PubMed

    Bult, Carol J; Eppig, Janan T; Blake, Judith A; Kadin, James A; Richardson, Joel E

    2016-01-01

    The Mouse Genome Database (MGD; http://www.informatics.jax.org) is the primary community model organism database for the laboratory mouse and serves as the source for key biological reference data related to mouse genes, gene functions, phenotypes and disease models with a strong emphasis on the relationship of these data to human biology and disease. As the cost of genome-scale sequencing continues to decrease and new technologies for genome editing become widely adopted, the laboratory mouse is more important than ever as a model system for understanding the biological significance of human genetic variation and for advancing the basic research needed to support the emergence of genome-guided precision medicine. Recent enhancements to MGD include new graphical summaries of biological annotations for mouse genes, support for mobile access to the database, tools to support the annotation and analysis of sets of genes, and expanded support for comparative biology through the expansion of homology data. PMID:26578600

  16. Haplotype mapping and sequence analysis of the mouse Nramp gene predict susceptibility to infection with intracellular parasites

    SciTech Connect

    Malo, D.; Hu, Jinxin; Schurr, E.

    1994-09-01

    The mouse chromosome 1 locus Bcg (Ity, Lsh) controls the capacity of the tissue macrophage to restrict the replication of antigenically unrelated intracellular parasites and therefore determines the natural resistance (BCG-R, dominant) or susceptibility (BCG-S, recessive) of inbred mouse strains to infection with diverse pathogens. We have used a positional cloning strategy based on genetic and physical mapping, YAC cloning, and exon trapping to isolate a candidate gene for Beg (Nramp) that encodes a predicted macrophage-specific transport protein. We have analyzed a total of 27 inbred mouse strains of BCG-R and BCG-S phenotypes for the presence of nucleotide sequence variations within the coding portion of Nramp and have carried out haplotype typing of the corresponding chromosome 1 region in these mice, using 11 additional polymorphic markers mapping in the immediate vicinity of Nramp. cDNA cloning and nucleotide sequencing identified 5 nucleotide sequence variations within Nramp in the inbred strains.

  17. Agro-economic impact of cattle cloning.

    PubMed

    Faber, D C; Ferre, L B; Metzger, J; Robl, J M; Kasinathan, P

    2004-01-01

    The purpose of this paper is to review the economic and social implications of cloned cattle, their products, and their offspring as related to production agriculture. Cloning technology in cattle has several applications outside of traditional production agriculture. These applications can include bio-medical applications, such as the production of pharmaceuticals in the blood or milk of transgenic cattle. Cloning may also be useful in the production of research models. These models may or may not include genetic modifications. Uses in agriculture include many applications of the technology. These include making genetic copies of elite seed stock and prize winning show cattle. Other purposes may range from "insurance" to making copies of cattle that have sentimental value, similar to cloning of pets. Increased selection opportunities available with cloning may provide for improvement in genetic gain. The ultimate goal of cloning has often been envisioned as a system for producing quantity and uniformity of the perfect dairy cow. However, only if heritability were 100%, would clone mates have complete uniformity. Changes in the environment may have significant impact on the productivity and longevity of the resulting clones. Changes in consumer preferences and economic input costs may all change the definition of the perfect cow. The cost of producing such animals via cloning must be economically feasible to meet the intended applications. Present inefficiencies limit cloning opportunities to highly valued animals. Improvements are necessary to move the applications toward commercial application. Cloning has additional obstacles to conquer. Social and regulatory acceptance of cloning is paramount to its utilization in production agriculture. Regulatory acceptance will need to address the animal, its products, and its offspring. In summary, cloning is another tool in the animal biotechnology toolbox, which includes artificial insemination, sexing of semen, embryo

  18. Agro-economic impact of cattle cloning.

    PubMed

    Faber, D C; Ferre, L B; Metzger, J; Robl, J M; Kasinathan, P

    2004-01-01

    The purpose of this paper is to review the economic and social implications of cloned cattle, their products, and their offspring as related to production agriculture. Cloning technology in cattle has several applications outside of traditional production agriculture. These applications can include bio-medical applications, such as the production of pharmaceuticals in the blood or milk of transgenic cattle. Cloning may also be useful in the production of research models. These models may or may not include genetic modifications. Uses in agriculture include many applications of the technology. These include making genetic copies of elite seed stock and prize winning show cattle. Other purposes may range from "insurance" to making copies of cattle that have sentimental value, similar to cloning of pets. Increased selection opportunities available with cloning may provide for improvement in genetic gain. The ultimate goal of cloning has often been envisioned as a system for producing quantity and uniformity of the perfect dairy cow. However, only if heritability were 100%, would clone mates have complete uniformity. Changes in the environment may have significant impact on the productivity and longevity of the resulting clones. Changes in consumer preferences and economic input costs may all change the definition of the perfect cow. The cost of producing such animals via cloning must be economically feasible to meet the intended applications. Present inefficiencies limit cloning opportunities to highly valued animals. Improvements are necessary to move the applications toward commercial application. Cloning has additional obstacles to conquer. Social and regulatory acceptance of cloning is paramount to its utilization in production agriculture. Regulatory acceptance will need to address the animal, its products, and its offspring. In summary, cloning is another tool in the animal biotechnology toolbox, which includes artificial insemination, sexing of semen, embryo

  19. MPHASYS: a mouse phenotype analysis system

    PubMed Central

    Calder, R Brent; Beems, Rudolf B; van Steeg, Harry; Mian, I Saira; Lohman, Paul HM; Vijg, Jan

    2007-01-01

    Background Systematic, high-throughput studies of mouse phenotypes have been hampered by the inability to analyze individual animal data from a multitude of sources in an integrated manner. Studies generally make comparisons at the level of genotype or treatment thereby excluding associations that may be subtle or involve compound phenotypes. Additionally, the lack of integrated, standardized ontologies and methodologies for data exchange has inhibited scientific collaboration and discovery. Results Here we introduce a Mouse Phenotype Analysis System (MPHASYS), a platform for integrating data generated by studies of mouse models of human biology and disease such as aging and cancer. This computational platform is designed to provide a standardized methodology for working with animal data; a framework for data entry, analysis and sharing; and ontologies and methodologies for ensuring accurate data capture. We describe the tools that currently comprise MPHASYS, primarily ones related to mouse pathology, and outline its use in a study of individual animal-specific patterns of multiple pathology in mice harboring a specific germline mutation in the DNA repair and transcription-specific gene Xpd. Conclusion MPHASYS is a system for analyzing multiple data types from individual animals. It provides a framework for developing data analysis applications, and tools for collecting and distributing high-quality data. The software is platform independent and freely available under an open-source license [1]. PMID:17553167

  20. Cloning

    MedlinePlus

    ... mammals. These twins are produced when a fertilized egg splits, creating two or more embryos that carry ... of the donor animal's somatic cell into an egg cell, or oocyte, that has had its own ...

  1. Detectability of Plasmodium falciparum clones

    PubMed Central

    2010-01-01

    Background In areas of high transmission people often harbour multiple clones of Plasmodium falciparum, but even PCR-based diagnostic methods can only detect a fraction (the detectability, q) of all clones present in a host. Accurate measurements of detectability are desirable since it affects estimates of multiplicity of infection, prevalence, and frequency of breakthrough infections in clinical drug trials. Detectability can be estimated by typing repeated samples from the same host but it has been unclear what should be the time interval between the samples and how the data should be analysed. Methods A longitudinal molecular study was conducted in the Kassena-Nankana district in northern Ghana. From each of the 80 participants, four finger prick samples were collected over a period of 8 days, and tested for presence of different Merozoite Surface Protein (msp) 2 genotypes. Implications for estimating q were derived from these data by comparing the fit of statistical models of serial dependence and over-dispersion. Results The distribution of the frequencies of detection for msp2 genotypes was close to binomial if the time span between consecutive blood samples was at least 7 days. For shorter intervals the probabilities of detection were positively correlated, i.e. the shorter the interval between two blood collections, the more likely the diagnostic results matched for a particular genotype. Estimates of q were rather insensitive to the statistical model fitted. Conclusions A simple algorithm based on analysing blood samples collected 7 days apart is justified for generating robust estimates of detectability. The finding of positive correlation of detection probabilities for short time intervals argues against imperfect detection being directly linked to the 48-hour periodicity of P. falciparum. The results suggest that the detectability of a given parasite clone changes over time, at an unknown rate, but fast enough to regard blood samples taken one week

  2. Cloning cattle: the methods in the madness.

    PubMed

    Oback, Björn; Wells, David N

    2007-01-01

    Somatic cell nuclear transfer (SCNT) is much more widely and efficiently practiced in cattle than in any other species, making this arguably the most important mammal cloned to date. While the initial objective behind cattle cloning was commercially driven--in particular to multiply genetically superior animals with desired phenotypic traits and to produce genetically modified animals-researchers have now started to use bovine SCNT as a tool to address diverse questions in developmental and cell biology. In this paper, we review current cattle cloning methodologies and their potential technical or biological pitfalls at any step of the procedure. In doing so, we focus on one methodological parameter, namely donor cell selection. We emphasize the impact of epigenetic and genetic differences between embryonic, germ, and somatic donor cell types on cloning efficiency. Lastly, we discuss adult phenotypes and fitness of cloned cattle and their offspring and illustrate some of the more imminent commercial cattle cloning applications.

  3. Unified universal quantum cloning machine and fidelities

    SciTech Connect

    Wang Yinan; Shi Handuo; Xiong Zhaoxi; Jing Li; Mu Liangzhu; Ren Xijun; Fan Heng

    2011-09-15

    We present a unified universal quantum cloning machine, which combines several different existing universal cloning machines together, including the asymmetric case. In this unified framework, the identical pure states are projected equally into each copy initially constituted by input and one half of the maximally entangled states. We show explicitly that the output states of those universal cloning machines are the same. One importance of this unified cloning machine is that the cloning procession is always the symmetric projection, which reduces dramatically the difficulties for implementation. Also, it is found that this unified cloning machine can be directly modified to the general asymmetric case. Besides the global fidelity and the single-copy fidelity, we also present all possible arbitrary-copy fidelities.

  4. Aup1, a novel gene on mouse Chromosome 6 and human Chromosome 2p13

    SciTech Connect

    Jang, Wonhee; Weber, J.S.; Meisler, M.H.

    1996-09-01

    We have cloned a novel mouse cDNA, Aup1, encoding a predicted protein of 410 amino acid residues. The 1.5-kb Aup1 transcript is ubiquitously expressed in mouse tissues. An evolutionary relationship to the Caenorhabditis elegans predicted protein F44b9.5 is indicated by the 35% identity and 53% conservation of the amino acid sequences. Nineteen related human ESTs spanning 80% of the protein have also been identified, with a predicted amino acid sequence identity of 86% between the human and the mouse proteins. The gene has been mapped to a conserved linkage group on human chromosome 2p13 and mouse Chromosome 6. Aup1 was eliminated as a candidate gene for two closely linked disorders, human LGMD2B and mouse mnd2. 15 refs., 2 figs.

  5. Synteny conservation of the Huntington's disease gene and surrounding loci on mouse Chromosome 5.

    PubMed

    Grosson, C L; MacDonald, M E; Duyao, M P; Ambrose, C M; Roffler-Tarlov, S; Gusella, J F

    1994-07-01

    The mouse homologs of the Huntington's disease (HD) gene and 17 other human Chromosome (Chr) 4 loci (including six previously unmapped) were localized by use of an interspecific cross. All loci mapped in a continuous linkage group on mouse Chr 5, distal to En2 and I16, whose human counterparts are located on Chr 7. The relative order of the loci on human Chr 4 and mouse Chr 5 was maintained, except for a break between D5H4S115E and Idua/rd, with relocation of the latter to the opposite end of the map. The mouse HD homolog (Hdh) mapped within a cluster of seven genes that were completely linked in our data set. In human these loci span a approximately 1.8 Mb stretch of human 4p16.3 that has been entirely cloned. To date, there is no phenotypic correspondence between human and mouse mutations mapping to this region of synteny conservation.

  6. MicroRNA expression profiling of elongated cloned and in vitro-fertilized bovine embryos.

    PubMed

    Castro, F O; Sharbati, S; Rodríguez-Alvarez, L L; Cox, J F; Hultschig, C; Einspanier, R

    2010-01-01

    The objective of this study was to identify microRNAs (miRNAs) expressed in bovine (Bos Taurus) cloned embryos at Day 17 of development (Day 0=day of nucleus transfer or in vitro fertilization) during elongation. Day 7 bovine expanded blastocysts produced by hand made cloning (HMC) or in vitro fertilization were bulk-transferred to synchronized recipient cattle (48 HMC embryos to 10 recipients and 28 in vitro-produced embryos to four recipients). Elongated embryos were retrieved at Day 17; miRNAs were isolated and subjected to microarray screening using custom composite slides spotted with human, mouse, and rat and in silico-predicted miRNAs. An initial profile of expressed miRNAs was determined in cloned embryos and somatic donor cells; this profile changed after somatic cell nucleus transfer, identifying differentially expressed miRNAs between cloned and in vitro-produced bovine embryos. Furthermore, microarray data were validated using a miRNA-specific quantitative reverse transcription-polymerase chain reaction (qRT-PCR) approach (miR-Q). There was an 83% correlation (P=0.01) between microarray and qPCR data. Based on qRT-PCR, correct reprogramming of some miRNAs from the donor cells was confirmed in cloned bovine embryos, whereas other somatic miRNAs were not appropriately reprogrammed. Some of the miRNAs that were equally reprogrammed clustered on the same chromosomal location in the bovine genome. In conclusion, reprogramming of miRNAs seemed to occur in cloned bovine embryos. This could have profound implications for elucidating nuclear reprogramming in somatic cloning, as well as for the role of miRNAs in preimplantation mammalian development.

