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Sample records for individual cloned mouse

  1. Therapeutic cloning in the mouse

    PubMed Central

    Mombaerts, Peter

    2003-01-01

    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. PMID:12949262

  2. Random cloning of genes from mouse chromosome 17.

    PubMed Central

    Kasahara, M; Figueroa, F; Klein, J

    1987-01-01

    We describe a method for isolating cosmid clones randomly from mouse chromosome 17. A cosmid library was constructed from the mouse-Chinese hamster cell line R4 4-1 that contains a limited amount of mouse DNA (chromosomes 17 and 18 and some other unidentified material) on a Chinese hamster background. The library was screened with the murine repetitive sequence probe pMBA14, which selectively hybridizes with mouse DNA. The mouse-derived cosmid clones thus identified were individually hybridized with DNA from the mouse-Syrian hamster cell line JS17 containing all mouse chromosomes except chromosome 17 on a Syrian hamster background. We deduced that the cosmid clones that contained sequences absent in JS17 were derived from mouse chromosome 17. One of the chromosome 17-derived cosmid clones, 3-4-1 (located proximal to the T122/T66C segment) was found to be highly polymorphic among European wild-mouse populations and may be a useful probe to elucidate the evolution and migration of Mus species. The randomly isolated mouse-derived cosmid clones can also be screened for the presence of functional genes. Using testicular cDNA as a probe, a testis-specific gene was cloned from mouse chromosome 17. Images PMID:3472212

  3. Molecular cloning and characterization of mouse Gipc3.

    PubMed

    Saitoh, Tetsuroh; Mine, Tetsuya; Katoh, Masaru

    2002-03-01

    GIPC1/GIPC interacts with GTPase-activating protein RGS-GAIP, transmembrane protein M-SemF, receptor tyrosine kinase TrkA, integrin alpha 6A subunit, and TGF beta type III receptor. Kermit, a Xenopus orthologue of human GIPC1, interacts with Frizzled-3 (FZD3) class of WNT receptor. We have recently cloned and characterized human GIPC2 and GIPC3. Here, we identified mouse Gipc3 gene fragments by using bioinformatics, and isolated mouse Gipc3 cDNAs by using cDNA-PCR. Mouse Gipc3 gene encoded a 297-amino-acid protein, showing 86.2% total-amino-acid identity with human GIPC3. In addition to the central PDZ domain, GIPC homologous domain 1 (GH1 domain) and GH2 domain were found to be conserved among mouse Gipc3, Gipc1, Gipc2, and Xenopus Kermit. Mouse Gipc3 gene was found to consist of 6 exons, and exon-intron structure was well conserved between mouse Gipc3 gene and human GIPC3 gene. Mouse Gipc3 mRNA was relatively highly expressed in adult lung, and was also expressed in brain and testis, but was almost undetectable in 7-, 11-, 15, and 17-day whole embryos. This is the first report on molecular cloning and initial characterization of mouse Gipc3.

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

    PubMed

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

    1998-10-01

    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.

  5. 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

  6. Sex-reversed somatic cell cloning in the mouse.

    PubMed

    Inoue, Kimiko; Ogonuki, Narumi; Mekada, Kazuyuki; Yoshiki, Atsushi; Sado, Takashi; Ogura, Atsuo

    2009-10-01

    Somatic cell nuclear transfer has many potential applications in the fields of basic and applied sciences. However, it has a disadvantage that can never be overcome technically-the inflexibility of the sex of the offspring. Here, we report an accidental birth of a female mouse following nuclear transfer using an immature Sertoli cell. We produced a batch of 27 clones in a nuclear transfer experiment using Sertoli cells collected from neonatal male mice. Among them, one pup was female. This "male-derived female" clone grew into a normal adult and produced offspring by natural mating with a littermate. Chromosomal analysis revealed that the female clone had a 39,X karyotype, indicating that the Y chromosome had been deleted in the donor cell or at some early step during nuclear transfer. This finding suggests the possibility of resuming sexual reproduction after a single male is cloned, which should be especially useful for reviving extinct or endangered species.

  7. Characterization and cloning of lgp110, a lysosomal membrane glycoprotein from mouse and rat cells.

    PubMed

    Granger, B L; Green, S A; Gabel, C A; Howe, C L; Mellman, I; Helenius, A

    1990-07-15

    lgp110 is a heavily glycosylated intrinsic protein of lysosomal membranes. Initially defined by monoclonal antibodies against mouse liver lysosomes, it consists of a 45-kilodalton core polypeptide with O-linked and 17 asparagine-linked oligosaccharide side chains in mouse cells. Sialic acid residues make the mature protein extremely acidic, with an isoelectric point of between 2 and 4 in both normal tissues and most cultured cell lines. Partial sequencing of mouse lgp110 allowed oligonucleotide probes to be constructed for the screening of several mouse cDNA libraries. A partial cDNA clone for mouse lgp110 was found and used for additional library screening, generating a cDNA clone covering all of the coding sequence of mature rat lgp110 as well as genomic clones covering most of the mouse gene. These new clones bring to seven the number of lysosomal membrane proteins whose amino acid sequences can be deduced, and two distinct but highly similar groups (designated lgp-A and lgp-B) can now be defined. Sequence comparisons suggest that differences within each group reflect species variations of the same protein and that lgp-A and lgp-B probably diverged from a common ancestor prior to the evolup4f1ary divergence of birds and mammals. Individual cells and individual lysosomes possess both lgp-A and lgp-B, suggesting that these two proteins have different functions. Mouse lgp110 is encoded by at least seven exons; intron positions suggest that the two homologous ectodomains of each lgp arose through gene duplication.

  8. Cloning, characterization, and localization of mouse and human SPO11.

    PubMed

    Romanienko, P J; Camerini-Otero, R D

    1999-10-15

    Spo11 is a meiosis-specific protein in yeast that has been found covalently bound to DNA double-strand breaks (DSBs) during the early stages of meiosis. These DSBs initiate homologous recombination, which is required for proper segregation of chromosomes and the generation of genetic diversity during meiosis. Here we report the cloning, characterization, tissue expression, and chromosomal localization of both mouse and human homologues of Spo11. The putative mouse and human proteins are 82% identical and share approximately 25% identity with other family members. Northern blot analysis revealed testis-specific expression for both genes, but RT-PCR results showed ubiquitous expression of at least a portion of Spo11 in mouse. Human SPO11 was also detected in several somatic tissues. Mouse Spo11 was localized to chromosome 2H4, and human SPO11 was localized to chromosome 20q13.2-q13.3, a region amplified in some breast and ovarian tumors.

  9. 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.

  10. Mouse cloning and somatic cell reprogramming using electrofused blastomeres

    PubMed Central

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

    2011-01-01

    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. PMID:21187860

  11. Cloning, characterization, and genomic structure of the mouse Ikbkap gene.

    PubMed

    Cuajungco, M P; Leyne, M; Mull, J; Gill, S P; Gusella, J F; Slaugenhaupt, S A

    2001-09-01

    Our laboratory recently reported that mutations in the human I-kappaB kinase-associated protein (IKBKAP) gene are responsible for familial dysautonomia (FD). Interestingly, amino acid substitutions in the IKAP correlate with increased risk for childhood bronchial asthma. Here, we report the cloning and genomic characterization of the mouse Ikbkap gene, the homolog of human IKBKAP. Like its human counterpart, Ikbkap encodes a protein of 1332 amino acids with a molecular weight of approximately 150 kDa. The Ikbkap gene product, Ikap, contains 37 exons that span approximately 51 kb. The protein shows 80% amino acid identity with human IKAP. It shows very high conservation across species and is homologous to the yeast Elp1/Iki3p protein, which is a member of the Elongator complex. The Ikbkap gene maps to chromosome 4 in a region that is syntenic to human chromosome 9q31.3. Because no animal model of FD currently exists, cloning of the mouse Ikbkap gene is an important first step toward creating a mouse model for FD. In addition, cloning of Ikbkap is crucial to the characterization of the putative mammalian Elongator complex.

  12. 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.

  13. Molecular cloning and characterization of mouse aquaporin 6.

    PubMed

    Nagase, Hiroaki; Agren, Johan; Saito, Akiko; Liu, Kun; Agre, Peter; Hazama, Akihiro; Yasui, Masato

    2007-01-05

    In the rat kidney, aquaporin (AQP) 6 is localized in the intracellular vesicle membranes of type-A intercalated cells of the collecting duct; mouse AQP6 (mAQP6) has not been characterized. Although mAQP6 was originally cloned from cDNA in a mouse cerebellum library (GenBank NM 175087), we have independently cloned a cDNA encoding mAQP6 from an adult kidney cDNA library (C57BL/6J strain). We identified two different spliced variants of mAQP6: mAQP6a and mAQP6b. The mAQP6a isoform is almost identical to that of rat AQP6, whereas mAQP6b is identical to that reported in the mouse cerebellum library mentioned above. We found that the mRNA expression of these two spliced variants is regulated in a tissue-specific and age-dependent manner. Functional analyses of water and ion permeation revealed that mAQP6a functions like rat AQP6 and that mAQP6b does not function as either a water channel or an ion channel under our experimental conditions.

  14. Molecular cloning and characterization of mouse aquaporin 6

    PubMed Central

    Nagase, Hiroaki; Agren, Johan; Saito, Akiko; Liu, Kun; Agre, Peter; Hazama, Akihiro; Yasui, Masato

    2007-01-01

    In the rat kidney, aquaporin (AQP) 6 is localized in the intracellular vesicle membranes of type-A intercalated cells of the collecting duct; mouse AQP6 (mAQP6) has not been characterized. Although mAQP6 was originally cloned from cDNA in a mouse cerebellum library (GenBank NM 175087), we have independently cloned a cDNA encoding mAQP6 from an adult kidney cDNA library (C57BL/6J strain). We identified two different spliced variants of mAQP6: mAQP6a and mAQP6b. The mAQP6a isoform is almost identical to rat AQP6, whereas mAQP6b is identical to that reported in the mouse cerebellum library mentioned above. We found that the mRNA expression of these two spliced variants is regulated in a tissue-specific and age-dependent manner. Functional analyses of water and ion permeation revealed that mAQP6a functions like rat AQP6 and that mAQP6b does not function as either a water channel or an ion channel under our experimental conditions. PMID:17112474

  15. 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.

  16. 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

  17. In vitro transcription of a cloned mouse ribosomal RNA gene.

    PubMed Central

    Mishima, Y; Yamamoto, O; Kominami, R; Muramatsu, M

    1981-01-01

    An in vitro transcription system which utilizes cloned mouse ribosomal RNA gene (rDNA) fragments and a mouse cell extract has been developed. RNA polymerases I is apparently responsible for this transcription as evidenced by the complete resistance to a high concentration (200 micrograms/ml) of alpha-amanitin. Run-off products obtained with three different truncated rDNA fragments indicated that RNA was transcribed from a unique site of rDNA. The S1 nuclease protection mapping of the in vitro product and of in vivo 45S RNA confirmed this site, indicating that, in this in vitro system, transcription of rDNA started from the same site as in vivo. This site is located at several hundred nucleotides upstream from the putative initiation site reported by us (1) and by others (2). Some sequence homology surrounding this region was noted among mouse, Xenopus laevis and Drosophila melanogaster. The data also suggest that some processing of the primary transcript occurs in this in vitro system. Images PMID:6278446

  18. Molecular cloning and expression of the mouse ornithine decarboxylase gene.

    PubMed Central

    McConlogue, L; Gupta, M; Wu, L; Coffino, P

    1984-01-01

    We used mRNA from a mutant S49 mouse lymphoma cell line that produces ornithine decarboxylase (OrnDCase) as its major protein product to synthesize and clone cDNA. Plasmids containing OrnDCase cDNA were identified by hybrid selection of OrnDCase mRNA and in vitro translation. The two of these with the largest inserts together span 2.05 kilobases of cDNA. Southern blot analysis of DNA from wild-type or mutant S49 cells, cleaved with EcoRI or with BamHI, revealed multiple bands homologous to OrnD-Case cDNA, only one of which was amplified in the mutant cells. RNA transfer blot analysis showed that the major OrnD-Case mRNA in the mouse lymphoma cells is 2.0 kilobases long. A similar size mRNA was found in mouse kidney and was more abundant in the kidneys of mice treated with testosterone, an inducer of OrnDCase activity in that tissue. Images PMID:6582509

  19. 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.

  20. Mouse ornithine decarboxylase gene: cloning, structure, and expression.

    PubMed Central

    Brabant, M; McConlogue, L; van Daalen Wetters, T; Coffino, P

    1988-01-01

    We used molecular cloning to isolate a functional gene for mouse ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) from a cell line in which that gene had been selectively amplified. The position of the 5' terminus of the mRNA was identified, and the coding sequence was shown to be preceded by a 312- or 313-nucleotide (nt) untranslated leader. The latter is highly G + C rich, particularly in its 5'-most portion. The leader can be anticipated to have extensive and stable secondary structure. The transcription unit of the gene is of relatively small size, approximately equal to 6.2 kilobases (kb) from the start site to the proximal site of polyadenylylation. Sequence analysis of DNA near the transcription start position demonstrated the presence of a "TATA" box, but no "CAAT" box. Functional properties of the cloned gene were tested by transfecting it into cultured cells. Expression of the putative full-length gene efficiently conferred ornithine decarboxylase activity on recipient mutant cells deficient in that activity. To assess the function and strength of the OrnDCase promoter region and to delimit its boundaries, we used a transient expression assay. Upstream of a bacterial chloramphenicol acetyltransferase gene was placed a portion of the OrnDCase gene, including the presumed promoter region, spanning a region from approximately equal to 3.0 kb 5' of the site of transcription initiation to the first 250 nt of the transcript. When expressed in mouse NIH 3T3 cells, this OrnDCase genomic element was comparable in strength to the Rous sarcoma virus long terminal repeat promoter. A similar construct, truncated so as to retain only 264 base pairs of the OrnDCase gene 5' to the site of transcription start, yielded undiminished levels of expression. Images PMID:3353375

  1. Measurement of background translocation frequencies in individuals with clones

    SciTech Connect

    Wade, Marcelle 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.

  2. Expression of cloned immunoglobulin genes introduced into mouse L cells.

    PubMed Central

    Gilles, S D; Tonegawa, S

    1983-01-01

    Functionally rearranged immunoglobulin heavy-chain (gamma 2b) and light-chain (lambda 1 and kappa) genes were introduced into mouse L tk- cells by co-transformation with the Herpes virus tk gene. Cloned cell lines were selected in HAT medium and tested for the presence of transfected immunoglobulin gene sequences by Southern blotting analysis. It was found that the gamma 2b gene was accurately transcribed at a low level in transfected mouse L cells and cytoplasmic gamma 2b, heavy-chain protein was detected by immunoprecipitation of cell extracts. Light-chain genes, on the other hand, were not accurately transcribed. Instead, lambda 1 or kappa RNA species were detected which were approximately 200 to 300 bases longer than the authentic mRNAs. These results suggest that the expression of rearranged heavy-chain and light-chain genes are controlled differently and that these differences can be seen in transfected, non-lymphoid cells. Images PMID:6316279

  3. Database for exchangeable gene trap clones: pathway and gene ontology analysis of exchangeable gene trap clone mouse lines.

    PubMed

    Araki, Masatake; Nakahara, Mai; Muta, Mayumi; Itou, Miharu; Yanai, Chika; Yamazoe, Fumika; Miyake, Mikiko; Morita, Ayaka; Araki, Miyuki; Okamoto, Yoshiyuki; Nakagata, Naomi; Yoshinobu, Kumiko; Yamamura, Ken-ichi; Araki, Kimi

    2014-02-01

    Gene trapping in embryonic stem (ES) cells is a proven method for large-scale random insertional mutagenesis in the mouse genome. We have established an exchangeable gene trap system, in which a reporter gene can be exchanged for any other DNA of interest through Cre/mutant lox-mediated recombination. We isolated trap clones, analyzed trapped genes, and constructed the database for Exchangeable Gene Trap Clones (EGTC) [http://egtc.jp]. The number of registered ES cell lines was 1162 on 31 August 2013. We also established 454 mouse lines from trap ES clones and deposited them in the mouse embryo bank at the Center for Animal Resources and Development, Kumamoto University, Japan. The EGTC database is the most extensive academic resource for gene-trap mouse lines. Because we used a promoter-trap strategy, all trapped genes were expressed in ES cells. To understand the general characteristics of the trapped genes in the EGTC library, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathway analysis and found that the EGTC ES clones covered a broad range of pathways. We also used Gene Ontology (GO) classification data provided by Mouse Genome Informatics (MGI) to compare the functional distribution of genes in each GO term between trapped genes in the EGTC mouse lines and total genes annotated in MGI. We found the functional distributions for the trapped genes in the EGTC mouse lines and for the RefSeq genes for the whole mouse genome were similar, indicating that the EGTC mouse lines had trapped a wide range of mouse genes. © 2014 The Authors Development, Growth & Differentiation © 2014 Japanese Society of Developmental Biologists.

  4. Cloned mouse cells with natural killer function and cloned suppressor T cells express ultrastructural and biochemical features not shared by cloned inducer T cells

    PubMed Central

    1983-01-01

    We have examined the morphology, cytochemistry, and biochemistry of mouse leukocyte subsets by analyzing cloned leukocyte populations specialized to perform different immunologic functions. Cloned cells expressing high-affinity plasma membrane receptors for IgE and mediating natural killer (NK) lysis and cloned antigen-specific suppressor T cells contained prominent osmiophilic cytoplasmic granules similar by ultrastructure to those of mouse basophils. Both clones also incorporated 35SO4 into granule-associated sulfated glycosaminoglycans, expressed a characteristic ultrastructural pattern of nonspecific esterase activity, incorporated exogenous [3H]5-hydroxytryptamine, and contained cytoplasmic deposits of particulate glycogen. By contrast, cloned inducer T cells lacked cytoplasmic granules and glycogen, incorporated neither 35SO4 nor [3H]5-hydroxytryptamine, and differed from the other clones in pattern of nonspecific esterase activity. These findings establish that certain cloned cells with NK activity and cloned suppressor T cells express morphologic and biochemical characteristics heretofore associated with basophilic granulocytes. However, these clones differ in surface glycoprotein expression and immunologic function, and the full extent of the similarities and differences among these populations and basophils remains to be determined. PMID:6220105

  5. 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.

  6. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Cloning: experience from the mouse and other animals.

    PubMed

    Yanagimachi, R

    2002-02-22

    Cloning mammals has been successful for many years by splitting an early embryo or transferring embryonic cell nuclei into enucleated oocytes. Cloning is now possible with adult somatic cells. At present, cloning efficiency--as determined by the proportion of live offspring developed from all oocytes that received donor cell nuclei--is low regardless of the cell type (including, embryonic stem (ES) cells) and animal species used. In all animals, except of Japanese black beef cattle, the vast majority (>97%) of cloned embryos perish before reaching full term. Even in the Japanese cattle, less than 20% of cloned embryos reach the adulthood. This low efficiency of cloning seems to be due largely to faulty epigenetic reprogramming of donor cell nuclei after transfer into recipient oocytes. Cloned embryos with major epigenetic errors die before or soon after implantation. Those with relatively 'minor' epigenetic errors may survive birth and reach adulthood. We found that almost all fetuses of inbred mice die at birth from respiratory problems, while those of hybrid mice do not, suggesting that genomic heterogeneity masks-to some extent-faulty epigenetic errors. Thus far, the majority of cloned mice that survived birth, had a normal life span and were fertile. However, these animals may not be totally free of health problems. Postpubertal obesity in certain strains of mice is one example. A trial and error approach may discover better cells for cloning, but it would be wiser to understand the molecular mechanisms of epigenetic nuclear programming and reprogramming to find the way to make cloning safer and more efficient. The relatively high cloning success rate in the Japanese black cattle may provide us a clue of solving the problem of high mortality of cloned offspring.

  8. Defective Chromatin Structure in Somatic Cell Cloned Mouse Embryos*

    PubMed Central

    Zhang, Miao; Wang, Fengchao; Kou, Zhaohui; Zhang, Yu; Gao, Shaorong

    2009-01-01

    Epigenetic reprogramming plays a central role in the development of cloned embryos generated by somatic cell nuclear transfer, and it is believed that aberrant reprogramming leads to the abnormal development of most cloned embryos. Recent studies show that trimethylation of H3K27 (H3K27me3) contributes to the maintenance of embryonic stem cell pluripotency because the differentiation genes are always occupied by nucleosomes trimethylated at H3K27, which represses gene expression. Here, we provide evidence that differential H3K27me3 modification exists between normal fertilization-produced blastocysts and somatic cell nuclear transfer cloned blastocysts; H3K27me3 was specifically found in cells of the inner cell mass (ICM) of normal blastocysts, whereas there was no modification of H3K27me3 in the ICM of cloned blastocysts. Subsequently, we demonstrated that the differentiation-related genes, which are marked by H3K27me3 in embryonic stem cells, were expressed at significantly higher levels in cloned embryos than in normal embryos. The polycomb repressive complex 2 (PRC2) component genes (Eed, Ezh2, and Suz12), which are responsible for the generation of H3K27me3, were expressed at lower levels in the cloned embryos. Our results suggest that reduced expression of PRC2 component genes in cloned embryos results in defective modification of H3K27me3 to the differentiation-related genes in pluripotent ICM cells. This results in premature expression of developmental genes and death of somatic cloned embryos shortly after implantation. Taken together, these studies suggest that H3K27me3 might be an important epigenetic marker with which to evaluate the developmental potential of cloned embryos. PMID:19602512

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Inferring Higher Functional Information for RIKEN Mouse Full-Length cDNA Clones With FACTS

    PubMed Central

    Nagashima, Takeshi; Silva, Diego G.; Petrovsky, Nikolai; Socha, Luis A.; Suzuki, Harukazu; Saito, Rintaro; Kasukawa, Takeya; Kurochkin, Igor V.; Konagaya, Akihiko; Schönbach, Christian