  7. No human cloning: a social ethics perspective.

    PubMed

    Cahill, L S

    1999-01-01

    This Essay addresses the negative impact of human cloning on the family, and argues further that market incentives to develop and implement cloning techniques exploit and exacerbate socioeconomic inequities. It suggests that cloning should be prohibited internationally and examines possible routes to that aim. To begin with, it offers some reflections on the nature of moral argument, and on the role of religion in public debate. PMID:12650145

  8. Cloning: pathways to a pluripotent future.

    PubMed

    McLaren, A

    2000-06-01

    In this month's essay, Anne McLaren traces the winding and pitted pathways that connect the early days of the cell theory of biology in the 1830s to the new and unfolding era of cloning science and technology that came to worldwide attention in 1997 with the announcement of the birth of Dolly, the Scottish cloned sheep. The possibilities, including the potential for new medical treatments and perhaps even human cloning, are fantastic ... and ethically charged.

  9. Telomeres and the ethics of human cloning.

    PubMed

    Allhoff, Fritz

    2004-01-01

    In search of a potential problem with cloning, I investigate the phenomenon of telomere shortening which is caused by cell replication; clones created from somatic cells will have shortened telomeres and therefore reach a state of senescence more rapidly. While genetic intervention might fix this problem at some point in the future, I ask whether, absent technological advances, this biological phenomenon undermines the moral permissibility of cloning.

  10. Cloning: pathways to a pluripotent future.

    PubMed

    McLaren, A

    2000-06-01

    In this month's essay, Anne McLaren traces the winding and pitted pathways that connect the early days of the cell theory of biology in the 1830s to the new and unfolding era of cloning science and technology that came to worldwide attention in 1997 with the announcement of the birth of Dolly, the Scottish cloned sheep. The possibilities, including the potential for new medical treatments and perhaps even human cloning, are fantastic ... and ethically charged. PMID:10877698

  11. Cloning of spin-coherent states

    SciTech Connect

    Demkowicz-Dobrzanski, Rafal; Kus, Marek; Wodkiewicz, Krzysztof

    2004-01-01

    We consider optimal cloning of the spin coherent states in Hilbert spaces of different dimensionality d. We give explicit form of optimal cloning transformation for spin coherent states in the three-dimensional space, analytical results for the fidelity of the optimal cloning in d=3 and d=4 as well as numerical results for higher dimensions. In the low-dimensional case we construct the corresponding completely positive maps and exhibit their structure with the help of Jamiolkowski isomorphism. This allows us to formulate some conjectures about the form of optimal coherent cloning completely positive maps in arbitrary dimension.

  12. Quantum cloning disturbed by thermal Davies environment

    NASA Astrophysics Data System (ADS)

    Dajka, Jerzy; Łuczka, Jerzy

    2016-06-01

    A network of quantum gates designed to implement universal quantum cloning machine is studied. We analyze how thermal environment coupled to auxiliary qubits, `blank paper' and `toner' required at the preparation stage of copying, modifies an output fidelity of the cloner. Thermal environment is described in terms of the Markovian Davies theory. We show that such a cloning machine is not universal any more but its output is independent of at least a part of parameters of the environment. As a case study, we consider cloning of states in a six-state cryptography's protocol. We also briefly discuss cloning of arbitrary input states.

  13. Species-specific challenges in dog cloning.

    PubMed

    Kim, G A; Oh, H J; Park, J E; Kim, M J; Park, E J; Jo, Y K; Jang, G; Kim, M K; Kim, H J; Lee, B C

    2012-12-01

    Somatic cell nuclear transfer (SCNT) is now an established procedure used in cloning of several species. SCNT in dogs involves multiple steps including the removal of the nuclear material, injection of a donor cell, fusion, activation of the reconstructed oocytes and finally transfer to a synchronized female recipient. There are therefore many factors that contribute to cloning efficiency. By performing a retrospective analysis of 2005-2012 published papers regarding dog cloning, we define the optimum procedure and summarize the specific feature for dog cloning.

  14. Human cloning: Eastern Mediterranean Region perspective.

    PubMed

    Abdur Rab, M; Khayat, M H

    2006-01-01

    Recent advances in genomics and biotechnology have ushered in a new era in health development. Therapeutic cloning possesses enormous potential for revolutionizing medical and therapeutic techniques. Cloning technology, however, is perceived as having the potential for reproductive cloning, which raises serious ethical and moral concerns. It is important that the Islamic countries come to a consensus on this vital issue. Developing science and technology for better health is a religious and moral obligation. There is an urgent need for Muslim scholars to discuss the issue of stem cell research and cloning rationally; such dialogue will not only consider the scientific merits but also the moral, ethical and legal implications.

  15. No end in sight to cloning debate.

    PubMed

    Graumann, Sigrid; Poltermann, Andreas

    2005-01-01

    Since last August, Great Britain has allowed the cloning for research purposes. This fact has re-generated an existing debate, taking into account the prohibition of cloning of the UN, the States are debating whether cloning should be prohibited or in the contrary, it should also be admitted for reproductive purposes. This situation has generated an international uneasiness due to the lack of a universal consensus. This article analyses this situation, bringing the reader closer to the very controversial texts, such as the European Constitution and the UN Convention on Cloning.

  16. 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?

  17. Microsatellite DNA fingerprinting, differentiation, and genetic relationships of clones, cultivars, and varieties of six poplar species from three sections of the genus Populus.

    PubMed

    Rahman, Muhammad H; Rajora, Om P

    2002-12-01

    Accurate identification of Populus clones and cultivars is essential for effective selection, breeding, and genetic resource management programs. The unit of cultivation and breeding in poplars is a clone, and individual cultivars are normally represented by a single clone. Microsatellite DNA markers of 10 simple sequence repeat loci were used for genetic fingerprinting and differentiation of 96 clones/cultivars and varieties belonging to six Populus species (P. deltoides, P. nigra, P. balsamifera, P. trichocarpa, P. grandidentata, and P maximowiczii) from three sections of the genus. All 96 clones/cultivars could be uniquely fingerprinted based on their single- or multilocus microsatellite genotypes. The five P. grandidentata clones could be differentiated based on their single-locus genotypes, while six clones of P. trichocarpa and 11 clones of P. maximowiczii could be identified by their two-locus genotypes. Twenty clones of P. deltoides and 25 clones of P. nigra could be differentiated by their multilocus genotypes employing three loci, and 29 clones of P. balsamifera required the use of multilocus genotypes at five loci for their genetic fingerprinting and differentiation. The loci PTR3, PTR5, and PTR7 were found to be the most informative for genetic fingerprinting and differentiation of the clones. The mean number of alleles per locus ranged from 2.9 in P. trichocarpa or P. grandidentata to 6.0 in P. balsamifera and 11.2 in 96 clones of the six species. The mean number of observed genotypes per locus ranged from 2.4 in P. grandidentata to 7.4 in P. balsamifera and 19.6 in 96 clones of the six species. The mean number of unique genotypes per locus ranged from 1.3 in P. grandidentata to 3.9 in P. deltoides and 8.8 in 96 clones of the six species. The power of discrimination of the microsatellite DNA markers in the 96 clones ranged from 0.726 for PTR4 to 0.939 for PTR7, with a mean of 0.832 over the 10 simple sequence repeat loci. Clones/cultivars from the same

  18. Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning.

    PubMed

    Steinborn, R; Schinogl, P; Zakhartchenko, V; Achmann, R; Schernthaner, W; Stojkovic, M; Wolf, E; Müller, M; Brem, G

    2000-07-01

    Mammals have been cloned from adult donor cells. Here we report the first cases of mitochondrial DNA (mtDNA) heteroplasmy in adult mammalian clones generated from fetal and adult donor cells. The heteroplasmic clones included a healthy cattle equivalent of the sheep Dolly, for which a lack of heteroplasmy was reported.

  19. Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning.

    PubMed

    Steinborn, R; Schinogl, P; Zakhartchenko, V; Achmann, R; Schernthaner, W; Stojkovic, M; Wolf, E; Müller, M; Brem, G

    2000-07-01

    Mammals have been cloned from adult donor cells. Here we report the first cases of mitochondrial DNA (mtDNA) heteroplasmy in adult mammalian clones generated from fetal and adult donor cells. The heteroplasmic clones included a healthy cattle equivalent of the sheep Dolly, for which a lack of heteroplasmy was reported. PMID:10888867

  20. STRU-cloning: a fast, inexpensive and efficient cloning procedure applicable to both small scale and structural genomics size cloning.

    PubMed

    Bellini, Dom; Fordham-Skelton, Anthony P; Papiz, Miroslav Z

    2011-05-01

    We have developed a Single-Tube Restriction-based Ultrafiltration (STRU) cloning procedure that updates traditional ligation-dependent cloning to challenge the newer, faster and more efficient ligation-free techniques and could make it the method of choice. STRU-cloning employs centrifugal filter units with membrane of suitable cut off to remove small unwanted DNA fragments created during restriction of plasmids or PCR products. Heat inactivation, of restriction enzymes, followed by DNA ligation is then performed on the filtrate. By removing the agarose gel electrophoresis DNA purification step from the traditional protocol, which is time consuming and is known to be the cause of ligation problems, STRU-cloning becomes fast, very efficient, inexpensive and offers the highest degree of cloning flexibility by using restriction sites and can be performed in a single tube. This novel agarose gel-free cloning procedure provides benefits for both small and large scale cloning projects. Unlike traditional cloning it can be easily implemented as a fully automated process at very low costs. PMID:21052867

  1. Children computer mouse use and anthropometry.

    PubMed

    Hughes, Erin E; Johnson, Peter W

    2012-01-01

    Studies have shown that increased computer use among adults in occupational settings is associated with the development of cumulative trauma disorders; however, the need to address how adult-sized mice and keyboards are affecting children is becoming increasingly important as both access to and use of computers is increasing among today's youth. To address the potential mismatch that exists between child stature and computer input device size and activation force, we have applied existing, age-specific, anthropometric data to elements of device design, including mouse size (length, width, height, switch location), and mouse-button activation forces. Trends supported the development of smaller computer input devices with lower activation forces for smaller statured individuals including children. Distinct and consistent trends in size delineations were seen across gender and age groups-trends that correlate well with grades and schooling in the United States education system . Three to four mouse sizes would be recommended: a mouse sized for adult and high school males; one for adult and high school females and junior high males; one for elementary school children, aged 6 to 10 years; and possibly a mouse for the smallest users who are less than six years old. PMID:22316827

  2. Memory-built-in quantum cloning in a hybrid solid-state spin register

    NASA Astrophysics Data System (ADS)

    Wang, Weibin; Zu, Chong; He, Li; Zhang, Wengang; Duan, Luming

    2015-05-01

    As a way to circumvent the quantum no-cloning theorem, approximate quantum cloning protocols have received wide attention with remarkable applications. Copying of quantum states to memory qubits provides an important strategy for eavesdropping in quantum cryptography. We report an experiment that realizes cloning of quantum states from an electron spin to a nuclear spin in a hybrid solid-state spin register with near-optimal fidelity. The nuclear spin provides an ideal memory qubit at room temperature, which stores the cloned quantum states for a millisecond under ambient conditions, exceeding the lifetime of the original quantum state carried by the electron spin by orders of magnitude, and making it an ideal memory qubit. Our experiment is based on control of an individual nitrogen vacancy (NV) center in the diamond, which is a diamond defect that attracts strong interest in recent years with great potential for implementation of quantum information protocols.

  3. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics.

    PubMed

    Ben Yehezkel, Tuval; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-02-29

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development. PMID:26481354

  4. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics.

    PubMed

    Ben Yehezkel, Tuval; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-02-29

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development.

  5. Bad copies. How popular media represent cloning as an ethical problem.

    PubMed

    Hopkins, P D

    1998-01-01

    The media, perhaps more than any other slice of culture, influence what we think and talk about, what we take to be important, what we worry about. And this was especially true when news of Dolly hit the airwaves and newstands. Most Americans received training in the ethics of cloning before they knew what cloning was. Media coverage fixed the content and outline of the public moral debate, both revealing and creating the dominant public worries about cloning humans. The primary characterization of cloning as an ethical issue centers around three connected concerns: the loss of human uniqueness and individuality, the pathological motivations of a cloner, and the fear of out-of-control scientists.

  6. Mouse Cleaning Apparatus and Method

    NASA Technical Reports Server (NTRS)

    Williams, Glenn L. (Inventor)

    2005-01-01

    The method of using the mouse pad cleaning apparatus is disclosed and claimed. The method comprises the steps of uncovering the mouse cleaning surface, applying the mouse and ball of the mouse to the cleaning surface, moving the mouse in a rotational pattern on the mouse cleaning surface, removing the mouse form the mouse cleaning surface, washing the cleaning surface, and covering the mouse cleaning surface. A mouse pad cleaning apparatus comprising a plurality of substrates, each said substrate having adhesive thereon, said plurality of substrates residing in and affixed to a receptacle. A single substrate having adhesive, which may be washable or non-washable, thereon may be employed. The washable adhesive may be an organopolysiloxane or gelatinous elastomer.