    2003-01-01

    FACTS (Functional Association/Annotation of cDNA Clones from Text/Sequence Sources) is a semiautomated knowledge discovery and annotation system that integrates molecular function information derived from sequence analysis results (sequence inferred) with functional information extracted from text. Text-inferred information was extracted from keyword-based retrievals of MEDLINE abstracts and by matching of gene or protein names to OMIM, BIND, and DIP database entries. Using FACTS, we found that 47.5% of the 60,770 RIKEN mouse cDNA FANTOM2 clone annotations were informative for text searches. MEDLINE queries yielded molecular interaction-containing sentences for 23.1% of the clones. When disease MeSH and GO terms were matched with retrieved abstracts, 22.7% of clones were associated with potential diseases, and 32.5% with GO identifiers. A significant number (23.5%) of disease MeSH-associated clones were also found to have a hereditary disease association (OMIM Morbidmap). Inferred neoplastic and nervous system disease represented 49.6% and 36.0% of disease MeSH-associated clones, respectively. A comparison of sequence-based GO assignments with informative text-based GO assignments revealed that for 78.2% of clones, identical GO assignments were provided for that clone by either method, whereas for 21.8% of clones, the assignments differed. In contrast, for OMIM assignments, only 28.5% of clones had identical sequence-based and text-based OMIM assignments. Sequence, sentence, and term-based functional associations are included in the FACTS database (http://facts.gsc.riken.go.jp/), which permits results to be annotated and explored through web-accessible keyword and sequence search interfaces. The FACTS database will be a critical tool for investigating the functional complexity of the mouse transcriptome, cDNA-inferred interactome (molecular interactions), and pathome (pathologies). PMID:12819151

  15. Cloning and characterization of a new type of mouse chemokine.

    PubMed

    Rossi, D L; Hardiman, G; Copeland, N G; Gilbert, D J; Jenkins, N; Zlotnik, A; Bazan, J F

    1998-01-15

    We report here the identification and characterization of the mouse homologue of a human CX3C chemokine described by F. Bazan et al. (1997, Nature 385, 640-644). Termed fractalkine, this molecule constitutes a fourth or delta chemokine structural type that displays a novel CX3C sequence fingerprint. Distinct from the alpha, beta, or gamma chemokine families, the polypeptide chain of CX3C predicts a 373-amino-acid type I transmembrane glycoprotein with the chemokine domain resting on top of an extended mucin-like stalk. Comparison of the mouse and human protein chains shows a high degree of conservation in all the globular segments with the exception of the stalk portion. The striking identity of an amino acid stretch encompassing a putative juxtamembrane cleavage site suggests the evolutionary conservation of both membrane-bound and processed CX3C forms. Northern analysis reveals the presence of mouse CX3C mRNA in heart, brain, lung, kidney, skeletal muscle, and testis tissues. The mouse CX3C gene was further localized to the central region of chromosome 8 by interspecific backcross mapping; a related locus was detected on chromosome 11. The novel location of this gene from other chemokine gene clusters adds to the notion that CX3C is a fundamentally new class of chemokine.

  16. Cloning

    MedlinePlus

    Cloning describes the processes used to create an exact genetic replica of another cell, tissue or organism. ... named Dolly. There are three different types of cloning: Gene cloning, which creates copies of genes or ...

  17. 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.

  18. Cloning and expression of the mouse histamine H3 receptor: evidence for multiple isoforms.

    PubMed

    Rouleau, Agnès; Héron, Anne; Cochois, Véronique; Pillot, Catherine; Schwartz, Jean-Charles; Arrang, Jean-Michel

    2004-09-01

    The existence of mouse H3-receptor isoforms was investigated by PCR analysis and cDNA cloning. Splicing mechanisms previously reported in various species are conserved in the mouse. The retention/deletion of a fragment in the third intracellular loop of the mouse receptor leads to the existence of three isoforms designated mH(3(445)), mH(3(413)) and mH(3(397)) according to the length of their deduced amino acid sequence. PCR analysis showed that mouse H3-receptor isoforms display different expression patterns in the brain. Following expression in Cos-1 cells, [125I]iodoproxyfan binding indicated similar pharmacological profiles of the mH(3(445)), mH(3(413)) and mH(3(397)) isoforms. The pharmacological profile of the mouse H3 receptor is more similar to the rat receptor than to the human receptor, although some differences were also observed between the mouse and rat receptors. For example, the potency of thioperamide and ciproxifan is slightly higher at the mouse receptor than at the rat receptor but 40-100-fold higher than at the human receptor. In situ hybridization histochemistry showed that the distribution of H3-receptor mRNAs in the mouse brain is rather similar to that previously reported in the rat brain. However, the autoradiographic and cellular expression patterns observed in several brain areas such as the thalamus or hippocampus reveal important differences between the two species.

  19. 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.

  20. Cloning and determination of the transcription termination site of ribosomal RNA gene of the mouse.

    PubMed Central

    Kominami, R; Mishima, Y; Urano, Y; Sakai, M; Muramatsu, M

    1982-01-01

    A Eco RI 6.6 kb DNA fragment containing the 3'-end of 28S ribosomal RNA gene of the mouse was detected by Southern blot hybridization, and cloned in a lambda-phage vector. The site of transcription termination and the processed 3'-end of 28S RNA were determined on the cloned fragment and the surrounding nucleotide sequence determined. The 3'-terminal nucleotides of mouse 28S RNA are similar to those of yeast, Drosophila and Xenopus although the homology was lost drastically beyond the 3'-end of 28S RNA. 45S precursor RNA terminated at 30 nucleotides downstream from the 3'-end of 28S RNA gene. A structure of a dyad symmetry with a loop was found immediately prior to the termination site of 45S RNA. The rDNA termination site thus shares some common features with termination sites recognized by other RNA polymerases. Images PMID:6281727

  1. Molecular cloning, genomic organization and cell‐binding characteristics of mouse Spα

    PubMed Central

    Gebe, J A; Llewellyn, M‐B C; Hoggatt, H; Aruffo, A

    2000-01-01

    Several group B scavenger receptor cysteine‐rich (SRCR) proteins have been shown to function as modulators in the immune response. Recently, we reported the cloning of a new member of this family, human Spα (hSpα). Herein we report the cloning and characterization of the mouse homologue of hSpα. Like its human counterpart, mouse Spα (mSpα), is a secreted protein containing three SRCR domains. Most lymphoid tissues express RNA transcripts encoding mSpα. Characterization of a genomic clone encoding the mature mSpα protein showed that each of the SRCR domains of mSpα is encoded by a single exon. Comparison of the sequence of mSPα with those of other published proteins indicates that it is the same as the recently reported protein named AIM (apoptosis inhibitor expressed by macrophages). Cell‐binding studies with a mSpα immunoglobulin (mSpα‐Rγ) fusion protein indicated that mSpα is capable of binding to spleen‐derived CD19+ B cells and minimally to peritoneal cavity‐derived CD19+ B cells but not to peripheral blood‐derived B cells. Spleen‐derived CD3+ T cells also bound mSpα‐Rγ; however, no binding was observed to either peripheral blood mononuclear cells or peritoneal cavity‐derived CD3+ T cells. The mSpα‐Rγ fusion protein was also shown to bind to the mouse cell lines WEHI3 (monocytic) and EL‐4 (thymoma, T cell). The cloning of cDNA and genomic clones encoding mSpα and the identification of cells expressing a putative mSpα receptor(s) should facilitate in vivo studies designed to investigate the function of Spα in the immune compartment. PMID:10651944

  2. Molecular cloning, genomic organization and cell-binding characteristics of mouse Spalpha.

    PubMed

    Gebe, J A; Llewellyn, M; Hoggatt, H; Aruffo, A

    2000-01-01

    Several group B scavenger receptor cysteine-rich (SRCR) proteins have been shown to function as modulators in the immune response. Recently, we reported the cloning of a new member of this family, human Spalpha (hSpalpha). Herein we report the cloning and characterization of the mouse homologue of hSpalpha. Like its human counterpart, mouse Spalpha (mSpalpha), is a secreted protein containing three SRCR domains. Most lymphoid tissues express RNA transcripts encoding mSpalpha. Characterization of a genomic clone encoding the mature mSpalpha protein showed that each of the SRCR domains of mSpalpha is encoded by a single exon. Comparison of the sequence of mSPalpha with those of other published proteins indicates that it is the same as the recently reported protein named AIM (apoptosis inhibitor expressed by macrophages). Cell-binding studies with a mSpalpha immunoglobulin (mSpalpha-Rgamma) fusion protein indicated that mSpalpha is capable of binding to spleen-derived CD19+ B cells and minimally to peritoneal cavity-derived CD19+ B cells but not to peripheral blood-derived B cells. Spleen-derived CD3+ T cells also bound mSpalpha-Rgamma; however, no binding was observed to either peripheral blood mononuclear cells or peritoneal cavity-derived CD3+ T cells. The mSpalpha-Rgamma fusion protein was also shown to bind to the mouse cell lines WEHI3 (monocytic) and EL-4 (thymoma, T cell). The cloning of cDNA and genomic clones encoding mSpalpha and the identification of cells expressing a putative mSpalpha receptor(s) should facilitate in vivo studies designed to investigate the function of Spalpha in the immune compartment.

  3. Microinjection of cloned DNA fragments into fertilized one-cell mouse eggs: I. Manual injection.

    PubMed

    Murphy, D

    1993-01-01

    Central to the process of making transgenic mice is the physical introduction of cloned DNA fragments into fertilized one-cell mouse eggs. First described 10 years ago by a number of investigators, microinjection remains the most popular and successful of the methods currently available for generating transgenic animals. Microinjection continues to be the method of choice, because the advantages of speed and reliability far outweigh the demands placed on the investigator for precise technical skill and expensive equipment.

  4. Cloning and characterization of a cDNA coding for mouse placental alkaline phosphatase

    SciTech Connect

    Terao, M.; Mintz, B.

    1987-10-01

    Mouse alkaline phosphatase was partially purified from placenta. Data obtained by immunoblotting analysis suggested that the primary structure of this enzyme has a much greater homology to that of human and bovine liver ALPs than to the human placental isozyme. Therefore, a full-length cDNA encoding human liver-type ALP was used as a probe to isolate the mouse placental ALP cDNA. The cloned mouse cDNA is 2459 base pairs long and is composed of an open reading frame encoding a 524-amino acid polypeptide that contains a putative signal peptide of 17 amino acids. Homology at the amino acid level of the mouse placental ALP is 90% to the human liver isozyme but only 55% to the human placental counterpart. RNA blot hybridization results indicate that the mouse placental ALP is encoded by a gene identical to the gene expressed in mouse liver, kidney, and teratocarcinoma stem cells. This gene is therefore evolutionarily highly conserved in mouse and human.

  5. 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)

  6. Cloning of the mouse dysferlin gene and genomic characterization of the SJL-Dysf mutation.

    PubMed

    Vafiadaki, E; Reis, A; Keers, S; Harrison, R; Anderson, L V; Raffelsberger, T; Ivanova, S; Hoger, H; Bittner, R E; Bushby, K; Bashir, R

    2001-03-05

    The SJL mouse strain has been widely used as an animal model for experimental autoimmune encephalitis (EAE), inflammatory muscle disease and lymphomas and has also been used as a background strain for the generation of animal models for a variety of diseases including motor neurone disease, multiple sclerosis and atherosclerosis. Recently the SJL mouse was shown to have myopathy due to dysferlin deficiency, so that it can now be considered a natural animal model for limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). We have cloned the mouse dysferlin cDNA and analysis of the sequence shows that the mouse dysferlin gene is characterized by six C2 domain sequences and a C-terminal anchoring domain, with the human and the mouse dysferlin genes sharing > 90% sequence homology overall. Genomic analysis of the SJL mutation confirms that the 171 bp RNA deletion has arisen by exon skipping resulting from a splice site mutation. The identification of this mutation has implications for the various groups using this widely available mouse stock.

  7. Cloning and expression of mouse peroxiredoxin I in IEC-6 Cells.

    PubMed

    Zhang, Bo; Su, Yong-Ping; Wang, Tao; Wang, Feng-Chao; Ai, Guo-Ping; Xu, Hui; Wang, Jun-Ping; Huang, Yue-Sheng; Jiang, Jian-Xin

    2004-07-15

    To clone and express mouse peroxiredoxin I in IEC-6 cells. Total RNAs were isolated from cultured IEC-6 cells, and the coding region of peroxiredoxin I was amplified by RT-PCR. After it was cloned into T-vector and sequenced, pSG5 was used to transiently express peroxiredoxin I in IEC-6 by liposome-mediated transfection, and the expression of peroxiredoxin I was evaluated by RT-PCR and Western blot. A DNA fragment about 750 bp was amplified from total RNAs of IEC-6 cells using specific primers of peroxiredoxin I. The sequencing confirmed the coding region was successfully cloned into T-vector, which was completely coincident with the sequence in GeneBank. After the EcoRI-BamHI fragment of T-vector containing peroxiredoxin I was inserted into pSG5, the recombinant plasmid was transferred to IEC-6 cells. RT-PCR assay showed that a DNA fragment of 930 bp could be amplified, which indicated the transcription of pSG5-Prx. Western blot confirmed the expression of peroxiredoxin I in IEC-6 cells. Mouse peroxiredoxin I can be successfully expressed in IEC-6 cells.

  8. Cloning of the repertoire of individual Plasmodium falciparum var genes using transformation associated recombination (TAR).

    PubMed

    Gaida, Annette; Becker, Marion M; Schmid, Christoph D; Bühlmann, Tobias; Louis, Edward J; Beck, Hans-Peter

    2011-03-07

    One of the major virulence factors of the malaria causing parasite is the Plasmodium falciparum encoded erythrocyte membrane protein 1 (PfEMP1). It is translocated to It the membrane of infected erythrocytes and expressed from approximately 60 var genes in a mutually exclusive manner. Switching of var genes allows the parasite to alter functional and antigenic properties of infected erythrocytes, to escape the immune defense and to establish chronic infections. We have developed an efficient method for isolating VAR genes from telomeric and other genome locations by adapting transformation-associated recombination (TAR) cloning, which can then be analyzed and sequenced. For this purpose, three plasmids each containing a homologous sequence representing the upstream regions of the group A, B, and C var genes and a sequence homologous to the conserved acidic terminal segment (ATS) of var genes were generated. Co-transfection with P. falciparum strain ITG2F6 genomic DNA in yeast cells yielded 200 TAR clones. The relative frequencies of clones from each group were not biased. Clones were screened by PCR, as well as Southern blotting, which revealed clones missed by PCR due to sequence mismatches with the primers. Selected clones were transformed into E. coli and further analyzed by RFLP and end sequencing. Physical analysis of 36 clones revealed 27 distinct types potentially representing 50% of the var gene repertoire. Three clones were selected for sequencing and assembled into single var gene containing contigs. This study demonstrates that it is possible to rapidly obtain the repertoire of var genes from P. falciparum within a single set of cloning experiments. This technique can be applied to individual isolates which will provide a detailed picture of the diversity of var genes in the field. This is a powerful tool to overcome the obstacles with cloning and assembly of multi-gene families by simultaneously cloning each member.

  9. The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and northern blot analysis.

    PubMed

    Katsuyama, M; Nishigaki, N; Sugimoto, Y; Morimoto, K; Negishi, M; Narumiya, S; Ichikawa, A

    1995-09-25

    A functional cDNA clone for the mouse prostaglandin (PG) E receptor EP2 subtype was isolated from a mouse cDNA library. The mouse EP2 receptor consists of 362 amino acid residues with seven putative transmembrane domains. [3H]PGE2 bound specifically to the membrane of Chinese hamster ovary cells stably expressing the cloned receptor. This binding was displaced by unlabeled prostanoids in the order of PGE2 = PGE1 > iloprost, a stable PGI2 agonist > PGF2 alpha > PGD2. Binding was also inhibited by butaprost (an EP2 agonist) and to a lesser extent by M&B 28767 (an EP3 agonist), but not by sulprostone (an EP1 and EP3 agonist) or SC-19220 (an EP1 antagonist). PGE2 and butaprost increased the cAMP level in the Chinese hamster ovary cells in a concentration-dependent manner. Northern blot analysis revealed that EP2 mRNA is expressed most abundantly in the uterus, followed by the spleen, lung, thymus, ileum, liver, and stomach.

  10. Cloning and recombinant expression of a novel mouse-secreted phospholipase A2.

    PubMed

    Valentin, E; Koduri, R S; Scimeca, J C; Carle, G; Gelb, M H; Lazdunski, M; Lambeau, G

    1999-07-02

    Secreted phospholipases A2 (sPLA2s) form a class of structurally related enzymes that are involved in a variety of physiological and pathological effects including inflammation and associated diseases, cell proliferation, cell adhesion, and cancer, and are now known to bind to specific membrane receptors. Here, we report the cloning and expression of a novel sPLA2 isolated from mouse thymus. Based on its structural features, this sPLA2 is most similar to the previously cloned mouse group IIA sPLA2 (mGIIA sPLA2). As for mGIIA sPLA2, the novel sPLA2 is made up of 125 amino acids with 14 cysteines, is basic (pI = 8.71) and its gene has been mapped to mouse chromosome 4. However, the novel sPLA2 has only 48% identity with mGIIA and displays similar levels of identity with the other mouse group IIC and V sPLA2s, indicating that the novel sPLA2 is not an isoform of mGIIA sPLA2. This novel sPLA2 has thus been called mouse group IID (mGIID) sPLA2. In further contrast with mGIIA, which is found mainly in intestine, transcripts coding for mGIID sPLA2 are found in several tissues including pancreas, spleen, thymus, skin, lung, and ovary, suggesting distinct functions for the two enzymes. Recombinant expression of mGIID sPLA2 in Escherichia coli indicates that the cloned sPLA2 is an active enzyme that has much lower specific activity than mGIIA and displays a distinct specificity for binding to various phospholipid vesicles. Finally, recombinant mGIID sPLA2 did not bind to the mouse M-type sPLA2 receptor, while mGIIA was previously found to bind to this receptor with high affinity.

  11. Cloning

    MedlinePlus

    ... 2001, researchers produced the first clone of an endangered species: a type of Asian ox known as a ... few days after its birth. In 2003, another endangered type of ox, called the ... many species that would otherwise disappear, others argue that cloning ...

  12. Construction of a cDNA clone corresponding to mouse alpha 1(IV) procollagen.

    PubMed Central

    dos Santos, C L; Villa, L L; Sonohara, S; Brentani, R R

    1984-01-01

    A new procedure for the synthesis of double stranded cDNA, based upon release of mRNA by "in vitro" translation, was used to clone type IV collagen. Collagen synthesizing polysomes selectively isolated from a mouse parietal yolk sac carcinoma (PYS-2) were used for translation in an heterologous cell-free system. Translation products were collagenase-sensitive and displayed an electrophoretic mobility correspondent to type IV collagen. Translation released mRNA was employed to construct a 100 base pairs long cDNA clone which hybridized to a 7,800 nucleotides long mRNA. Peptides synthesized by "in vitro" translation of hybrid selected mRNA displayed an electrophoretic mobility compatible with that of alpha 1 (IV) collagen, were sensitive to collagenase and were immunoprecipitated by anti-type IV collagen antibody. Images PMID:6546618

  13. Expression of Functional MHC Class II Molecules By a Mouse Pro-B Cell Clone

    PubMed Central

    Fisher, Amanda G.; Meyer, Valérie; Ceredig, Rhodri

    1995-01-01

    We describe here the G12 pro-B cell clone that has been isolated from an IL-7 transgenic mouse. This clone has the phenotype B220+, BP-1+ , HSA +, CD43+ λ5+ , and CD25-, and has its Ig locus in a germline configuration. G12 cells spontaneously express cell-surface MHC class II molecules, although to a much lesser extent than the mature M12.4.1 B-cell lymphoma. G12 cells can process and present the native Hen Egg Lysozyme (HEL) to an MHC class II-restricted T-cell hybridoma. The efficiency of presentation is inferior to that obtained with M12.4.1 cells. This is the first report where a pro-B cell can serve as APC in an MHC class II-restricted presentation. PMID:9700358

  14. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype.

    PubMed

    Honda, A; Sugimoto, Y; Namba, T; Watabe, A; Irie, A; Negishi, M; Narumiya, S; Ichikawa, A

    1993-04-15

    A functional cDNA clone encoding mouse EP2 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse EP3 subtype PGE receptor cDNA. The mouse EP2 receptor consists of 513 amino acid residues with putative seven-transmembrane domains. In contrast to EP3 receptor, this receptor possesses long third intracellular loop and carboxyl-terminal tail. [3H] PGE2 specifically bound to the membrane of mammalian COS cells transfected with the cDNA. The binding to the membrane was displaced with unlabeled PG in the order of PGE2 = PGE1 > iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist, and SC-19220, an EP1 antagonist. PGE2 markedly increased cAMP level in COS cells transfected with the cDNA. These results suggest that this receptor is EP2 subtype. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the abundant expression being observed in ileum, thymus, and mastocytoma P-815 cells.

  15. Cloning, expression and characterization of mouse (Mus musculus) nicotinamide 5'-mononucleotide adenylyltransferase-2.