  7. A brain-specific gene cluster isolated from the region of the mouse obesity locus is expressed in the adult hypothalamus and during mouse development

    SciTech Connect

    Laig-Webster, M.; Lim, M.E.; Chehab, F.F.

    1994-09-01

    The molecular defect underlying an autosomal recessive form of genetic obesity in a classical mouse model C57 BL/6J-ob/ob has not yet been elucidated. Whereas metabolic and physiological disturbances such as diabetes and hypertension are associated with obesity, the site of expression and the nature of the primary lesion responsible for this cascade of events remains elusive. Our efforts aimed at the positional cloning of the ob gene by YAC contig mapping and gene identification have resulted in the cloning of a brain-specific gene cluster from the ob critical region. The expression of this gene cluster is remarkably complex owing to the multitude of brain-specific mRNA transcripts detected on Northern blots. cDNA cloning of these transcripts suggests that they are expressed from different genes as well as by alternate splicing mechanisms. Furthermore, the genomic organization of the cluster appears to consist of at least two identical promoters displaying CpG islands characteristic of housekeeping genes, yet clearly involving tissue-specific expression. Sense and anti-sense synthetic RNA probes were derived from a common DNA sequence on 3 cDNA clones and hybridized to 8-16 days mouse embryonic stages and mouse adult brain sections. Expression in development was noticeable as of the 11th day of gestation and confined to the central nervous system mainly in the telencephalon and spinal cord. Coronal and sagittal sections of the adult mouse brain showed expression only in 3 different regions of the brain stem. In situ hybridization to mouse hypothalamus sections revealed the presence of a localized and specialized group of cells expressing high levels of mRNA, suggesting that this gene cluster may also be involved in the regulation of hypothalamic activities. The hypothalamus has long been hypothesized as a primary candidate tissue for the expression of the obesity gene mainly because of its well-established role in the regulation of energy metabolism and food intake.

  8. BodyMap: a human and mouse gene expression database.

    PubMed

    Hishiki, T; Kawamoto, S; Morishita, S; Okubo, K

    2000-01-01

    BodyMap is a human and mouse gene expression database that has been maintained since 1993. It is based on site-directed 3'-ESTs collected from non-biased cDNA libraries constructed at Osaka University and contains >270 000 sequences from 60 human and 38 mouse tissues. The site-directed nature of the sequence tags allows unequivocal grouping of tags representing the same transcript and provides abundance information for each transcript in different parts of the body. Our collection of ESTs was compared periodically with other public databases for cross referencing. The histological resolution of source tissues and unique cloning strategy that minimized cloning bias enabled BodyMap to support three unique mRNA based experiments in silico. First, the recurrence information for clones in each library provides a rough estimate of the mRNA composition of each source tissue. Second, a user can search the entire data set with nucleotide sequences or keywords to assess expression patterns of particular genes. Third, and most important, BodyMap allows a user to select genes that have a desired expression pattern in humans and mice. BodyMap is accessible through the WWW at http://bodymap.ims.u-tokyo.ac.jp PMID:10592203

  9. Potassium channels cloned from neuroblastoma cells display slowly inactivating outward currents in Xenopus oocytes.

    PubMed

    Ito, Y; Yokoyama, S; Higashida, H

    1992-05-22

    Messenger RNAs (mRNAs) specific for NGK1 and NGK2 potassium channels were synthesized from complementary DNAs (cDNAs) that had been cloned from mouse neuroblastoma x rat glioma hybrid NG108-15 cells. Outward pottasium currents were evoked by 5 s depolarizing voltage commands in Xenopus oocytes injected with NGK1- or NGK2-specific mRNAs. The NGK1 or NGK2 currents showed different activation and inactivation kinetics, and different pharmacological sensitivities. The threshold potential for activation of the NGK2 current (-14 mV) was more positive than that for the NGK1 (-36 mV). The NGK2 current showed faster inactivation during a 5 s depolarizing pulse than did the NGK1 current. Inactivation was best fit by time constants of 0.37, 1.5 and 19 s for the NGK2 current and 4.4 and 19 s for NGK1. Extracellularly applied tetraethylammonium chloride (TEA) was 1000 times more potent on the NGK2 current than the NGK1 current. Furthermore we examined outward current following co-injection of an equal amount of mRNAs for NGK1 and NGK2. The timecourse of inactivation differed from either alone or from a simple sum of the two individual currents. TEA sensitivity could not be explained by summation of the two homomultimeric channels. These findings suggest that both NGK1 and NGK2 proteins assemble to form heteromultimeric K+ channels in addition to homomultimeric K+ channels. NGK2 channels and the heteromultimeric channels may be responsible for the native transient outward current with slow inactivation in NG108-15 hybrid cells.

  10. Positional Cloning by Linkage Disequilibrium

    PubMed Central

    Maniatis, Nikolas; Collins, Andrew; Gibson, Jane; Zhang, Weihua; Tapper, William; Morton, Newton E.

    2004-01-01

    Recently, metric linkage disequilibrium (LD) maps that assign an LD unit (LDU) location for each marker have been developed (Maniatis et al. 2002). Here we present a multiple pairwise method for positional cloning by LD within a composite likelihood framework and investigate the operating characteristics of maps in physical units (kb) and LDU for two bodies of data (Daly et al. 2001; Jeffreys et al. 2001) on which current ideas of blocks are based. False-negative indications of a disease locus (type II error) were examined by selecting one single-nucleotide polymorphism (SNP) at a time as causal and taking its allelic count (0, 1, or 2, for the three genotypes) as a pseudophenotype, Y. By use of regression and correlation, association between every pseudophenotype and the allelic count of each SNP locus (X) was based on an adaptation of the Malecot model, which includes a parameter for location of the putative gene. By expressing locations in kb or LDU, greater power for localization was observed when the LDU map was fitted. The efficiency of the kb map, relative to the LDU map, to describe LD varied from a maximum of 0.87 to a minimum of 0.36, with a mean of 0.62. False-positive indications of a disease locus (type I error) were examined by simulating an unlinked causal SNP and the allele count was used as a pseudophenotype. The type I error was in good agreement with Wald’s likelihood theorem for both metrics and all models that were tested. Unlike tests that select only the most significant marker, haplotype, or haploset, these methods are robust to large numbers of markers in a candidate region. Contrary to predictions from tagging SNPs that retain haplotype diversity, the sample with smaller size but greater SNP density gave less error. The locations of causal SNPs were estimated with the same precision in blocks and steps, suggesting that block definition may be less useful than anticipated for mapping a causal SNP. These results provide a guide to

  11. Reversibility of continuous-variable quantum cloning

    SciTech Connect

    Filip, Radim; Marek, Petr; Fiurasek, Jaromir

    2004-01-01

    We analyze a reversibility of optimal Gaussian 1{yields}2 quantum cloning of a coherent state using only local operations on the clones and classical communication between them and propose a feasible experimental test of this feature. Performing Bell-type homodyne measurement on one clone and anticlone, an arbitrary unknown input state (not only a coherent state) can be restored in the other clone by applying appropriate local unitary displacement operation. We generalize this concept to a partial reversal of the cloning using only local operations and classical communication (LOCC) and we show that this procedure converts the symmetric cloner to an asymmetric cloner. Further, we discuss a distributed LOCC reversal in optimal 1{yields}M Gaussian cloning of coherent states which transforms it to optimal 1{yields}M{sup '} cloning for M{sup '}cloning as a possible eavesdropping attack on quantum communication link, the reversibility can be utilized to improve the security of the link even after the attack.

  12. Cloning of endangered mammalian species: any progress?

    PubMed

    Loi, Pasqualino; Galli, Cesare; Ptak, Grazyna

    2007-05-01

    Attempts through somatic cell nuclear transfer to expand wild populations that have shrunk to critical numbers is a logical extension of the successful cloning of mammals. However, although the first mammal was cloned 10 years ago, nuclear reprogramming remains phenomenological, with abnormal gene expression and epigenetic deregulation being associated with the cloning process. In addition, although cloning of wild animals using host oocytes from different species has been successful, little is known about the implication of partial or total mitochondrial DNA heteroplasmy in cloned embryos, fetuses and offspring. Finally, there is a need for suitable foster mothers for inter-intra specific cloned embryos. Considering these issues, the limited success achieved in cloning endangered animals is not surprising. However, optimism comes from the rapid gain in the understanding of the molecular clues underlying nuclear reprogramming. If it is possible to achieve a controlled reversal of the differentiated state of a cell then it is probable that other issues that impair the cloning of endangered animals, such as the inter-intra species oocyte or womb donor, will be overcome in the medium term.

  13. Impact of cloning on cattle breeding systems.

    PubMed

    McClintock, A E

    1998-01-01

    The concept of clone-family testing is compared with existing progeny testing systems. The critical factors that will decide how cloning is utilized are the potential size of cloned families, and the cost per embryo (or per calf born). If family sizes of 100,000 become routinely achievable (cheaply), then clone testing becomes viable. In rough figures, cloned embryos costing $30 with a 50% calving rate would be attractive to farmers and would be cheap enough that farmers would buy more (crossbred) embryos in order to breed further replacement cows. At $300 per embryo, farmers would be more inclined to buy a number of cloned pure-bred female embryos and then to use conventional artificial insemination to breed further replacements from these superior cows. At $3000 per embryo, farmers would probably only be interested in very small numbers of cloned animals, most of which would be males. The relative importance of adult versus fetal cloning is discussed. The need for gene banks to preserve genetic variation is emphasized; both gametes and somatic tissue cultures should be considered.

  14. The ethics of human reproductive cloning.

    PubMed

    Strong, Carson

    2005-03-01

    This article addresses the question of whether human reproductive cloning could be ethically justifiable in at least some cases involving infertile couples who would choose cloning as a way to have a genetically related child. At present, the risk of congenital anomalies constitutes a compelling argument against human reproductive cloning. The article explores whether reproductive cloning could be ethically justifiable if, at some future time, cloning becomes possible without an elevated risk of anomalies. It is argued that freedom to use cloning is a form of procreative freedom and, as such, deserves respect. All of the objections that have been raised against human reproductive cloning fall under three main categories: those that appeal to the interests of the child, those based on consequences for society, and those arising from teleological views. Objections that appeal to the child's interests are, in turn, of two main kinds: consequentialist and deontological. All of these types of objections are examined, and it is found that each involves serious problems that prevent it from being a reasonable objection in the context of the infertility cases considered. It is concluded that human reproductive cloning would be ethically justifiable in at least some cases involving infertile couples, provided that it could be performed without an elevated risk of anomalies.

  15. Molecular cloning and characterization of an N-acetylglucosamine-6-O-sulfotransferase.

    PubMed

    Uchimura, K; Muramatsu, H; Kadomatsu, K; Fan, Q W; Kurosawa, N; Mitsuoka, C; Kannagi, R; Habuchi, O; Muramatsu, T

    1998-08-28

    We isolated a cDNA clone encoding mouse N-acetylglucosamine-6-O-sulfotransferase based on sequence homology to the previously cloned mouse chondroitin 6-sulfotransferase. The cDNA clone contained an open reading frame that predicts a type II transmembrane protein composed of 483 amino acid residues. The expressed enzyme transferred sulfate to the 6 position of nonreducing GlcNAc in GlcNAcbeta1-3Galbeta1-4GlcNAc. Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc and various glycosaminoglycans did not serve as acceptors. Expression of the cDNA in COS-7 cells resulted in production of a cell-surface antigen, the epitope of which was NeuAcalpha2-3Galbeta1-4(SO4-6)GlcNAc; double transfection with fucosyltransferase IV yielded Galbeta1-4(Fucalpha1-3)(SO4-6)GlcNAc antigen. The sulfotransferase mRNA was strongly expressed in the cerebrum, cerebellum, eye, pancreas, and lung of adult mice. In situ hybridization revealed that the mRNA was localized in high endothelial venules of mesenteric lymph nodes. The sulfotransferase was concluded to be involved in biosynthesis of glycoconjugates bearing the 6-sulfo N-acetyllactosamine structure such as 6-sulfo sialyl Lewis X. The products of the sulfotransferase probably include glycoconjugates with intercellular recognition signals; one candidate of such a glycoconjugate is an L-selectin ligand.

  16. Future and applications of cloning.

    PubMed

    Trounson, Alan O

    2006-01-01

    The birth of viable offspring from somatic cell nuclear transfer (SCNT) in mammals caused a major re-examination of the understanding of the commitment of cells to specific tissue lineages during differentiation. The questions of whether cells undergo dedifferentiation or transdifferentiation during the development of offspring and how these changes are controlled is a source of ongoing debate that is yet to be resolved. Irrespective of the outcome of this debate, it is clear that cloning using SCNT has a place and purpose in the future of research and animal breeding. The future uses of SCNT could include the production of transgenic mice, the production of transgenic livestock and assisting with the re-establishment of endangered species. Human medicine also would benefit from future use of SCNT because it would allow the production of patient-specific embryonic stem cells.