    PubMed

    Raches, David W; Xiao, Suhong; Kusumanchi, Praveen; Yalowitz, Joel; Sanghani, Paresh; Long, Eric C; Antony, Aśok C; Jayaram, Hiremagalur N

    2011-11-01

    Nicotinamide adenine dinucleotide (NAD+) is synthesized by the action of nicotinamide mononucleotide adenylyltransferase (NMNAT) from NMN and ATP. The mouse homolog of NMNAT-2 (mmNMNAT-2) was cloned, expressed, and subsequently identified using MALDI-TOF in conjunction with the ProFound database. Circular dichroism analyses of recombinant mmNMNAT-2 showed α helical and β sheet secondary structures, consistent with the known structure of the human isoform. Competition experiments using mouse pancreatic tissue lysates with recombinant mmNMNAT-2 demonstrated that the activity of the expressed protein was similar to the human isoform. Immunohistochemistry of mouse embryonic tissues with hNMNAT-2 also showed a tissue- and cellular-specific expression of this isoform. Therefore, our studies demonstrate for the first time the clear biological evidence for the existence of a mouse isoform of hNMNAT-2. These studies may help in future investigations aimed at understanding the regulation of this gene and its pathway, and in turn, will spur the development of novel therapies for diseases such as cancer and diabetes since mice are the most frequently used experimental system for in vivo studies.

  16. Genomic cloning of mouse MIF (macrophage inhibitory factor) and genetic mapping of the human and mouse expressed gene and nine mouse pseudogenes

    SciTech Connect

    Kozak, C.A.; Adamson, M.C.; Buckler, C.E.

    1995-06-10

    The single functional mouse gene for MIF (macrophage migration inhibitory factor) has been cloned from a P1 library, and its exon/intron structure determined and shown to resemble that of the human gene. The gene was mapped to chromosome 10 using two multilocus crosses between laboratory strains and either Mus musculus or Mus spretus. Nine additional loci containing related sequences, apparently all processed pseudogenes, were also mapped to chromosomes 1, 2, 3, 7, 8, 9, 12, 17, and 19. While most of these pseudogenes were also found in inbred mice and M. spretus, some are species specific. This suggests that there have been active phases of pseudogene formation in Mus both before and after the separation of musculus and spretus. The human gene contains no pseudogene; we assigned the human gene to chromosome 19, consistent with the location of mouse and human functional genes for MIF in a region of conserved linkage. 43 refs., 4 figs., 1 tab.

  17. 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.

  18. Improved Development of Somatic Cell Cloned Mouse Embryos by Vitamin C and Latrunculin A

    PubMed Central

    Mallol, Anna; Santaló, Josep; Ibáñez, Elena

    2015-01-01

    Impaired development of embryos produced by somatic cell nuclear transfer (SCNT) is mostly associated with faulty reprogramming of the somatic nucleus to a totipotent state and can be improved by treatment with epigenetic modifiers. Here we report that addition of 100 μM vitamin C (VitC) to embryo culture medium for at least 16 h post-activation significantly increases mouse blastocyst formation and, when combined with the use of latrunculin A (LatA) during micromanipulation and activation procedures, also development to term. In spite of this, no significant effects on pluripotency (OCT4 and NANOG) or nuclear reprogramming markers (H3K14 acetylation, H3K9 methylation and DNA methylation and hydroxymethylation) could be detected. The use of LatA alone significantly improved in vitro development, but not full-term development. On the other hand, the simultaneous treatment of cloned embryos with VitC and the histone deacetylase inhibitor psammaplin A (PsA), in combination with the use of LatA, resulted in cloning efficiencies equivalent to those of VitC or PsA treatments alone, and the effects on pluripotency and nuclear reprogramming markers were less evident than when only the PsA treatment was applied. These results suggest that although both epigenetic modifiers improve cloning efficiencies, possibly through different mechanisms, they do not show an additive effect when combined. Improvement of SCNT efficiency is essential for its applications in reproductive and therapeutic cloning, and identification of molecules which increase this efficiency should facilitate studies on the mechanism of nuclear reprogramming and acquisition of totipotency. PMID:25749170

  19. Mouse cloning with nucleus donor cells of different age and type.

    PubMed

    Wakayama, T; Yanagimachi, R

    2001-04-01

    We have tested different cell types as sources for nucleus donors to determine differences in cloning efficiency. When donor nuclei were isolated from cumulus cells and injected into recipient oocytes from adult hybrid mice (B6D2F1 and B6C3F1), the success rate of cloning was 1.5-1.9%. When cumulus cell donor nuclei were isolated from adult inbred mice (C57BL/6, C3H/He, DBA/2, 129/SvJ, and 129/SvEvTac), reconstructed oocytes did not develop to full term or resulted in a very low success rate (0-0.3%) with the exception of 129 strains which yielded 0.7-1.4% live young. When fetal (13.5-15.5 dpc), ovarian, and testicular cells were used as nucleus donors, 2.2 and 1.0% of reconstructed oocytes developed into live offspring, respectively. When various types of adult somatic cells (fibroblasts, thymocytes, spleen cells, and macrophages) were used, oocytes receiving thymocyte nuclei never developed beyond implantation, whereas those receiving the nuclei of other cell types did. These results indicate that adult somatic cells are not necessarily inferior to younger cells (fetal and ES cells) in the context of mouse cloning. Although fetal cells are believed to have less genetic damage than adult somatic cells, the success rate of cloning using any cell types were very low. This may largely be due to technical problems and/or problems of genomic reprogramming by oocytes rather than the accumulation of mutational damage in adult somatic cells.

  20. Cloning and characterization of the gene for mouse macrophage migration inhibitory factor (MIF)

    SciTech Connect

    Mitchell, R.; Bacher, M.; Bernhagen, J.

    1995-04-15

    An emerging body of data indicates that the protein mediator described originally as macrophage migration inhibitory factor (MIF) exerts a central and wide ranging role in host inflammatory responses. MIF is a major constituent of corticotrophic cells within the anterior pituitary gland and is secreted into the circulation in a hormone-like fashion. MIF also exists preformed in monocytes/macrophages and is a pivotal mediator in the host response to endotoxic shock. To gain further insight into the biologic expression of this protein that encompasses components of both the immune and the endocrine systems, we have cloned the mouse MIF gene and identified potential regulatory sequences present within the 5{prime}-proximal promoter region. The gene for mouse MIF is located on chromosome 10, spans approximately 1 kb, and shares a high degree of structural homology with its human counterpart. Of note, the consensus enhancer/promoter motifs identified include both inflammatory/growth factor-related elements and sites associated with the genes for certain peptide hormones. We also report the structures of two MIF pseudogenes that account for early observations suggesting that mouse MIF is encoded by a highly homologous multigene family. 38 refs., 5 figs., 1 tab.

  1. Cloning and characterization of mouse brush border myosin-I in adult and embryonic intestine.

    PubMed

    Skowron, J F; Mooseker, M S

    1999-02-15

    Brush border myosin-I is a class I myosin with calmodulin light chains that has been identified in several vertebrate species. In chicken, it is exclusively expressed in intestinal epithelial cells where it forms spirally arrayed bridges that tether the microvillar actin bundle to the membrane. To facilitate future knockout strategies, we have isolated mouse brush border myosin-I cDNA and genomic clones. The deduced primary structure of mouse brush border myosin-I is homologous to other known brush border myosins-I. Northern blot, immunoblot, and immunolocalization studies indicate that the intestine-specific and subcellular localization profile of mouse brush border myosin-I are comparable to that determined for other brush border myosins-I. Northern analysis during embryogenesis revealed a 3.9-kb transcript first detected in 15-day embryos. This is in marked contrast to chicken, where brush border myosin-I expression begins early in embryogenesis. In situ localization in 17-day embryos indicated that RNA expression is restricted to the intestine. Protein expression is first detected in 16-day embryos with decreasing levels observed in a proximal to distal fashion. Immunolocalization in embryonic intestine revealed that brush border myosin-I is evenly distributed on both apical and basolateral membrane domains. There is also pronounced localization to a supranuclear region, presumably the Golgi apparatus. This suggests that brush border myosin-I may be targeted to the plasma membrane on Golgi-derived vesicles rather than by direct targeting to microvillar actin cores.

  2. 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.

  3. Molecular cloning, expression, and characterization of mouse amine N-sulfotransferases

    SciTech Connect

    Takahashi, Saki |; Sakakibara, Yoichi | Mishiro, Emi |; Kouriki, Haruna; Nobe, Rika |; Kurogi, Katsuhisa; Yasuda, Shin |; Liu, M.-C.; Suiko, Masahito |

    2008-10-31

    By searching the GenBank database, we recently identified a novel mouse cytosolic sulfotransferase (SULT) cDNA (IMAGE Clone ID 679629) and a novel mouse SULT gene (LOC 215895). Sequence analysis revealed that both mouse SULTs belong to the cytosolic SULT3 gene family. The recombinant form of these two newly identified SULTs, designated SULT3A1 and SULT3A2, were expressed using the pGEX-4T-1 glutathione S-transferase fusion system and purified from transformed BL21 Escherichia coli cells. Both purified SULT3A1 and SULT3A2 exhibited strong amine N-sulfonating activities toward 1-naphthylamine among a variety of endogenous and xenobiotic compounds tested as substrates. Kinetic constants of the sulfation of 1-naphthylamine and 1-naphthol by these two enzymes were determined. Collectively, these results imply that these two amine-sulfonating SULT3s may play essential roles in the metabolism and detoxification of aromatic amine compounds in the body.

  4. [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.

  5. Assembly, Verification, and Initial Annotation of the NIA Mouse 7.4K cDNA Clone Set

    PubMed Central

    VanBuren, Vincent; Piao, Yulan; Dudekula, Dawood B.; Qian, Yong; Carter, Mark G.; Martin, Patrick R.; Stagg, Carole A.; Bassey, Uwem C.; Aiba, Kazuhiro; Hamatani, Toshio; Kargul, George J.; Luo, Amber G.; Kelso, Janet; Hide, Winston; Ko, Minoru S.H.

    2002-01-01

    A set of 7407 cDNA clones (NIA mouse 7.4K) was assembled from >20 cDNA libraries constructed mainly from early mouse embryos, including several stem cell libraries. The clone set was assembled from embryonic and newborn organ libraries consisting of ∼120,000 cDNA clones, which were initially re-arrayed into a set of ∼11,000 unique cDNA clones. A set of tubes was constructed from the racks in this set to prevent contamination and potential mishandling errors in all further re-arrays. Sequences from this set (11K) were analyzed further for quality and clone identity, and high-quality clones with verified identity were re-arrayed into the final set (7.4K). The set is freely available, and a corresponding database was built to provide comprehensive annotation for those clones with known identity or homology, and has been made available through an extensive Web site that includes many link-outs to external databases and analysis servers. [The sequence data from this study have been submitted to GenBank under accession nos. BQ550036–BQ563104.] PMID:12466305

  6. Retrotransposon expression and incorporation of cloned human and mouse retroelements in human spermatozoa.

    PubMed

    Lazaros, Leandros; Kitsou, Chrysoula; Kostoulas, Charilaos; Bellou, Sofia; Hatzi, Elissavet; Ladias, Paris; Stefos, Theodoros; Markoula, Sofia; Galani, Vasiliki; Vartholomatos, Georgios; Tzavaras, Theodore; Georgiou, Ioannis

    2017-03-01

    To investigate the expression of long interspersed element (LINE) 1, human endogenous retrovirus (HERV) K10, and short interspersed element-VNTR-Alu element (SVA) retrotransposons in ejaculated human spermatozoa by means of reverse-transcription (RT) polymerase chain reaction (PCR) analysis as well as the potential incorporation of cloned human and mouse active retroelements in human sperm cell genome. Laboratory study. University research laboratories and academic hospital. Normozoospermic and oligozoospermic white men. RT-PCR analysis was performed to confirm the retrotransposon expression in human spermatozoa. Exogenous retroelements were tagged with a plasmid containing a green fluorescence (EGFP) retrotransposition cassette, and the de novo retrotransposition events were tested with the use of PCR, fluorescence-activated cell sorting analysis, and confocal microscopy. Retroelement expression in human spermatozoa, incorporation of cloned human and mouse active retroelements in human sperm genome, and de novo retrotransposition events in human spermatozoa. RT-PCR products of expressed human LINE-1, HERV-K10, and SVA retrotransposons were observed in ejaculated human sperm samples. The incubation of human spermatozoa with either retrotransposition-active human LINE-1 and HERV-K10 or mouse reverse transcriptase-deficient VL30 retrotransposons tagged with an EGFP-based retrotransposition cassette led to EGFP-positive spermatozo; 16.67% of the samples were positive for retrotransposition. The respective retrotransposition frequencies for the LINE-1, HERV-K10, and VL30 retrotransposons in the positive samples were 0.34 ± 0.13%, 0.37 ± 0.17%, and 0.30 ± 0.14% per sample of 10,000 spermatozoa. Our results show that: 1) LINE-1, HERV-K10, and SVA retrotransposons are transcriptionally expressed in human spermatozoa; 2) cloned active retroelements of human and mammalian origin can be incorporated in human sperm genome; 3) active reverse transcriptases exist in human

  7. Isolation and characterization of mouse-human microcell hybrid cell clones permissive for infectious HIV particle release

    PubMed Central

    Coskun, Ayse K.; van Maanen, Marc; Janka, David; Stockton, David; Stankiewicz, Pawel; Yatsenko, Svetlana; Sutton, Richard E.

    2007-01-01

    Mouse cells are non-permissive to human immunodeficiency virus type 1 (HIV) in that there is a pronounced post-integration block to viral replication. We have recently demonstrated that mouse-human somatic cell hybrids that contain human chromosome 2 increase both HIV Capsid (CA) production and infectious virus release. Here we report on the isolation of three mouse-human microcell hybrids (MCHs) that behave similarly, starting from a pool of 500 MCH clones. Release of virus was specific to HIV and cell revertants that no longer contained any human chromosome fragments did not release CA or infectious virus. Two of the three cell clones were identical as judged by PCR STS content and fluorescence in situ hybridization (FISH) and contained a single 2-12 human chromosome chimera. The third cell clone only contained human chromosome 12, as determined by PCR, FISH, and microarray analyses. There were no consistent differences in Gag protein and spliced/unspliced viral RNA levels between mouse cell lines. CMV promoter-driven, codon-optimized gag-pol had no effect on infectious HIV release from these mouse cells, despite allowing Gag targeting and increasing CA production. These permissive mouse-human MCHs and their corresponding non-permissive revertants may prove useful for mechanistic studies and also for identifying the responsible gene(s) or factor(s) involved in the production of HIV. PMID:17270231

  8. Cloning and characterization of a cdc25 phosphatase from mouse lymphocytes.

    PubMed

    Nargi, J L; Woodford-Thomas, T A

    1994-01-01

    Members of the cdc25 phosphatase family are proposed to function as important regulators of the eukaryotic cell cycle, particularly in the induction of mitotic events. A new cdc25 tyrosine phosphatase, cdc25M1, has been cloned from a mouse pre-B cell cDNA library and characterized. The cdc25M1 protein consists of 465 amino acids with a predicted relative molecular mass (M(r)) of 51,750. Over the highly conserved carboxyl terminal region, the amino acid sequence similarity to the human cdc25 C or Hs1 isoform is 89%, while the overall similarity is 67%. The phosphatase active site is located within residues 367-374. Tissue expression of the cdc25M1 was highest in mouse spleen and thymus by northern blot analysis. The cdc25M1 mRNA was detected in a number of cloned mouse lymphocyte cell lines including both CD8+ and CD4+ cells. cdc25M1 mRNA was shown to be cell cycle-regulated in T cells following interleukin-2 (IL-2)-stimulation. Accumulation of cdc25M1 mRNA occurred at 48 h after IL-2 stimulation, when lymphocytes were progressing from S phase to G2/M phase of the cell cycle. This pattern of expression is in contrast to that observed for other protein tyrosine phosphatases expressed in T lymphocytes including CD45, LRP, SHP, and PEP. The elevation in cdc25M1 mRNA level occurred concomittant to the appearance of the hyperphosphorylated form of p34cdc2 protein kinase. A purified, bacterial-expressed recombinant cdc25M1 phosphatase domain catalyzed the dephosphorylation of p-nitrophenol phosphate, as well as [32P-Tyr] and [32P-Ser/Thr]-containing substrates. Preincubation of p34cdc2 kinase with cdc25M1 activated its histone H1 kinase activity in vitro. These results suggest that cdc25M1 may be involved in regulating the proliferation of mouse T lymphocytes following cytokine stimulation, through its action on p34cdc2 kinase.

  9. Cloning and Characterization of Inducible Nitric Oxide Synthase from Mouse Macrophages

    NASA Astrophysics Data System (ADS)

    Xie, Qiao-Wen; Cho, Hearn J.; Calaycay, Jimmy; Mumford, Richard A.; Swiderek, Kristine M.; Lee, Terry D.; Ding, Aihao; Troso, Tiffany; Nathan, Carl

    1992-04-01

    Nitric oxide (NO) conveys a variety of messages between cells, including signals for vasorelaxation, neurotransmission, and cytotoxicity. In some endothelial cells and neurons, a constitutive NO synthase is activated transiently by agonists that elevate intracellular calcium concentrations and promote the binding of calmodulin. In contrast, in macrophages, NO synthase activity appears slowly after exposure of the cells to cytokines and bacterial products, is sustained, and functions independently of calcium and calmodulin. A monospecific antibody was used to clone complementary DNA that encoded two isoforms of NO synthase from immunologically activated mouse macrophages. Liquid chromatography-mass spectrometry was used to confirm most of the amino acid sequence. Macrophage NO synthase differs extensively from cerebellar NO synthase. The macrophage enzyme is immunologically induced at the transcriptional level and closely resembles the enzyme in cytokine-treated tumor cells and inflammatory neutrophils.

  10. Astrocyte cytolysis by MHC class II-specific mouse T cell clones.

    PubMed

    Reder, A T; Lascola, C D; Flanders, S A; Maimone, D; Jensen, M A; Skias, D D; Lancki, D W

    1993-08-01

    The brain is "immunologically privileged," in part because class I and II MHC antigens are not normally present on glia or neurons. However, under certain conditions such as transplantation, glial cells express MHC proteins at levels sufficient for glia to become targets of immune responses. Cultured astrocytes expressing very low levels of MHC class I protein are killed efficiently by MHC class I antigen-specific CTL. Mouse brain allografts, however, are rejected by CD4+ T cells that are likely to be class II MHC-specific. The level of expression of MHC class II antigen needed to trigger specific killing of astrocytes by CD4+ T cells, independent of exogenous antigen, has not been studied. We examined the role of glial class II MHC in the lysis of cultured neonatal mouse astrocytes by an alloreactive murine CD4+ CTL alone. IFN-gamma induced functionally relevant increases in MHC class II antigen on target cells. Astrocytes were lysed by the CD4+ clone only when class II MHC antigens reached levels readily detectable by flow cytometry. MHC class II expression and lysis increased when astrocytes were coincubated with IFN-gamma and TNF-alpha. Conversely, lysis decreased when class II expression was downregulated by IFN-alpha/beta or dbcAMP. Cytolysis by CD4+ clones was blocked by antibodies to CD4 and LFA-1 on T cells, and to ICAM-1 and class II molecules on astrocytes. The role of LFA-1 in CD4+ cell-mediated lysis was greater than that of LFA-1/ICAM-1 in CD8+ T cell-mediated lysis. CD4+ cells may lyse activated astrocytes when the immune privilege of the brain is compromised as in transplantation, tumors, and inflammatory diseases.

  11. 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.

  12. 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.

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

    SciTech Connect

    Kuo, C.J.; Conley, P.B.; Hsieh, Chihlin; Francke, U.; Crabtree, G.R. )

    1990-12-01

    The homeodomain-containing transcription factor hepatocyte nuclear factor 1 (HNF-1) most likely plays and essential role during liver organogenesis by transactivating a family of {gt}15 predominantly hepatic genes. The authors 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 NHF-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.

  14. Cloning of the genes encoding mouse cardiac and skeletal calsequestrins: expression pattern during embryogenesis.

    PubMed

    Park, K W; Goo, J H; Chung, H S; Kim, H; Kim, D H; Park, W J

    1998-09-14

    Calsequestrin is a low-affinity and high-capacity calcium-binding protein in the sarcoplasmic reticulum (SR). In the present study, we have cloned and sequenced mouse cardiac and skeletal calsequestrin cDNAs. The deduced amino acid sequences are highly homologous to those of other mammalian calsequestrins. As expected, the cardiac and skeletal calsequestrins are expressed specifically and exclusively in adult heart and skeletal muscles, respectively. In-situ hybridization was performed to examine the expression pattern of the calsequestrins in the developing mouse and rat embryos. During early organogenesis, the cardiac and skeletal calsequestrin transcripts were detected exclusively in the heart primordium and the myotome of somites, respectively. The cardiac calsequestrin transcripts were later detected in fetal heart and skeletal muscles, whereas the skeletal calsequestrin transcripts were only found in fetal skeletal muscles. These data suggest that the cardiac calsequestrin plays a role in the differentiation and function of heart, and in the function of fetal skeletal muscles in conjunction with the skeletal calsequestrin, but not in the early differentiation of the myotome of somites. The expression of the skeletal calsequestrin in the myotome is regulated probably by myogenin, a myogenic regulatory gene.

  15. Toxoplasma gondii sexual cross in a single naturally infected feline host: Generation of highly mouse-virulent and avirulent clones, genotypically different from clonal types I, II and III

    PubMed Central

    2012-01-01

    Tachyzoite clones obtained from a single Toxoplasma gondii oocyst field sample were genotyped and characterized regarding mouse virulence. PCR-RFLP genotyping of tachyzoites initially isolated from interferon-γ-knockout (GKO) mice, BALB/c mice and VERO cell culture using the nine independent, unlinked genetic markers nSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico revealed mixed T. gondii infections showing combinations of type II and type III alleles at different loci. Forty-five individual clones were obtained from all mixed T. gondii tachyzoite cell cultures by limiting dilution. Sixteen T. gondii clones showed type III alleles at all loci and 29 clones displayed a combination of type II and type III alleles at different loci. Five clone groups were identified in total, four of which include T. gondii clones that showed a non-canonical allele pattern and have never been described in natural infections before. All tested clones, except two, were highly virulent in BALB/c mice. The isolation of different non-canonical T. gondii clones originating from an oocyst sample of a single naturally infected cat demonstrate that sexual recombination as well as re-assortment of chromosomes via a sexual cross of T. gondii occur under natural conditions and result in the emergence of clones with increased virulence in mice. PMID:22546040

  16. 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.