  17. Meat and milk compositions of bovine clones.

    PubMed

    Tian, X Cindy; Kubota, Chikara; Sakashita, Kunihito; Izaike, Yoshiaki; Okano, Ryoichi; Tabara, Norio; Curchoe, Carol; Jacob, Lavina; Zhang, Yuqin; Smith, Sadie; Bormann, Charles; Xu, Jie; Sato, Masumi; Andrew, Sheila; Yang, Xiangzhong

    2005-05-01

    The technology is now available for commercial cloning of farm animals for food production, but is the food safe for consumers? Here, we provide data on >100 parameters that compare the composition of meat and milk from beef and dairy cattle derived from cloning to those of genetic- and breed-matched control animals from conventional reproduction. The cloned animals and the comparators were managed under the same conditions and received the same diet. The composition of the meat and milk from the clones were largely not statistically different from those of matched comparators, and all parameters examined were within the normal industry standards or previously reported values. The data generated from our match-controlled experiments provide science-based information desired by regulatory agencies to address public concerns about the safety of meat and milk from somatic animal clones.

  18. [Human clone or a delayed twin?].

    PubMed

    Szybalski, W

    2001-01-01

    Cloning is a natural mode of asexual reproduction for many organisms, which results in nearly identical copies of cells or organisms. In animals, including humans, identical twins are an example of natural cloning. In the case of sheep, scientists succeeded to produce the "delayed" identical twin. Dolly, of a mature animal by a rather complex and inefficient procedure. However, if this procedure is perfected, it will be useful to clone beloved pets and important laboratory animals. It will be much less suited for making (cloning) "delayed twin" of mature persons because of high costs together with present experimental uncertainties. The only required regulation for human cloning is that somebody must be legally, including financially, responsible for the results of such novel reproductive technique.

  19. [Human cloning in Muslim and Arab law].

    PubMed

    Aldeeb Abu-Sahlieh, Sami A

    2009-01-01

    Cloning is a modern medical procedure that Muslim religious authorities treat en resorting to the general principles established by classical Muslim law based on the Koran and the Sunnah of Muhhamad as the messenger of God. In this regard, human beings are not capable of deciding what is or what is not lawful without resorting to divine norms. Cloning clashes with several principles. Firstly, the principle of the respect for life in relation to surpernumeraries, but Muslim authors are not in unanimous agreement on the determination of the moment at which life begins. Secondly, is the respect of progeny: cloning could only take place between a married couple. But even if these two principles are respected, cloning poses two major problems: the diversity of species expounded by the Koran and the Sunnah and a lack of interest. Which explains the quasi-unanimous opposition of Muslim writings regarding cloning.

  20. "Goodbye Dolly?" The ethics of human cloning.

    PubMed

    Harris, J

    1997-12-01

    The ethical implications of human clones have been much alluded to, but have seldom been examined with any rigour. This paper examines the possible uses and abuses of human cloning and draws out the principal ethical dimensions, both of what might be done and its meaning. The paper examines some of the major public and official responses to cloning by authorities such as President Clinton, the World Health Organisation, the European parliament, UNESCO, and others and reveals their inadequacies as foundations for a coherent public policy on human cloning. The paper ends by defending a conception of reproductive rights of "procreative autonomy" which shows human cloning to be not inconsistent with human rights and dignity.

  1. "Goodbye Dolly?" The ethics of human cloning.

    PubMed Central

    Harris, J

    1997-01-01

    The ethical implications of human clones have been much alluded to, but have seldom been examined with any rigour. This paper examines the possible uses and abuses of human cloning and draws out the principal ethical dimensions, both of what might be done and its meaning. The paper examines some of the major public and official responses to cloning by authorities such as President Clinton, the World Health Organisation, the European parliament, UNESCO, and others and reveals their inadequacies as foundations for a coherent public policy on human cloning. The paper ends by defending a conception of reproductive rights of "procreative autonomy" which shows human cloning to be not inconsistent with human rights and dignity. PMID:9451604

  2. Normal telomere lengths found in cloned cattle.

    PubMed

    Tian, X C; Xu, J; Yang, X

    2000-11-01

    Success of cloning using adult somatic cells has been reported in sheep, mice and cattle. The report that 'Dolly' the sheep, the first clone from an adult mammal, inherited shortened telomeres from her cell donor and that her telomeres were further shortened by the brief culture of donor cells has raised serious scientific and public concerns about the 'genetic age' and potential developmental problems of cloned animals. This observation was challenged by a recent report that showed calves cloned from fetal cells have longer telomeres than their age-matched controls. The question remains whether Dolly's short telomeres were an exception or a general fact, which would differ from the telomeres of fetal-derived clones. PMID:11062462

  3. [Human clone or a delayed twin?].

    PubMed

    Szybalski, W

    2001-01-01

    Cloning is a natural mode of asexual reproduction for many organisms, which results in nearly identical copies of cells or organisms. In animals, including humans, identical twins are an example of natural cloning. In the case of sheep, scientists succeeded to produce the "delayed" identical twin. Dolly, of a mature animal by a rather complex and inefficient procedure. However, if this procedure is perfected, it will be useful to clone beloved pets and important laboratory animals. It will be much less suited for making (cloning) "delayed twin" of mature persons because of high costs together with present experimental uncertainties. The only required regulation for human cloning is that somebody must be legally, including financially, responsible for the results of such novel reproductive technique. PMID:11684762

  4. AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach

    PubMed Central

    Beyer, Hannes M.; Gonschorek, Patrick; Samodelov, Sophia L.; Meier, Matthias; Weber, Wilfried; Zurbriggen, Matias D.

    2015-01-01

    Assembly cloning is increasingly replacing conventional restriction enzyme and DNA-ligase-dependent cloning methods for reasons of efficiency and performance. Here, we describe AQUA (advanced quick assembly), a simple and versatile seamless assembly cloning approach. We demonstrate the applicability and versatility of AQUA Cloning in selected proof-of-principle applications including targeted insertion-, deletion- and site-directed point-mutagenesis, and combinatorial cloning. Furthermore, we show the one pot de novo assembly of multiple DNA fragments into a single circular plasmid encoding a complex light- and chemically-regulated Boolean A NIMPLY B logic operation. AQUA Cloning harnesses intrinsic in vivo processing of linear DNA fragments with short regions of homology of 16 to 32 bp mediated by Escherichia coli. It does not require any kits, enzymes or preparations of reagents and is the simplest assembly cloning protocol to date. PMID:26360249

  5. Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries.

    PubMed

    Schramm, Andreas; Fuchs, Bernhard M; Nielsen, Jeppe L; Tonolla, Mauro; Stahl, David A

    2002-11-01

    A method is presented for fluorescence in situ hybridization (FISH) of 16S rRNA gene clones targeting in vivo transcribed plasmid inserts (Clone-FISH). Several different cloning approaches and treatments to generate target-rRNA in the clones were compared. Highest signal intensities of Clone-FISH were obtained using plasmids with a T7 RNA polymerase promoter and host cells with an IPTG-inducible T7 RNA polymerase. Combined IPTG-induction and chloramphenicol treatment of those clones resulted in FISH signals up to 2.8-fold higher than signals of FISH with probe EUB338 to cells of Escherichia coli. Probe dissociation curves for three oligonucleotide probes were compared for reference cells containing native (FISH) or cloned (Clone-FISH) target sequences. Melting behaviour and calculated T(d) values were virtually identical for clones and cells, providing a format to use 16S rRNA gene clones instead of pure cultures for probe validation and optimization of hybridization conditions. The optimized Clone-FISH protocol was also used to screen an environmental clone library for insert sequences of interest. In this application format, 13 out of 82 clones examined were identified to contain sulphate-reducing bacterial rRNA genes. In summary, Clone-FISH is a simple and fast technique, compatible with a wide variety of cloning vectors and hosts, that should have general utility for probe validation and screening of clone libraries. PMID:12460279

  6. The ethics of human reproductive cloning: when world views collide.

    PubMed

    Cohen, Cynthia B

    2004-01-01

    Two camps in bioethics with seemingly opposing world views have staked out conflicting positions regarding the ethics of human reproductive cloning. These camps do not appear to share common concepts or ways of reasoning through which to exchange views and come to a meeting of minds about uses of this technology. Yet analysis of their respective approaches to several issues surrounding reproductive cloning, such as where the ethical limits of individual reproductive choice lie, whether the use of this technology would violate human dignity, whether it would create risks to the resulting fetuses and children that would make its use intolerable, and whether it would challenge certain core social values, reveals that they are not wholly opposed to one another. Indeed, it displays that they hold certain beliefs, values, and concerns in common. Moreover, it indicates that the different world views that they each presuppose, while flawed in certain respects, do not collide in every respect, but can be reconciled in significant ways that provide fertile ground for agreement about several issues related to human reproductive cloning.

  7. Streptococcus pneumoniae serogroup 6 clones over two decades.

    PubMed

    Payne, D B; Gray, B M

    2014-12-01

    The major evolutionary stresses on Streptococcus pneumoniae are thought to be the widespread use of antibiotics and the deployment of effective vaccines against the capsular polysaccharides. Our current knowledge of genetic lineages among pneumococcal isolates comes largely from investigations just before and after the introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) introduced in 2000. We examined 66 serogroup 6 isolates from the 1970s, long before the introduction of PCV7 and before widespread penicillin resistance was common in Birmingham, Alabama, to look for ancestors of the clones that came into play around the introduction of the PCV7 vaccine. The hypothesis was that some clonal complexes, if not individual clones, would be stable enough to persist over this period of time. We compared the 1970s isolates with 122 isolates from the 1990s in US and worldwide collections. Genotyping with pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) revealed that while some clones were probably localized to our area, others have persisted within groups that have expanded or diminished over the years.

  8. Targeted disruption of the murine Facc gene: Towards the establishment of a mouse model for Fanconi anemia

    SciTech Connect

    Chen, M.; Auerbach, W.; Buchwald, M.

    1994-09-01

    Fanconi anemia (FA) is an autosomal recessive disease characterized by bone marrow failure, congenital malformations and predisposition to malignancies. The gene responsible for the defect in FA group C has been cloned and designated the Fanconi Anemia Complementation Group C gene (FACC). A murine cDNA for this gene (Facc) was also cloned. Here we report our progress in the establishment of a mouse model for FA. The mouse Facc cDNA was used as probe to screen a genomic library of mouse strain 129. More than twenty positive clones were isolated. Three of them were mapped and found to be overlapping clones, encompassing the genomic region from exon 8 to the end of the 3{prime} UTR of the mouse cDNA. A targeting vector was constructed using the most 5{prime} mouse genomic sequence available. The end result of the homologous recombination is that exon 8 is deleted and the neo gene is inserted. The last exon, exon 14, is essential for the complementing function of the FACC gene product; the disruption in the middle of the murine Facc gene should render this locus biologically inactive. This targeting vector was linearized and electroporated into R1 embryonic stem (ES) cells which were derived from the 129 mouse. Of 102 clones screened, 19 positive cell lines were identified. Four targeted cell lines have been used to produce chimeric mice. 129-derived ES cells were aggregated ex vivo into the morulas derived from CD1 mice and then implanted into foster mothers. 22 chimeras have been obtained. Moderately and strongly chimeric mice have been bred to test for germline transmission. Progeny with the expected coat color derived from 2 chimeras are currently being examined to confirm transmission of the targeted allele.

  9. Principles and application of LIMS in mouse clinics.

    PubMed

    Maier, Holger; Schütt, Christine; Steinkamp, Ralph; Hurt, Anja; Schneltzer, Elida; Gormanns, Philipp; Lengger, Christoph; Griffiths, Mark; Melvin, David; Agrawal, Neha; Alcantara, Rafael; Evans, Arthur; Gannon, David; Holroyd, Simon; Kipp, Christian; Raj, Navis Pretheeba; Richardson, David; LeBlanc, Sophie; Vasseur, Laurent; Masuya, Hiroshi; Kobayashi, Kimio; Suzuki, Tomohiro; Tanaka, Nobuhiko; Wakana, Shigeharu; Walling, Alison; Clary, David; Gallegos, Juan; Fuchs, Helmut; de Angelis, Martin Hrabě; Gailus-Durner, Valerie

    2015-10-01

    Large-scale systemic mouse phenotyping, as performed by mouse clinics for more than a decade, requires thousands of mice from a multitude of different mutant lines to be bred, individually tracked and subjected to phenotyping procedures according to a standardised schedule. All these efforts are typically organised in overlapping projects, running in parallel. In terms of logistics, data capture, data analysis, result visualisation and reporting, new challenges have emerged from such projects. These challenges could hardly be met with traditional methods such as pen & paper colony management, spreadsheet-based data management and manual data analysis. Hence, different Laboratory Information Management Systems (LIMS) have been developed in mouse clinics to facilitate or even enable mouse and data management in the described order of magnitude. This review shows that general principles of LIMS can be empirically deduced from LIMS used by different mouse clinics, although these have evolved differently. Supported by LIMS descriptions and lessons learned from seven mouse clinics, this review also shows that the unique LIMS environment in a particular facility strongly influences strategic LIMS decisions and LIMS development. As a major conclusion, this review states that there is no universal LIMS for the mouse research domain that fits all requirements. Still, empirically deduced general LIMS principles can serve as a master decision support template, which is provided as a hands-on tool for mouse research facilities looking for a LIMS.

  10. Molecular and cytogenetic characterization of expanded B-cell clones from multiclonal versus monoclonal B-cell chronic lymphoproliferative disorders

    PubMed Central

    Henriques, Ana; Rodríguez-Caballero, Arancha; Criado, Ignacio; Langerak, Anton W.; Nieto, Wendy G.; Lécrevisse, Quentin; González, Marcos; Cortesão, Emília; Paiva, Artur; Almeida, Julia; Orfao, Alberto

    2014-01-01

    Chronic antigen-stimulation has been recurrently involved in the earlier stages of monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The expansion of two or more B-cell clones has frequently been reported in individuals with these conditions; potentially, such coexisting clones have a greater probability of interaction with common immunological determinants. Here, we analyzed the B-cell receptor repertoire and molecular profile, as well as the phenotypic, cytogenetic and hematologic features, of 228 chronic lymphocytic leukemia-like and non-chronic lymphocytic leukemia-like clones comparing multiclonal (n=85 clones from 41 cases) versus monoclonal (n=143 clones) monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The B-cell receptor of B-cell clones from multiclonal cases showed a slightly higher degree of HCDR3 homology than B-cell clones from mono clonal cases, in association with unique hematologic (e.g. lower B-lymphocyte counts) and cytogenetic (e.g. lower frequency of cytogenetically altered clones) features usually related to earlier stages of the disease. Moreover, a subgroup of coexisting B-cell clones from individual multiclonal cases which were found to be phylogenetically related showed unique molecular and cytogenetic features: they more frequently shared IGHV3 gene usage, shorter HCDR3 sequences with a greater proportion of IGHV mutations and del(13q14.3), than other unrelated B-cell clones. These results would support the antigen-driven nature of such multiclonal B-cell expansions, with potential involvement of multiple antigens/epitopes. PMID:24488564

  11. 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

  12. Economical quantum cloning in any dimension

    SciTech Connect

    Durt, Thomas; Fiurasek, Jaromir; Cerf, Nicolas J.

    2005-11-15

    The possibility of cloning a d-dimensional quantum system without an ancilla is explored, extending on the economical phase-covariant cloning machine for qubits found in Phys. Rev. A 60, 2764 (1999). We prove the impossibility of constructing an economical version of the optimal universal 1{yields}2 cloning machine in any dimension. We also show, using an ansatz on the generic form of cloning machines, that the d-dimensional 1{yields}2 phase-covariant cloner, which optimally clones all balanced superpositions with arbitrary phases, can be realized economically only in dimension d=2. The used ansatz is supported by numerical evidence up to d=7. An economical phase-covariant cloner can nevertheless be constructed for d>2, albeit with a slightly lower fidelity than that of the optimal cloner requiring an ancilla. Finally, using again an ansatz on cloning machines, we show that an economical version of the 1{yields}2 Fourier-covariant cloner, which optimally clones the computational basis and its Fourier transform, is also possible only in dimension d=2.

  13. Who is the parent in cloning?

    PubMed

    Elster, N

    1999-01-01

    In July 1996, a sheep named Dolly was born in Scotland. What makes Dolly's birth noteworthy is that she is the result of the first successful cloning attempt using the nucleus of an adult cell. The technique that led to Dolly's birth involved transferring the nucleus of a mammary cell from an adult sheep to the enucleated egg cell of an unrelated sheep with gestation occurring in a third sheep. The possibility of applying this technique to human reproduction raised concerns worldwide with several countries moving for an immediate bans on human cloning. In the United States, President Clinton requested that the National Bioethics Advisory Commission ("NBAC"), a multidisciplinary group composed of scientists, lawyers, educators, theologians, and ethicists study the implications of cloning and issue recommendations. The Commission consulted other scientists, ethicists, theologians, lawyers, and citizens with interests in this advancing technology and concluded that, "at this time it is morally unacceptable for anyone in the public or private sector, whether in a research or clinical setting, to attempt to create a child using somatic cell nuclear transfer cloning." This Article was included in a larger work prepared at the request of, and submitted to the Commission by, law professor Lori B. Andrews. Cloning through nuclear transfer will change the way we create and define families. This Article explores how existing law relating to parentage, surrogacy, egg donation, and artificial insemination may apply in the cloning context to clarify the parent-child relationship established through cloning.

  14. Human embryo cloning prohibited in Hong Kong.

    PubMed

    Liu, Athena

    2005-12-01

    Since the birth of Dolly (the cloned sheep) in 1997, debates have arisen on the ethical and legal questions of cloning-for-biomedical-research (more commonly termed "therapeutic cloning") and of reproductive cloning using human gametes. Hong Kong enacted the Human Reproductive Technology Ordinance (Cap 561) in 2000. Section 15(1)(e) of this Ordinance prohibits the "replacing of the nucleus of a cell of an embryo with a nucleus taken from any other cell," i.e., nucleus substitution. Section 15(1)(f) prohibits the cloning of any embryo. The scope of the latter, therefore, is arguably the widest, prohibiting all cloning techniques such as cell nucleus replacement, embryo splitting, parthenogenesis, and cloning using stem cell lines. Although the Human Reproductive Technology Ordinance is not yet fully operative, this article examines how these prohibitions may adversely impact on basic research and the vision of the Hong Kong scientific community. It concludes that in light of recent scientific developments, it is time to review if the law offers a coherent set of policies in this area.

  15. Who is the parent in cloning?

    PubMed

    Elster, N

    1999-01-01

    In July 1996, a sheep named Dolly was born in Scotland. What makes Dolly's birth noteworthy is that she is the result of the first successful cloning attempt using the nucleus of an adult cell. The technique that led to Dolly's birth involved transferring the nucleus of a mammary cell from an adult sheep to the enucleated egg cell of an unrelated sheep with gestation occurring in a third sheep. The possibility of applying this technique to human reproduction raised concerns worldwide with several countries moving for an immediate bans on human cloning. In the United States, President Clinton requested that the National Bioethics Advisory Commission ("NBAC"), a multidisciplinary group composed of scientists, lawyers, educators, theologians, and ethicists study the implications of cloning and issue recommendations. The Commission consulted other scientists, ethicists, theologians, lawyers, and citizens with interests in this advancing technology and concluded that, "at this time it is morally unacceptable for anyone in the public or private sector, whether in a research or clinical setting, to attempt to create a child using somatic cell nuclear transfer cloning." This Article was included in a larger work prepared at the request of, and submitted to the Commission by, law professor Lori B. Andrews. Cloning through nuclear transfer will change the way we create and define families. This Article explores how existing law relating to parentage, surrogacy, egg donation, and artificial insemination may apply in the cloning context to clarify the parent-child relationship established through cloning. PMID:12650149

  16. Human embryo cloning prohibited in Hong Kong.

    PubMed

    Liu, Athena

    2005-12-01

    Since the birth of Dolly (the cloned sheep) in 1997, debates have arisen on the ethical and legal questions of cloning-for-biomedical-research (more commonly termed "therapeutic cloning") and of reproductive cloning using human gametes. Hong Kong enacted the Human Reproductive Technology Ordinance (Cap 561) in 2000. Section 15(1)(e) of this Ordinance prohibits the "replacing of the nucleus of a cell of an embryo with a nucleus taken from any other cell," i.e., nucleus substitution. Section 15(1)(f) prohibits the cloning of any embryo. The scope of the latter, therefore, is arguably the widest, prohibiting all cloning techniques such as cell nucleus replacement, embryo splitting, parthenogenesis, and cloning using stem cell lines. Although the Human Reproductive Technology Ordinance is not yet fully operative, this article examines how these prohibitions may adversely impact on basic research and the vision of the Hong Kong scientific community. It concludes that in light of recent scientific developments, it is time to review if the law offers a coherent set of policies in this area. PMID:16331533

  17. FX cloning: a simple and robust high-throughput cloning method for protein expression.

    PubMed

    Geertsma, Eric R

    2014-01-01

    The immense amount of gene sequences available nowadays allows scientist to screen broadly for extraordinary proteins. Reliable cloning tools that allow the parallel processing of many targets are vital for the success of this strategy. The FX cloning procedure detailed here is such a straightforward and efficient tool. It is dedicated to the cloning of open reading frames (ORFs) with the final aim of expressing the corresponding proteins. FX cloning combines attractive features of established high-throughput cloning methods that were thus far not unified in one single method. It facilitates the subcloning of a sequence-verified ORF to a variety of expression vectors, but is sufficiently versatile to accept PCR products as well. Moreover, the common, but undesirable feature of extending target ORFs with long cloning-related sequences is avoided. It leads to the addition of only one amino acid to each side of the protein. As a consequence, only one primer pair or PCR product suffices to generate expression vectors for both N- and C-terminal translational fusions. FX cloning is highly efficient and economical in its use. The method is suited for high-throughput cloning projects and also for everyday cloning of single targets. FX cloning is based on the use of type IIS restriction enzymes and negative selection markers. The full procedure takes place in one pot in less than 3 h and does not require intermediate purification steps nor extensive handling. The method has proven to be very robust and suitable for all common expression systems.

  18. The UniTrap resource: tools for the biologist enabling optimized use of gene trap clones

    PubMed Central

    Roma, Guglielmo; Sardiello, Marco; Cobellis, Gilda; Cruz, Pedro; Lago, Giampiero; Sanges, Remo; Stupka, Elia

    2008-01-01

    We have developed a comprehensive resource devoted to biologists wanting to optimize the use of gene trap clones in their experiments. We have processed 300 602 such clones from both public and private projects to generate 28 199 ‘UniTraps’, i.e. distinct collections of unambiguous insertions at the same subgenic region of annotated genes. The UniTrap resource contains data relative to 9583 trapped genes, which represent 42.3% of the mouse gene content. Among the trapped genes, 7 728 have a counterpart in humans, and 677 are known to be involved in the pathogenesis of human diseases. The aim of this analysis is to provide the wet lab researchers with a comprehensive database and curated tools for (i) identifying and comparing the clones carrying a trap into the genes of interest, (ii) evaluating the severity of the mutation to the protein function in each independent trapping event and (iii) supplying complete information to perform PCR, RT-PCR and restriction experiments to verify the clone and identify the exact point of vector insertion. To share this unique resource with the scientific community, we have designed and implemented a web interface that is freely accessible at http://unitrap.cbm.fvg.it/. PMID:17942430

  19. Expression cloning of a rat liver Na(+)-independent organic anion transporter.

    PubMed Central

    Jacquemin, E; Hagenbuch, B; Stieger, B; Wolkoff, A W; Meier, P J

    1994-01-01

    Using expression cloning in Xenopus laevis oocytes, we have isolated a cDNA encoding a rat liver organic anion-transporting polypeptide (oatp). The cloned oatp mediated Na(+)-independent uptake of sulfobromophthalein (BSP) which was Cl(-)-dependent in the presence of bovine serum albumin (BSA) at low BSP concentrations (e.g., 2 microM). Addition of increasing amounts of BSA had no effects on the maximal velocity of initial BSP uptake, but it increased the Km value from 1.5 microM (no BSA) to 24 microM (BSA/BSP molar ratio, 3.7) and 35 microM (BSA/BSP ratio, 18.4). In addition to BSP, the cloned oatp also mediated Na(+)-independent uptake of conjugated (taurocholate) and unconjugated (cholate) bile acids. Sequence analysis of the cDNA revealed an open reading frame of 2010 nucleotides coding for a protein of 670 amino acids (calculated molecular mass, 74 kDa) with four possible N-linked glycosylation sites and 10 putative transmembrane domains. Translation experiments in vitro indicated that the transporter was indeed glycosylated and that its polypeptide backbone had an apparent molecular mass of 59 kDa. Northern blot analysis with the cloned probe revealed crossreactivity with several mRNA species from rat liver, kidney, brain, lung, skeletal muscle, and proximal colon as well as from liver tissues of mouse and rabbit, but not of skate (Raja erinacea) and human. Images Fig. 5 PMID:8278353

  20. Systems Genetics Implicates Cytoskeletal Genes in Oocyte Control of Cloned Embryo Quality

    PubMed Central

    Cheng, Yong; Gaughan, John; Midic, Uros; Han, Zhiming; Liang, Cheng-Guang; Patel, Bela G.; Latham, Keith E.

    2013-01-01

    Cloning by somatic cell nuclear transfer is an important technology, but remains limited due to poor rates of success. Identifying genes supporting clone development would enhance our understanding of basic embryology, improve applications of the technology, support greater understanding of establishing pluripotent stem cells, and provide new insight into clinically important determinants of oocyte quality. For the first time, a systems genetics approach was taken to discover genes contributing to the ability of an oocyte to support early cloned embryo development. This identified a primary locus on mouse chromosome 17 and potential loci on chromosomes 1 and 4. A combination of oocyte transcriptome profiling data, expression correlation analysis, and functional and network analyses yielded a short list of likely candidate genes in two categories. The major category—including two genes with the strongest genetic associations with the traits (Epb4.1l3 and Dlgap1)—encodes proteins associated with the subcortical cytoskeleton and other cytoskeletal elements such as the spindle. The second category encodes chromatin and transcription regulators (Runx1t1, Smchd1, and Chd7). Smchd1 promotes X chromosome inactivation, whereas Chd7 regulates expression of pluripotency genes. Runx1t1 has not been associated with these processes, but acts as a transcriptional repressor. The finding that cytoskeleton-associated proteins may be key determinants of early clone development highlights potential roles for cytoplasmic components of the oocyte in supporting nuclear reprogramming. The transcriptional regulators identified may contribute to the overall process as downstream effectors. PMID:23307892

  1. A lotka-volterra model of coexistence between a sexual population and multiple asexual clones.

    PubMed

    Pound, Graeme E; Doncaster, C Patrick; Cox, Simon J

    2002-08-21

    At carrying capacity, small advantages in competitive ability can compensate a sexual population for its two-fold disadvantage in growth capacity when facing invasion by asexual mutants. In this paper, we develop a generic analytical model to consider the ecology of a sexual population comprising equal numbers of males and females, competing for shared prey resources with multiple female-only clones. We assume that the clones arise from the sexual population and are distinguished from it only by having narrower resource niches and twice the growth capacity. For sexual populations, at density-dependent carrying capacity, intra-specific competition between clonal individuals prevents them from realizing their two-fold advantage in intrinsic growth. This prediction leads to three novel outcomes: (i) a sexual population can coexist with any number of clones, provided their combined competitive impact remains less than the impact of the clones on each other; (ii) a sexual species can immediately exclude asexual invaders if it is a fast growing and strong competitor of shared resources and also has refuge in an abundant alternative resource; (iii) the rate of accumulation of clones in a sexual population will be slowed by intra and inter-specific competition amongst the clones themselves, in addition to the competitive impact from the original sexual population.