  17. 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

  18. Molecular cloning of a mouse DNA repair gene that complements the defect of group-A xeroderma pigmentosum.

    PubMed Central

    Tanaka, K; Satokata, I; Ogita, Z; Uchida, T; Okada, Y

    1989-01-01

    For isolation of the gene responsible for xeroderma pigmentosum (XP) complementation group A, plasmid pSV2gpt and genomic DNA from a mouse embryo were cotransfected into XP2OSSV cells, a group-A XP cell line. Two primary UV-resistant XP transfectants were isolated from about 1.6 X 10(5) pSV2gpt-transformed XP colonies. pSV2gpt and genomic DNA from the primary transfectants were again cotransfected into XP2OSSV cells and a secondary UV-resistant XP transfectant was obtained by screening about 4.8 X 10(5) pSV2gpt-transformed XP colonies. The secondary transfectant retained fewer mouse repetitive sequences. A mouse gene that complements the defect of XP2OSSV cells was cloned into an EMBL3 vector from the genome of a secondary transfectant. Transfections of the cloned DNA also conferred UV resistance on another group-A XP cell line but not on XP cell lines of group C, D, F, or G. Northern blot analysis of poly(A)+ RNA with a subfragment of cloned mouse DNA repair gene as the probe revealed that an approximately 1.0 kilobase mRNA was transcribed in the donor mouse embryo and secondary transfectant, and approximately 1.0- and approximately 1.3-kilobase mRNAs were transcribed in normal human cells, but none of these mRNAs was detected in three strains of group-A XP cells. These results suggest that the cloned DNA repair gene is specific for group-A XP and may be the mouse homologue of the group-A XP human gene. Images PMID:2748601

  19. Molecular cloning of a mouse DNA repair gene that complements the defect of group-A xeroderma pigmentosum

    SciTech Connect

    Tanaka, K.; Satokata, I.; Ogita, Z.; Uchida, T.; Okada, Y.

    1989-07-01

    For isolation of the gene responsible for xeroderma pigmentosum (XP) complementation group A, plasmid pSV2gpt and genomic DNA from a mouse embryo were cotransfected into XP2OSSV cells, a group-A XP cell line. Two primary UV-resistant XP transfectants were isolated from about 1.6 X 10(5) pSV2gpt-transformed XP colonies. pSV2gpt and genomic DNA from the primary transfectants were again cotransfected into XP2OSSV cells and a secondary UV-resistant XP transfectant was obtained by screening about 4.8 X 10(5) pSV2gpt-transformed XP colonies. The secondary transfectant retained fewer mouse repetitive sequences. A mouse gene that complements the defect of XP2OSSV cells was cloned into an EMBL3 vector from the genome of a secondary transfectant. Transfections of the cloned DNA also conferred UV resistance on another group-A XP cell line but not on XP cell lines of group C, D, F, or G. Northern blot analysis of poly(A)+ RNA with a subfragment of cloned mouse DNA repair gene as the probe revealed that an approximately 1.0 kilobase mRNA was transcribed in the donor mouse embryo and secondary transfectant, and approximately 1.0- and approximately 1.3-kilobase mRNAs were transcribed in normal human cells, but none of these mRNAs was detected in three strains of group-A XP cells. These results suggest that the cloned DNA repair gene is specific for group-A XP and may be the mouse homologue of the group-A XP human gene.

  20. Cloning and characterization of mouse growth hormone-releasing hormone (GRH) complementary DNA: increased GRH messenger RNA levels in the growth hormone-deficient lit/lit mouse.

    PubMed

    Frohman, M A; Downs, T R; Chomczynski, P; Frohman, L A

    1989-10-01

    We have isolated and cloned the full length cDNA for mouse GH-releasing hormone (mGRH) from mouse hypothalamus using a recently described strategy involving the polymerase chain reaction technique (PCR). Degenerate oligonucleotide primers were selected based on short (six amino acids) conserved regions in the human and rat GRH peptides that would recognize DNA sequences encoding similar amino acids regardless of codon usage. Primer-extended cDNA was amplified by PCR on cDNA templates prepared by reverse transcribing total mouse hypothalamic RNA. After cloning and sequencing the initial product, the 3' and 5' ends of mGRH were generated using a separate PCR strategy (RACE protocol). The mGRH cDNA encodes a 103-amino acid reading frame, structurally similar to the human and rat GRH genes, containing a signal sequence, a 42-residue GRH peptide, and a 31-residue C-terminal region. Although the structures of mouse and rat GRH are highly conserved in the signal peptide and C-terminal region, there is considerable diversity in the GRH region, which exhibits nearly comparable homology with the rat (68%) and human (62%) structures. Differences between mouse and rat GRH were also found in the amino acid cleavage sites at the 5' and 3' ends of the mature peptide and at the polyadenylation signal.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Molecular cloning and analysis of the endogenous retrovirus chemically induced from RFM/Un mouse cell cultures

    SciTech Connect

    Liou, R.S.; Boone, L.R.; Kiggans, J.O.; Yang, D.M.; Wang, T.W.; Tennant, R.W.; Yang, W.K.

    1983-04-01

    An N-tropic ecotropic retrovirus induced with iododeoxyuridine from RFM/Un mouse cell cultures was molecularly cloned and analyzed. Based on the restriction map, the RFM/Un virus appears to be indistinguishable from other induced N-tropic retroviruses. A nucleotide sequence analysis of the long terminal repeat of an infectious clone revealed structural features characteristic of murine type C retrovirus long terminal repeats. The U3 region of the RFM/Un virus long terminal repeat, however, contained no short sequence duplication or insertion found in other murine leukemia virus isolates.

  2. Mouse microsomal triglyceride transfer protein large subunit: cDNA cloning, tissue-specific expression, and chromosomal localization

    SciTech Connect

    Nakamuta, Makoto; Chang, Benny Hung-Junn; Hoogeveen, R.

    1996-04-15

    Microsomal triglyceride transfer protein (MTP) catalyzes the transfer of triglyceride, cholesteryl ester, and phospholipid between membranes. It is essential for the secretion of apolipoprotein B from the cell. Mutations in MTP are a major cause of abetalipoproteinemia. The mouse is a popular animal model for lipoprotein metabolism. We have cloned and sequenced mouse MTP cDNA. The DNA-deduced amino acid sequence indicates that mouse protein shows 93, 86, and 83% sequence indicates that mouse MTP contains 894 amino acids; the mouse protein shows 93, 86, and 83% sequence identity to the hamster, human, and bovine sequences, respectively. Northern blot analysis indicates that mouse MTP mRNA is expressed at high levels in the small intestine and at substantially lower levels in the liver and that it is not detectable in six other tissues examined. The mouse MTP gene has been localized to the distal region of chromosome 3 by Southern blots of interspecific backcross panels using progeny derived from matings of (C57BL/6J x SPRET/Ei)F1 x SPRET/Ei. Comparison of MTP sequences from human, bovine, hamster, and mouse indicates that the C-terminal region of MTP is better conserved than its N-terminal region. 21 refs., 2 figs.

  3. Immortalization and Characterization of Mouse Temporomandibular Joint Disc Cell Clones with Capacity for Multi-lineage Differentiation

    PubMed Central

    Park, Young; Hosomichi, Jun; Ge, Chunxi; Xu, Jinping; Franceschi, Renny; Kapila, Sunil

    2015-01-01

    Objective Despite the importance of TMJ disc in normal function and disease, studying the responses of its cells has been complicated by the lack of adequate characterization of the cell subtypes. The purpose of our investigation was to immortalize, clone, characterize and determine the multi-lineage potential of mouse TMJ disc cells. Design Cells from 12-week-old female mice were cultured and immortalized by stable transfection with human telomerase reverse transcriptase. The immortalized cell clones were phenotyped for fibroblast- or chondrocyte-like characteristics and ability to undergo adipocytic, osteoblastic and chondrocytic differentiation. Results Of 36 isolated clones, four demonstrated successful immortalization and maintenance of stable protein expression for up to 50 passages. Two clones each were initially characterized as fibroblast-like and chondrocyte-like on the basis of cell morphology and growth rate. Further the chondrocyte-like clones had higher mRNA expression levels of cartilage oligomeric matrix protein (>3.5-fold), collagen × (>11-fold), collagen II expression (2-fold) and collagen II:I ratio than the fibroblast-like clones. In contrast, the fibroblast-like clones had higher mRNA expression level of vimentin (>1.5-fold), and fibroblastic specific protein 1 (>2.5-fold) than he chondrocyte-like clones. Both cell types retained multi-lineage potential as demonstrated by their capacity to undergo robust adipogenic, osteogenic and chondrogenic differentiation. Conclusions These studies are the first to immortalize TMJ disc cells and characterize chondrocyte-like and fibroblast-like clones with retained multi-differentiation potential that would be a valuable resource in studies to dissect the behavior of specific cell types in health and disease and for tissue engineering. PMID:25887369

  4. Functional properties of a cloned 5-hydroxytryptamine ionotropic receptor subunit: comparison with native mouse receptors.

    PubMed Central

    Hussy, N; Lukas, W; Jones, K A

    1994-01-01

    1. A comparative study of the whole-cell and single-channel properties of cloned and native mouse 5-hydroxytryptamine ionotropic receptors (5-HT3) was undertaken using mammalian cell lines expressing the cloned 5-HT3 receptor subunit A (5-HT3R-A), superior cervical ganglia (SCG) neurones and N1E-115 cells. 2. No pharmacological difference was found in the sensitivity to the agonists 5-HT and 2-methyl-5-HT, or to the antagonists d-tubocurare and 3-tropanyl-3,5-dichlorobenzoate (MDL-72222). 3. Current-voltage (I-V) relationships of whole-cell currents showed inward rectification in the three preparations. Rectification was stronger both in cells expressing the 5-HT3R-A subunit and in N1E-115 cells when compared with SCG neurones. 4. No clear openings could be resolved in 5-HT-activated currents in patches excised from cells expressing the 5-HT3R-A subunit or N1E-115 cells. Current fluctuation analysis of whole-cell and excised-patch records revealed a slope conductance of 0.4-0.6 pS in both preparations. Current-voltage relationships of these channels showed strong rectification that fully accounted for the whole-cell voltage dependence. 5. In contrast, single channels of about 10 pS were activated by 5-HT in patches excised from SCG neurones. The weak voltage dependence of their conductance did not account completely for the rectification of whole-cell currents. A lower unitary conductance (3.4 pS) was inferred from whole-cell noise analysis. 6. We conclude that the receptor expressed from the cloned cDNA is indistinguishable from the 5-HT3 receptor of N1E-115 cells, suggesting an identical structure for these two receptors. The higher conductance and different voltage dependence of the 5-HT3 receptor in SCG neurones might indicate the participation of an additional subunit in the structure of native ganglionic 5-HT3 receptors. Homo-oligomeric 5-HT3R-A channels may also be present as suggested by the lower conductance estimated by whole-cell noise analysis. PMID

  5. Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells.