  2. Mouse bladder wall injection.

    PubMed

    Fu, Chi-Ling; Apelo, Charity A; Torres, Baldemar; Thai, Kim H; Hsieh, Michael H

    2011-07-12

    Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

  3. Mouse Palmitoyl Protein Thioesterase: Gene Structure and Expression of cDNA

    PubMed Central

    Salonen, Tarja; Hellsten, Elina; Horelli-Kuitunen, Nina; Peltonen, Leena; Jalanko, Anu

    1998-01-01

    Palmitoyl protein thioesterase (PPT) is the defective enzyme in infantile neuronal ceroid lipofuscinosis (INCL), which is a recessively inherited, progressive neurodegenerative disorder. We present here the cloning, chromosomal mapping, genomic structure, and the expression of the cDNA of mouse PPT. The mouse PPT gene spans >21 kb of genomic DNA and contains nine exons with a coding sequence of 918 bp. Fluorescence in situ hybridization to metaphase chromosomes localized the mouse PPT gene to the chromosome 4 conserved syntenic region with human chromosome 1p32 where the human PPT is located. PPT is expressed widely in a variety of mouse tissues. The mouse PPT cDNA is conserved highly with the human and rat PPT both at the nucleotide and amino acid sequence level. Transient expression of mouse PPT in COS-1 cells yielded a 38/36-kD differentially glycosylated polypeptide that was also secreted into culture media. Immunofluorescence analysis of transiently transfected HeLa cells indicated lysosomal localization of mouse PPT. Based on the high conservation of the gene and polypeptide structure as well as similar processing and intracellular localization, the function of PPT in mouse and human are likely to be very similar. [The sequence data described in this paper have been submitted to GenBank under accession no. AF071O25.] PMID:9685319

  4. Characterization of Bovine NANOG5′-flanking Region during Differentiation of Mouse Embryonic Stem Cells

    PubMed Central

    Jang, Hye-Jeong; Park, Hwan Hee; Linh, Tran Thi Thuy; Lee, Hak-Kyo; Song, Ki-Duk; Lee, Woon Kyu

    2015-01-01

    Embryonic stem cells (ESCs) have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (−420/+181) bovine NANOG 5′-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP) as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (−420/+181) promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells. PMID:26580439

  5. (New hosts and vectors for genome cloning)

    SciTech Connect

    Not Available

    1991-01-01

    The main goal of our project remains the development of new bacterial hosts and vectors for the stable propagation of human DNA clones in E. coli. During the past six months of our current budget period, we have (1) continued to develop new hosts that permit the stable maintenance of unstable features of human DNA, and (2) developed a series of vectors for (a) cloning large DNA inserts, (b) assessing the frequency of human sequences that are lethal to the growth of E. coli, and (c) assessing the stability of human sequences cloned in M13 for large-scale sequencing projects.

  6. Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a) P-Glycoprotein cDNA.

    PubMed

    Pluchino, Kristen M; Esposito, Dominic; Moen, Janna K; Hall, Matthew D; Madigan, James P; Shukla, Suneet; Procter, Lauren V; Wall, Vanessa E; Schneider, Thomas D; Pringle, Ian; Ambudkar, Suresh V; Gill, Deborah R; Hyde, Steven C; Gottesman, Michael M

    2015-01-01

    The efflux transporter P-glycoprotein (P-gp) is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.

  7. Phylogenetic conservation of immunoglobulin heavy chains: direct comparison of hamster and mouse Cmu genes.

    PubMed Central

    McGuire, K L; Duncan, W R; Tucker, P W

    1985-01-01

    We have analyzed the JH-Cmu locus of the Syrian hamster by DNA cloning and sequencing. The single Cmu gene is highly homologous to that of the mouse. The hamster equivalents of the JH and switch (S) recombination regions are arranged as in the mouse, but surprisingly are not highly conserved. Also unlike its close murine relative, the Smu regions among inbred hamster strains are not polymorphic. The complete nucleotide sequence of hamster and mouse Cmu genes have been compared to partial Cmu sequences of other species. Conservation within a portion of the 3' untranslated region may signify functional requirements for 3' end processing. Mutational frequencies within exons and introns of hamster and mouse do not support the theory that the rate of DNA transitions to transversions decreases with evolutionary distance. Images PMID:2994005

  8. Characterizing a distal muscle enhancer in the mouse Igf2 locus.

    PubMed

    Alzhanov, Damir; Rotwein, Peter

    2016-02-01

    Insulin-like growth factor-2 (IGF2) is highly expressed in skeletal muscle and was identified as a quantitative trait locus for muscle mass. Yet little is known about mechanisms of its regulation in muscle. Recently, a DNA segment found ∼100 kb from the Igf2 gene was identified as a possible muscle transcriptional control element. Here we have developed an in vivo reporter system to assess this putative enhancer by substituting nuclear (n) EGFP for Igf2 coding exons in a bacterial artificial chromosome containing the mouse Igf2 - H19 chromosomal locus. After stable transfection into a mesenchymal stem cell line, individual clones were converted to myoblasts and underwent progressive muscle-specific gene expression and myotube formation in differentiation medium. Transgenic mRNA and nuclear-targeted enhanced green fluorescent protein were produced coincident with endogenous Igf2 mRNA, but only in lines containing an intact distal conserved DNA element. Our results show that a 294 bp DNA fragment containing two E-boxes is a necessary and sufficient long-range enhancer for induction of Igf2 gene transcription during skeletal muscle differentiation and provides a robust experimental platform for its further functional dissection. PMID:26645089

  9. Differentiation of human B lymphocyte subpopulations induced by an alloreactive helper T-cell clone

    SciTech Connect

    Anderson, S.J.; Hummell, D.S.; Lawton, A.R.

    1988-07-01

    We have used cloned alloreactive helper T cells to determine if direct T cell-B cell interaction can induce differentiation of human peripheral blood B cells which do not respond to pokeweed mitogen (PWM). T-cell clone 2F8 was derived from a one-way mixed lymphocyte reaction. 2F8 cells are T3+T4+T8-IL-2R+ and proliferate in response to irradiated stimulator cells, but not autologous cells, in the absence of exogenous interleukin-2. 2F8 cells provide allospecific help for polyclonal proliferation and differentiation of B cells in the absence of any other stimulus. The magnitude of this response is comparable to that of the response of the same B cells to PWM and fresh autologous T cells. 2F8 cells could also provide nonspecific help for unrelated donor B cells in the presence of PWM, with no requirement for costimulation by irradiated stimulator cells. Allospecific stimulation of B cells was completely inhibited by antibodies to class II major histocompatibility complex (MHC) framework determinants and was abrogated by 1000-rad irradiation. Cloned 2F8 T cells stimulated differentiation of both small, high-density B cells and larger B cells, generating up to 30% plasma cells with either fraction. B cells forming rosettes with mouse erythrocytes were also induced to differentiate by the helper T cell clone. As found previously, neither small, high-density B cells nor mouse rosette+ B cells responded well to PWM. Direct interaction with allospecific T cells induces differentiation of a broader spectrum of B cells than soluble growth and differentiation factors in conjunction with polyclonal activators such as PWM and protein A containing staphylococci.

  10. Calpain 11 Is Unique to Mouse Spermatogenic Cells

    PubMed Central

    Ben-Aharon, Irit; Brown, Paula R.; Shalgi, Ruth; Eddy, Edward M.

    2007-01-01

    The calpains are a family of calcium-dependent thiol proteases involved in intracellular processing of proteins. They occur as heterodimers containing one of various large subunits and a common small subunit. Some of the large subunits are expressed ubiquitously and others are expressed in a restricted set of tissues. We have cloned the cDNA for mouse calpain 11 and demonstrated that it is expressed specifically in the mouse testis. The mRNA begins to accumulate in the testis between days 14 and 16 days after birth, corresponding to the period of pachytene spermatocyte development. The protein is detected by day 18 after birth, during mid to late pachytene spermatocyte development, and is present in the acrosomal region of spermatozoa from the cauda epididymis. The expression of calpain 11 during spermatogenesis and its localization is spermatozoa suggest that it is involved in regulating calcium-dependent signal transduction events during meiosis and sperm functional processes. PMID:16541461

  11. Translating Mouse Vocalizations: Prosody and Frequency Modulation

    PubMed Central

    Lahvis, Garet P.; Alleva, Enrico; Scattoni, Maria Luisa

    2010-01-01

    Mental illness can include impaired abilities to express emotions or respond to the emotions of others. Speech provides a mechanism for expressing emotions, by both what words are spoken and by the melody or intonation of speech (prosody). Through the perception of variations in prosody, an individual can detect changes in another's emotional state. Prosodic features of mouse ultrasonic vocalizations (USVs), indicated by changes in frequency and amplitude, also convey information. Dams retrieve pups that emit separation calls, females approach males emitting solicitous calls, and mice can become fearful of a cue associated with the vocalizations of a distressed conspecific. Since acoustic features of mouse USVs respond to drugs and genetic manipulations that influence reward circuits, USV analysis can be employed to examine how genes influence social motivation, affect regulation, and communication. The purpose of this review is to discuss how genetic and developmental factors influence aspects of the mouse vocal repertoire and how mice respond to the vocalizations of their conspecifics. To generate falsifiable hypotheses about the emotional content of particular calls, this review addresses USV analysis within the framework of affective neuroscience (e.g. measures of motivated behavior such as conditioned place preference tests, brain activity, and systemic physiology). Suggested future studies include employment of an expanded array of physiological and statistical approaches to identify the salient acoustic features of mouse vocalizations. We are particularly interested in rearing environments that incorporate sufficient spatial and temporal complexity to familiarize developing mice with a broader array of affective states. PMID:20497235

  12. MausDB: An open source application for phenotype data and mouse colony management in large-scale mouse phenotyping projects

    PubMed Central

    Maier, Holger; Lengger, Christoph; Simic, Bruno; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabé de Angelis, Martin

    2008-01-01

    Background Large-scale, comprehensive and standardized high-throughput mouse phenotyping has been established as a tool of functional genome research by the German Mouse Clinic and others. In all these projects, vast amounts of data are continuously generated and need to be stored, prepared for data-mining procedures and eventually be made publicly available. Thus, central storage and integrated management of mouse phenotype data, genotype data, metadata and linked external data are highly important. Requirements most probably depend on the individual mouse housing unit or project and the demand for either very specific individual database solutions or very flexible solutions that can be easily adapted to local demands. Not every group has the resources and/or the know-how to develop software for this purpose. A database application has been developed for the German Mouse Clinic in order to meet all requirements mentioned above. Results We present MausDB, the German Mouse Clinic web-based database application that integrates standard mouse colony management, phenotyping workflow scheduling features and mouse phenotyping result data management. It links mouse phenotype data with genotype data, metadata and external data such as public web databases, which is a prerequisite for comprehensive data analysis and mining. We describe how this can be achieved with a lean and user-friendly system built on open standards. Conclusion MausDB is suited for large-scale, high-throughput phenotyping facilities but can also be used exclusively for mouse colony management within smaller units or projects. The system is successfully used as the primary mouse and data management tool of the German Mouse Clinic and other mouse facilities. We offer MausDB to the scientific community as open source software to provide a system for storage of data from functional genomics projects in a well-structured, easily accessible form. PMID:18366799

  13. Optimal cloning of mixed Gaussian states

    SciTech Connect

    Guta, Madalin; Matsumoto, Keiji

    2006-09-15

    We construct the optimal one to two cloning transformation for the family of displaced thermal equilibrium states of a harmonic oscillator, with a fixed and known temperature. The transformation is Gaussian and it is optimal with respect to the figure of merit based on the joint output state and norm distance. The proof of the result is based on the equivalence between the optimal cloning problem and that of optimal amplification of Gaussian states which is then reduced to an optimization problem for diagonal states of a quantum oscillator. A key concept in finding the optimum is that of stochastic ordering which plays a similar role in the purely classical problem of Gaussian cloning. The result is then extended to the case of n to m cloning of mixed Gaussian states.

  14. Generation of phase-covariant quantum cloning

    SciTech Connect

    Karimipour, V.; Rezakhani, A.T.

    2002-11-01

    It is known that in phase-covariant quantum cloning, the equatorial states on the Bloch sphere can be cloned with a fidelity higher than the optimal bound established for universal quantum cloning. We generalize this concept to include other states on the Bloch sphere with a definite z component of spin. It is shown that once we know the z component, we can always clone a state with a fidelity higher than the universal value and that of equatorial states. We also make a detailed study of the entanglement properties of the output copies and show that the equatorial states are the only states that give rise to a separable density matrix for the outputs.

  15. Dolly, the so-called clone.

    PubMed

    Mittwoch, Ursula

    2003-02-01

    The word 'clone' is in the forefront of scientific terms that are familiar to the general public. But what exactly does it mean? Suprisingly, there is no simple answer, for different scientists are using the term for different entities.

  16. Cloning and characterization of new bioluminescent proteins

    NASA Astrophysics Data System (ADS)

    Szent-Gyorgyi, Christopher; Ballou, Byron T.; Dagnal, Erich; Bryan, Bruce

    1999-07-01

    Over the past two years Prolume has undertaken a comprehensive program to clone luciferases and associated 'green fluorescent proteins' (GFPs) from marine animals that use coelenterazine as the luciferin. To data we have cloned several bioluminescent proteins, including two novel copepod luciferases and two anthozoan GFPs. These four proteins have sequences that differ greatly form previously cloned analogous proteins; the sequence diversity apparently is due to independent evolutionary origins and unusual evolutionary constraints. Thus coelenterazine-based bioluminescent systems may also manifest a variety of useful properties. We discuss form this taxonomic perspective the initial biochemical and spectral characterization of our cloned proteins. Emphasis is placed on the anthozoan luciferase-GFP systems, whose efficient resonance energy transfer has elicited much current interest.

  17. Nuclear cloning, epigenetic reprogramming and cellular differentiation.

    PubMed

    Jaenisch, Rudolf; Hochedlinger, Konrad; Eggan, Kevin

    2005-01-01

    The full-term development of sheep, cows, goats, pigs and mice has been achieved through the transfer of somatic cell nuclei into enucleated oocytes. Despite these successes, mammalian cloning remains an inefficient process, with a preponderance of reconstructed embryos failing at early- to mid-gestation stages of development. The small percentage of conceptuses that survive to term are characterized by a high mortality rate and frequently display grossly increased placental and birth weights. It is likely that inappropriate expression of key developmental genes may contribute to lethality of cloned embryos. One of the most interesting issues of nuclear cloning is the question of genomic reprogramming, i.e. whether successful cloning requires the resetting of epigenetic modifications which are characteristic of the adult donor nucleus. Processes such as X-inactivation and genomic imprinting are known to depend on epigenetic modifications of the genome. The classical nuclear transfer experiments with frogs have suggested that the source of the donor nucleus affects the phenotype of the clone. We have, using expression profiling, compared gene expression in clones derived from embryonic stem (ES) cells and from somatic donor cell nuclei and find substantial gene dysregulation. Our results suggest that faulty reprogramming is caused by the nuclear cloning procedure itself. In addition, the type of donor nucleus contributes to the abnormal expression pattern seen in cloned animals. One of the major unresolved issues has been whether nuclei of terminally differentiated cells can be reprogrammed by transfer into the oocyte. To address this question we have derived monoclonal mice from B and T cells and used the genetic rearrangements of the immunoglobulin and T cell receptor genes to retrospectively verify the differentiation state of the donor nucleus. Finally, we discuss our recent studies on the reprogramming of nuclei from terminally differentiated neurons and from

  18. Cloning in America: constitutional rights and limits.

    PubMed

    Erwin, C

    2000-01-01

    As readers of science fiction are well aware, the term "clone" refers to asexually produced offspring, that is, produced by a process of cell-division which does not begin with the union of two sex cells. A clone would be the genetic twin of the cell donor. Propagation of plants by this method is, of course, commonplace, but mammalian reproduction in this fashion would be indeed a revolutionary accomplishment, with profound and disturbing implications. PMID:14627028

  19. The Mouse SAGE Site: database of public mouse SAGE libraries.

    PubMed

    Divina, Petr; Forejt, Jirí

    2004-01-01

    The Mouse SAGE Site is a web-based database of all available public libraries generated by the Serial Analysis of Gene Expression (SAGE) from various mouse tissues and cell lines. The database contains mouse SAGE libraries organized in a uniform way and provides web-based tools for browsing, comparing and searching SAGE data with reliable tag-to-gene identification. A modified approach based on the SAGEmap database is used for reliable tag identification. The Mouse SAGE Site is maintained on an ongoing basis at the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic and is accessible at the internet address http://mouse.biomed.cas.cz/sage/.

  20. The mouse and human genes encoding the recognition component of the N-end rule pathway

    PubMed Central

    Kwon, Yong Tae; Reiss, Yuval; Fried, Victor A.; Hershko, Avram; Yoon, Jeong Kyo; Gonda, David K.; Sangan, Pitchai; Copeland, Neal G.; Jenkins, Nancy A.; Varshavsky, Alexander

    1998-01-01

    The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3α. Mouse UBR1p (E3α) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans ≈120 kilobases of genomic DNA and contains ≈50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway. PMID:9653112

  1. Genomic structure and chromosomal assignment of the mouse Ku70 gene

    SciTech Connect

    Takiguchi, Yuichi |; Kurimasa, Akihiro; Chen, Fanqing

    1996-07-01

    DNA-dependent protein kinase (DNA-PK) consists of three polypeptide subunits: Ku70, Ku80, and the DNA-PK catalytic subunit (DNA-PKcs). Mammalian mutants deficient in either Ku80 or DNA-PKcs function have been shown to be lacking in DNA double-strand break repair and V(D)J recombination, respectively. The precise role of the Ku70 gene in this process has not yet been determined, in part because no cell lines, animals, or human diseases involved with deficiencies in this gene have yet been identified. Both the human and the mouse Ku70 cDNAs have been cloned, and the human gene has been mapped to chromosome 22q13. The original mouse cDNA clones, however, lacked a complete 5{prime}-region, and none of the mammalian Ku70 genomic sequences have been characterized. This report contains an analysis of the 5{prime}-region of the mouse cDNA sequence, a characterization of the mouse Ku70 genomic structure, and fluorescence in situ hybridization data that map the mouse gene to chromosome 15. The deduced amino acid sequence of the mouse gene consists of 608 amino acids compared to 609 for the human gene. The genomic sequence is 24 kb and consists of 13 exons, including an untranslated first exon. Sequences form the upstream region of exon 1 revealed four consensus GC box sequences and a strong transcription initiation site at a reasonable location. The assignment of the mouse Ku70 gene to chromosome 15 is consistent with the syntenic relationship of this gene in human (chromosome 22q13) and mouse and adds to the comparative mapping data for the genes involved in the SCID phenotype. 39 refs., 3 figs.

  2. Reproductive cloning combined with genetic modification.

    PubMed

    Strong, C

    2005-11-01

    Although there is widespread opposition to reproductive cloning, some have argued that its use by infertile couples to have genetically related children would be ethically justifiable. Others have suggested that lesbian or gay couples might wish to use cloning to have genetically related children. Most of the main objections to human reproductive cloning are based on the child's lack of unique nuclear DNA. In the future, it may be possible safely to create children using cloning combined with genetic modifications, so that they have unique nuclear DNA. The genetic modifications could be aimed at giving such children genetic characteristics of both members of the couple concerned. Thus, cloning combined with genetic modification could be appealing to infertile, lesbian, or gay couples who seek genetically related children who have genetic characteristics of both members. In such scenarios, the various objections to human reproductive cloning that are based on the lack of genetic uniqueness would no longer be applicable. The author argues that it would be ethically justifiable for such couples to create children in this manner, assuming these techniques could be used safely.

  3. Cloning: Past, Present, and the Exciting Future. Breakthroughs in Bioscience.

    ERIC Educational Resources Information Center

    Di Berardino, Marie A.

    This document explores the history of cloning by focusing on Dolly the Sheep, one of the first large animal clonings. The disadvantages and advantages of transgenic clones are discussed as well as the future implications of cloning from the perspective of human health. (Contains 10 resources.) (YDS)

  4. Mouse and human FcR effector functions.

    PubMed

    Bruhns, Pierre; Jönsson, Friederike

    2015-11-01

    Mouse and human FcRs have been a major focus of attention not only of the scientific community, through the cloning and characterization of novel receptors, and of the medical community, through the identification of polymorphisms and linkage to disease but also of the pharmaceutical community, through the identification of FcRs as targets for therapy or engineering of Fc domains for the generation of enhanced therapeutic antibodies. The availability of knockout mouse lines for every single mouse FcR, of multiple or cell-specific--'à la carte'--FcR knockouts and the increasing generation of hFcR transgenics enable powerful in vivo approaches for the study of mouse and human FcR biology. This review will present the landscape of the current FcR family, their effector functions and the in vivo models at hand to study them. These in vivo models were recently instrumental in re-defining the properties and effector functions of FcRs that had been overlooked or discarded from previous analyses. A particular focus will be made on the (mis)concepts on the role of high-affinity IgG receptors in vivo and on results from antibody engineering to enhance or abrogate antibody effector functions mediated by FcRs. PMID:26497511

  5. Cloning the entanglement of a pair of quantum bits

    SciTech Connect

    Lamoureux, Louis-Philippe; Navez, Patrick; Cerf, Nicolas J.; Fiurasek, Jaromir

    2004-04-01

    It is shown that any quantum operation that perfectly clones the entanglement of all maximally entangled qubit pairs cannot preserve separability. This 'entanglement no-cloning' principle naturally suggests that some approximate cloning of entanglement is nevertheless allowed by quantum mechanics. We investigate a separability-preserving optimal cloning machine that duplicates all maximally entangled states of two qubits, resulting in 0.285 bits of entanglement per clone, while a local cloning machine only yields 0.060 bits of entanglement per clone.

  6. Structure of the TCR expressed on a gastritogenic T cell clone, II-6, and frequent appearance of similar clonotypes in mice bearing autoimmune gastritis.

    PubMed

    Katakai, T; Agata, Y; Shimizu, A; Ohshima, C; Nishio, A; Inaba, M; Kasakura, S; Mori, K J; Masuda, T

    1997-12-01

    A parietal cell-specific Th1 clone, II-6, which was established from a BALB/c mouse bearing post-thymectomy autoimmune gastritis (AIG), recognizes a peptide of the alpha subunit (alpha891-905) of H+/K+-ATPase and induces gastritis in nu/nu BALB/c mice by adoptive cell transfer. In the present study, the primary structure of the TCR of II-6 was determined as Valpha10-Jalpha c5a-Calpha and Vbeta14-Jbeta2.3-Cbeta2 by cDNA cloning. Using PCR with specific primers, we defined the use of this II-6 TCR in nu/nu mice with transferred II-6 cells and in mice that spontaneously developed AIG by thymectomy on day 3 after birth (d3-Tx). II-6 TCR mRNAs were detected in the gastric mucosa of all of the nu/nu mice, suggesting that II-6 cells indeed home to the gastric mucosa and thereby were directly involved in the destruction of target parietal cells. TCR beta chain mRNAs encoding CDR3 region sequences almost identical with that of II-6 were also found in the gastric mucosa in 43% (six of 14 mice tested) of the d3-Tx AIG mice at 4-12 weeks old by nested RT-PCR. Such a frequent appearance of similar clonotypes in independent individuals suggests that T cells bearing II-6-like TCR including the II-6 itself might be directly involved in, although not essential for, the pathogenesis of AIG in 3d-Tx mice.

  7. Identification and chromosomal localization of Atm, the mouse homolog of the ataxia-telangiectasia gene

    SciTech Connect

    Pecker, I.; Savitsky, K.; Rotman, G.

    1996-07-01

    Atm, the mouse homolog of the human ATM gene defective in ataxia-telangiectasia (A-T), has been identified. The entire coding sequence of the Atm transcript was cloned and found to contain an open reading frame encoding a protein of 3066 amino acids with 84% overall identity and 91% similarity to the human ATM protein. Variable levels of expression of Atm were observed in different tissues. Fluorescence in situ hybridization and linkage analysis located the Atm gene on mouse chromosome 9, band 9C, in a region homologous to the ATM region on human chromosome 11q22-q23. 32 refs., 6 figs.

  8. Mapping of the Tuple1 gene to mouse chromosome 16A-B1

    SciTech Connect

    Mattei, M.G.; Halford, S.; Scambler, P.J.

    1994-10-01

    The human TUPLE1 gene encodes a putative transcriptional regulator and maps to chromosome 22, and therefore may play a role in Di-George syndrome (DGS), relo-cardio-facial syndrome (VCFS), or a related pathology. The murine TUPLE1 gene has also been cloned and shows strong sequence similarity to TUPLE1. Comparative mapping is useful in the study of chromosome evolution and is sometimes able to indicate possible mouse mutations that are potential models of human genetic disorders. As TIPLE1 is a candidate gene for the haploinsufficient phenotype in DGS, we mapped TUPLE1 to mouse chromosome 16A-B1. 6 refs., 1 fig.

  9. Mouse ataxin-3 functional knock-out model.

    PubMed

    Switonski, Pawel M; Fiszer, Agnieszka; Kazmierska, Katarzyna; Kurpisz, Maciej; Krzyzosiak, Wlodzimierz J; Figiel, Maciej

    2011-03-01

    Spinocerebellar ataxia 3 (SCA3) is a genetic disorder resulting from the expansion of the CAG repeats in the ATXN3 gene. The pathogenesis of SCA3 is based on the toxic function of the mutant ataxin-3 protein, but the exact mechanism of the disease remains elusive. Various types of transgenic mouse models explore different aspects of SCA3 pathogenesis, but a knock-in humanized mouse has not yet been created. The initial aim of this study was to generate an ataxin-3 humanized mouse model using a knock-in strategy. The human cDNA for ataxin-3 containing 69 CAG repeats was cloned from SCA3 patient and introduced into the mouse ataxin-3 locus at exon 2, deleting it along with exon 3 and intron 2. Although the human transgene was inserted correctly, the resulting mice acquired the knock-out properties and did not express ataxin-3 protein in any analyzed tissues, as confirmed by western blot and immunohistochemistry. Analyses of RNA expression revealed that the entire locus consisting of human and mouse exons was expressed and alternatively spliced. We detected mRNA isoforms composed of exon 1 spliced with mouse exon 4 or with human exon 7. After applying 37 PCR cycles, we also detected a very low level of the correct exon 1/exon 2 isoform. Additionally, we confirmed by bioinformatic analysis that the structure and power of the splicing site between mouse intron 1 and human exon 2 (the targeted locus) was not changed compared with the native mouse locus. We hypothesized that these splicing aberrations result from the deletion of further splicing sites and the presence of a strong splicing site in exon 4, which was confirmed by bioinformatic analysis. In summary, we created a functional ataxin-3 knock-out mouse model that is viable and fertile and does not present a reduced life span. Our work provides new insights into the splicing characteristics of the Atxn3 gene and provides useful information for future attempts to create knock-in SCA3 models.

  10. The murine ufo receptor: molecular cloning, chromosomal localization and in situ expression analysis.

    PubMed

    Faust, M; Ebensperger, C; Schulz, A S; Schleithoff, L; Hameister, H; Bartram, C R; Janssen, J W

    1992-07-01

    We have cloned the mouse homologue of the ufo oncogene. It encodes a novel tyrosine kinase receptor characterized by a unique extracellular domain containing two immunoglobulin-like and two fibronectin type III repeats. Comparison of the predicted ufo amino acid sequences of mouse and man revealed an overall identity of 87.6%. The ufo locus maps to mouse chromosome 7A3-B1 and thereby extends the known conserved linkage group between mouse chromosome 7 and human chromosome 19. RNA in situ hybridization analysis established the onset of specific ufo expression in the late embryogenesis at day 12.5 post coitum (p.c.) and localized ufo transcription to distinct substructures of a broad spectrum of developing tissues (e.g. subepidermal cells of the skin, mesenchymal cells of the periosteum). In adult animals ufo is expressed in cells forming organ capsules as well as in connective tissue structures. ufo may function as a signal transducer between specific cell types of mesodermal origin.

  11. Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof

    PubMed Central

    Nolden, T.; Pfaff, F.; Nemitz, S.; Freuling, C. M.; Höper, D.; Müller, T.; Finke, Stefan

    2016-01-01

    Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache’s reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level. PMID:27046474

  12. Specific inhibition of lymphokine biosynthesis and autocrine growth using antisense oligonucleotides in Th1 and Th2 helper T cell clones

    PubMed Central

    1988-01-01

    T helper cells have recently been divided into two subsets. The Th1 subset secretes and responds to IL-2 in an autocrine manner. The Th2 subset upon mitogen or antigen stimulation releases IL-4. Here we describe a novel technology that allowed us to confirm this distinction. We have used synthetic oligonucleotides complementary to the 5' end of mouse IL-2 and IL-4 to specifically block the biosynthesis of IL-2 or IL-4 in two murine helper T cell clones from the Th1 or Th2 subset. We show that the antisense IL-2 oligonucleotide inhibited the proliferation of the Th1 clone and had no effect on the Th2 clone. In parallel experiments, the antisense IL-4 oligonucleotide blocked the proliferation of the Th2 clone and not the proliferation of the Th1 clone. The inhibition was significantly reversed in both cases by the addition of the relevant lymphokine (IL-2 in the case of the Th1 clone, IL-4 in the case of the Th2 clone). Northern analysis, using cDNA probes specific for the two lymphokines, showed a decrease in the steady-state level of the relevant lymphokine mRNA, suggesting the specific degradation of the mRNA by an RNase H-like enzymatic activity. This strategy, which allows the specific blockade of the biosynthesis of a lymphokine, could be useful for future studies on the role of each T helper subset in physiological immune responses. PMID:2974066

  13. Within-Host Selection of Drug Resistance in a Mouse Model of Repeated Incomplete Malaria Treatment: Comparison between Atovaquone and Pyrimethamine.

    PubMed

    Nuralitha, Suci; Siregar, Josephine E; Syafruddin, Din; Roelands, Jessica; Verhoef, Jan; Hoepelman, Andy I M; Marzuki, Sangkot

    2015-10-26

    The evolutionary selection of malaria parasites within individual hosts is an important factor in the emergence of drug resistance but is still not well understood. We have examined the selection process for drug resistance in the mouse malaria agent Plasmodium berghei and compared the dynamics of the selection for atovaquone and pyrimethamine. Resistance to these drugs has been shown to be associated with genetic lesions in the dihydrofolate reductase gene in the case of pyrimethamine and in the mitochondrial cytochrome b gene for atovaquone. A mouse malaria model for the selection of drug resistance, based on repeated incomplete treatment (RICT) with a therapeutic dose of antimalarial drugs, was established. The number of treatment cycles for the development of stable resistance to atovaquone (2.47 ± 0.70; n = 19) was found to be significantly lower than for pyrimethamine (5.44 ± 1.46; n = 16; P < 0.0001), even when the parental P. berghei Leiden strain was cloned prior to the resistance selection. Similar results were obtained with P. berghei Edinburgh. Mutational changes underlying the resistance were identified to be S110N in dihydrofolate reductase for pyrimethamine and Y268N, Y268C, Y268S, L271V-K272R, and G280D in cytochrome b for atovaquone. These results are consistent with the rate of mitochondrial DNA mutation being higher than that in the nucleus and suggest that mutation leading to pyrimethamine resistance is not a rare event.

  14. Nonlocal quantum cloning via quantum dots trapped in distant cavities

    NASA Astrophysics Data System (ADS)

    Yu, Tao; Zhu, Ai-Dong; Zhang, Shou

    2012-05-01

    A scheme for implementing nonlocal quantum cloning via quantum dots trapped in cavities is proposed. By modulating the parameters of the system, the optimal 1 → 2 universal quantum cloning machine, 1 → 2 phase-covariant cloning machine, and 1 → 3 economical phase-covariant cloning machine are constructed. The present scheme, which is attainable with current technology, saves two qubits compared with previous cloning machines.

  15. Cloning of the canine glucose-6-phosphatase gene

    SciTech Connect

    Kishnani, P.; Bao, Y.; Brix, A.E.

    1994-09-01

    Two Maltese puppies with massive hepatomegaly and failure to thrive were found to have a markedly reduced Glucose-6-phosphatase (G-6-Pase) activity in the liver and kidney. Deficiency of G-6-Pase activity causes type 1a glycogen storage disease in humans. To further study the mutation responsible for the disease in dog, we cloned G-6-Pase canine cDNA from normal mixed breed dog liver RNA using reverse transcriptase and PCR amplification using primers derived from the published murine G-6-Pase gene sequence. Sequencing revealed an open reading frame of 1071 nucleotides that encodes a predicted 357 amino acid polypeptide in the canine G-6-Pase gene, same as mouse and human. We found more than 90% sequence homology between dog and human G-6-Pase sequence. Hydropathy analysis of the deduced canine G-6-Pase polypeptide shows six transmembrane-spanning segments similar to those seen in human and mouse. Endoplasmic reticulum (ER) localization is similarly predicted by the presence of the ER protein retention signal KK positioned 3 and 4 amino acids from the carboxy terminal. Potential asparagine-linked glycosylation sites are identified at positions 96, 203, and 276. Northern blot analysis revealed increased G-6-Pase mRNA in the deficient dog liver compared to control. This could possibly reflect upregulation of transcription due to the persistent hypoglycemic state. Further studies are directed at the identification of the mutation involved in this deficient dog strain. Characterization of the G-6-Pase gene and protein in the deficient dog model can pave the way for new understanding in the pathophysiology of this disease and for the trials of novel therapeutic approaches including gene therapy.

  16. Cloning and chromosomal localization of the three human syntrophin genes

    SciTech Connect

    Feener, C.A.; Anderson, M.D.S.; Selig, S.

    1994-09-01

    Dystrophin, the protein product the Duchenne muscular dystrophy locus, is normally found to be associated with a complex of proteins. Among these dystrophin-associated proteins are the syntrophins, a group of 59 kDa membrane-associated proteins. When the syntrophins are purified based upon their association with dystrophin, they have been shown previously to form two distinct groups, the acidic ({alpha}) and basic ({beta}) forms. Based on peptide and rodent cDNA sequences, three separate syntrophin genes have been cloned and characterized from human tissues. The predicted amino acid sequences from these cDNA reveal that these proteins are related but are distinct with respect to charge, as predicted from their biochemistry. The family consists of one acidic ({alpha}-syntrophin, analogous to mouse syntrophin-1) and two basic ({beta}{sub 1}-syntrophin; and {beta}{sub 2}-syntrophin, analogous to mouse syntrophin-2) genes. Each of the three genes are widely expressed in a variety of human tissues, but the relative abundance of the three are unique with respect to each other. {alpha}-syntrophin is expressed primarily in skeletal muscle and heart as a single transcript. {beta}{sub 1}-syntrophin is expressed widely in up to five distinct transcript sizes, and is most abundant in brain. The human chromosomal locations of the three syntrophins are currently being mapped. {beta}{sub 1}-syntrophin maps to chromosome 8q23-24 and {beta}{sub 2}-syntrophin to chromosome 16. The {alpha}-syntrophin gene will be mapped accordingly. Although all three genes are candidates for neuromuscular diseases, the predominant expression of {alpha}-syntrophin in skeletal muscle and heart makes it a strong candidate to be involved in a neuromuscular disease.

  17. 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.

  18. The complete primary structure of the T-cell receptor genes from an alloreactive cytotoxic human T-lymphocyte clone.

    PubMed

    Leiden, J M; Fraser, J D; Strominger, J L

    1986-01-01

    The complete primary structure of the cDNAs encoding the alpha and beta chains of the T-lymphocyte receptor for antigen from a human alloreactive, cytotoxic T-cell clone, L17, is presented. Sequence analysis of these genes reveals that both are related to immunoglobulins and are composed of variable, diversity (at least in the case of the Ti beta clone), joining, and constant region sequences. Comparison of the sequence of the alpha-chain cDNA to that of previously sequenced mouse and human alpha cDNAs suggests the presence of human T-cell receptor alpha D-region sequences. Southern blot analysis confirms the finding that these cDNAs represent the functional receptor genes expressed by the L17 cytotoxic T-cell clone. The availability of these full-length T-cell receptor cDNA clones from a human T-lymphocyte clone of known antigen specificity should allow an analysis of the relationship between T-cell receptor structure and function. PMID:2426193

  19. Molecular cloning and analysis of cDNA encoding a plant tryptophan decarboxylase: comparison with animal dopa decarboxylases.

    PubMed Central

    De Luca, V; Marineau, C; Brisson, N

    1989-01-01

    The sequence of a cDNA clone that includes the complete coding region of tryptophan decarboxylase (EC 4.1.1.28, formerly EC 4.1.1.27) from periwinkle (Catharanthus roseus) is reported. The cDNA clone (1747 base pairs) was isolated by antibody screening of a cDNA expression library produced from poly(A)+ RNA found in developing seedlings of C. roseus. The clone hybridized to a 1.8-kilobase mRNA from developing seedlings and from young leaves of mature plants. The identity of the clone was confirmed when extracts of transformed Escherichia coli expressed a protein containing tryptophan decarboxylase enzyme activity. The tryptophan decarboxylase cDNA clone encodes a protein of 500 amino acids with a calculated molecular mass of 56,142 Da. The amino acid sequence shows a high degree of similarity with the aromatic L-amino acid decarboxylase (dopa decarboxylase) and the alpha-methyldopa-hypersensitive protein of Drosophila melanogaster. The tryptophan decarboxylase sequence also showed significant similarity to feline glutamate decarboxylase and mouse ornithine decarboxylase, suggesting a possible evolutionary link between these amino acid decarboxylases. Images PMID:2704736

  20. Towards high-throughput single cell/clone cultivation and analysis.

    PubMed

    Lindström, Sara; Larsson, Rolf; Svahn, Helene Andersson

    2008-03-01

    In order to better understand cellular processes and behavior, a controlled way of studying high numbers of single cells and their clone formation is greatly needed. Numerous ways of ordering single cells into arrays have previously been described, but platforms in which each cell/clone can be addressed to an exact position in the microplate, cultivated for weeks and treated separately in a high-throughput manner have until now been missing. Here, a novel microplate developed for high-throughput single cell/clone cultivation and analysis is presented. Rapid single cell seeding into microwells, using conventional flow cytometry, allows several thousands of single cells to be cultivated, short-term (72 h) or long-term (10-14 days), and analyzed individually. By controlled sorting of individual cells to predefined locations in the microplate, analysis of single cell heterogeneity and clonogenic properties related to drug sensitivity can be accomplished. Additionally, the platform requires remarkably low number of cells, a major advantage when screening limited amounts of patient cell samples. By seeding single cells into the microplate it is possible to analyze the cells for over 14 generations, ending up with more than 10 000 cells in each well. Described here is a proof-of-concept on compartmentalization and cultivation of thousands of individual cells enabling heterogeneity analysis of various cells/clones and their response to different drugs.