    PubMed

    Marquardt, D L; Walker, L L; Heinemann, S

    1994-05-01

    Adenosine potentiates the stimulated release of mast cell mediators. Pharmacologic studies suggest the presence of two adenosine receptors, one positively coupled to adenylate cyclase and the other coupled to phospholipase C activation. To identify mast cell adenosine receptor subtypes, cDNAs for the A1 and A2a adenosine receptors were obtained by screening a mouse brain cDNA library with the use of PCR-derived probes. Mouse bone marrow-derived mast cell cDNA libraries were constructed and screened with the use of A1 and A2a cDNA probes, which revealed the presence of A2a, but not A1, receptor clones. A putative A2b receptor was identified by using low stringency mast cell library screening. Northern blotting of mast cell poly(A)+ RNA with the use of receptor subtype probes labeled single mRNA bands of 2.4 kb and 1.8 kb for the A2a and A2b receptors, respectively. In situ cells. An A2a receptor-specific agonist failed to enhance mast cell mediator release, which suggests that the secretory process is modulated through the A2b and/or another receptor subtype. By using RNase protection assays, we found that mast cells that had been cultured in the presence of N-ethylcarboxamidoadenosine for 24 h exhibited a decrease in both A2a and A2b receptor RNA levels. Cells that had been cultured for 1 to 2 days in the presence of dexamethasone demonstrated increased amounts of A2a receptor mRNA, but no identifiable change in A2b receptor mRNA. Mast cells possess at least two adenosine receptor subtypes that may be differentially regulated.

  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. 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.

  8. Cloning, expression, purification and preliminary X-ray crystallographic analysis of mouse protein arginine methyltransferase 7.

    PubMed

    Cura, Vincent; Troffer-Charlier, Nathalie; Lambert, Marie-Annick; Bonnefond, Luc; Cavarelli, Jean

    2014-01-01

    Protein arginine methyltransferase 7 (PRMT7) is a unique but less characterized member of the family of protein arginine methyltransferases (PRMTs) that plays a role in male germline gene imprinting. PRMT7 is the only known PRMT member that catalyzes the monomethylation but not the dimethylation of the target arginine residues and harbours two catalytic domains in tandem. PRMT7 genes from five different species were cloned and expressed in Escherichia coli and Sf21 insect cells. Four gave soluble proteins from Sf21 cells, of which two were homogeneous and one gave crystals. The mouse PRMT7 structure was solved by the single anomalous dispersion method using a crystal soaked with thimerosal that diffracted to beyond 2.1 Å resolution. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 97.4, c = 168.1 Å and one PRMT7 monomer in the asymmetric unit. The structure of another crystal form belonging to space group I222 was solved by molecular replacement.

  9. 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

  10. 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

  11. Establishment of mouse-immortalized hybrid clones expressing characteristics of differentiated neurons derived from the cerebellar and brain stem regions.

    PubMed

    Satoh, J; Gallyas, F; Endoh, M; Yamamura, T; Kunishita, T; Kobayashi, T; Tabira, T

    1992-09-01

    Two clonal immortalized neurons designated CL8c4.7 and CL8a5.2 were established by somatic cell fusion between a hypoxanthine phosphoribosyltransferase-(HPRT-) deficient neuroblastoma N18TG2 and newborn mouse cerebellar/brain stem neurons. In the serum-containing medium without extra differentiating agents, both clones exhibited a morphology of differentiated neurons. They contained high levels of glutamate but no gamma-aminobutyric acid (GABA). The CL8a5.2 clone synthesized choline acetyltransferase and serotonin. In immunocytochemical studies, both clones expressed 200 kD neurofilament protein, neuron-specific enolase, microtubule-associated protein 2 (MAP2), tau protein, neuronal cell adhesion molecule (N-CAM), HNK-1, Thy-1.2, saxitoxin-binding sodium channel protein, and glutamate. Synaptophysin immunoreactivity was identified in the neuritic terminals of CL8c4.7 cells. Most of these antigens were barely detectable on N18TG2 cells. Electrophysiologically, both clones generated action potentials in response to electrical stimuli. The hybrid clones that express characteristics of differentiated neurons derived from the cerebellar and brain stem regions might be invaluable for the study of the molecular basis of neuronal differentiation and degeneration in these regions.

  12. The nucleotide sequence of the putative transcription initiation site of a cloned ribosomal RNA gene of the mouse.

    PubMed Central

    Urano, Y; Kominami, R; Mishima, Y; Muramatsu, M

    1980-01-01

    Approximately one kilobase pairs surrounding and upstream the transcription initiation site of a cloned ribosomal DNA (rDNA) of the mouse were sequenced. The putative transcription initiation site was determined by two independent methods: one nuclease S1 protection and the other reverse transcriptase elongation mapping using isolated 45S ribosomal RNA precursor (45S RNA) and appropriate restriction fragments of rDNA. Both methods gave an identical result; 45S RNA had a structure starting from ACTCTTAG---. Characteristically, mouse rDNA had many T clusters (greater than or equal to 5) upstream the initiation site, the longest being 21 consecutive T's. A pentadecanucleotide, TGCCTCCCGAGTGCA, appeared twice within 260 nucleotides upstream the putative initiation site. No such characteristic sequences were found downstream this site. Little similarity was found in the upstream of the transcription initiation site between the mouse, Xenopus laevis and Saccharomyces cerevisiae rDNA. Images PMID:6162156

  13. Molecular cloning of mouse erythrocyte protein 4.2: a membrane protein with strong homology with the transglutaminase supergene family.

    PubMed

    Rybicki, A C; Schwartz, R S; Qiu, J J; Gilman, J G

    1994-07-01

    We report the molecular cloning and characterization of mouse erythrocyte protein 4.2 (P4.2). Mouse erythrocyte P4.2 is a 691-amino-acid protein with a predicted MW of 77 kDa. Northern blot analysis detected a 2.2-kb transcript in mouse reticulocytes, compared with a 2.4- to 2.5-kb transcript in human reticulocytes, which is consistent with the absence of the 30-amino-acid splicing insert in mouse erythrocyte P4.2 that is found in the human protein (isoform I). Like the human erythrocyte P4.2, mouse erythrocyte P4.2 contains regions strikingly homologous with the transglutaminase (TGase) proteins although it too most likely lacks TGase crosslinking activity. Mouse P4.2 is on average 73% identical with human erythrocyte P4.2, although regional variations exist, with greatest conservation in the regions of the molecule that contain the TGase active site, the TGase calcium-binding site, and a band 3 binding site. Hydropathy analysis reveals a protein containing a series of hydrophobic domains, similar to the situation for human P4.2 and consistent with its tight binding to the membrane, although the mouse P4.2 is missing both the strongly hydrophilic region and adjacent highly charged region that are present in the human protein, suggesting that the two proteins could differ in their physical characteristics, binding associations, or functional properties. The availability of the complete mouse erythrocyte P4.2 cDNA should help in the design of P4.2-deficient animal models (for example, ribozyme or homologous recombinant "knockout" models) that should accelerate the understanding of P4.2 function in both erythroid and non-erythroid cells.

  14. Cloning and characterization of the mouse homolog (D12H14S564E) of a novel human cochlear gene (D14S564E)

    SciTech Connect

    Kovatch, K.A.; Robertson, N.G.; Brody, T.H.

    1994-09-01

    We have isolated mouse cDNA clones homologous to a novel human cochlear cDNA (D14S564E) identified by subtractive hybridization of a human fetal cochlear cDNA library with human fetal brain RNA. The human gene is preferentially expressed in cochlea, with low level expression in brain and eye, and has three transcripts of 2.0, 2.3, and 2.9 kb. To investigate the structure and function of the human cochlear gene and its role in the biology of hearing and deafness, we have cloned the mouse homolog (D12H14S564E). Mouse cDNAs, ranging from 2.0 to 3.5 kb, were identified with a mouse brain cDNA library screened by hybridization with the human cDNA. Analysis of mouse clones by DNA sequencing revealed significant homology with the human cochlear gene; in one region analyzed, the mouse clone possessed 88% homology over 525 bases with the human clone. In the area of homology, there is an open reading frame of approximately 125 amino acids. Comparison of the homologous sequence with those entered in GenBank identifies similarity with a von Willebrand factor type A-like domain in the area of sequence conservation, a feature consistent with the human clone. Known proteins containing the von Willebrand type A-like domain have diverse functions including extracellular matrix assembly, hemostasis, cellular adhesion and defense mechanisms. Further homology of mouse and human clones is supported by hybridization of the mouse clone to a Northern blot of human fetal cochlea and brain RNA; results show that the mouse clone hybridizes with the same three messages in the human cochlea RNA as does the novel human cochlear gene. D12H14S564E maps to mouse chromosome 12 in a region to which asp-1 (audiogenic seizure prone) is assigned. Further sequencing and expression studies are in progress to fully characterize the mouse gene and its homology with the novel human cochlear gene.

  15. Detection and characterization of a mouse. alpha. -spectrin cDNA clone by its expression in Escherichia coli

    SciTech Connect

    Cioe, L.; Curtis, P.

    1985-03-01

    A cloned segment of mouse ..alpha..-spectrin mRNA has been identified by radioimmunoassay and immunoautoradiography. Double-stranded cDNA derived from spleens of anemic mice was introduced into a bacterial expression vector, pUC, and transformed Escherichia coli colonies were screened by using an /sup 125/I-labeled antiserum to erythrocyte membrane ghost proteins. Of 17 positive colonies, 2 bound antibody to mouse spectrin, and these 2 colonies contained 750-base-pair inserts that cross-hybridized. Transfer of the 750-base-pair insert to an expression vector containing the P/sub L/ promoter of phage lambda produced larger amounts of peptides that were bound by antibody to mouse spectrin. The spectrin-like peptides made in E. coli elicited antibody that reacted only with the ..alpha..-spectrin subunit of erythrocyte membranes. This clone will be useful for the study of the structure and expression of the spectrin gene, particularly in understanding the role of spectrin in human inherited hemolytic anemias.

  16. 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.

  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

  18. 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…

  19. 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…

  20. 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…

  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. Cloning, comparative mapping, and RNA expression of the mouse homologues of the Saccharomyces cerevisiae nucleotide excision repair gene RAD23

    SciTech Connect

    Spek, P.J. van der; Visser, C.E.; Bootsma, D.

    1996-01-01

    The Saccharomyces cerevisiae RAD23 gene is involved in nucleotide excision repair (NER). Two human homologs of RAD23, HHR23A and HHR23B (HGMW-approved symbols RAD23A and RAD23B), were previously isolated. The HHR23B protein is complexed with the protein defective in the cancer-prone repair syndrome xeroderma pigmentosum, complementation group C, and is specifically involved in the global genome NER subpathway. The cloning of both mouse homologs (designated MHR23A and MHR23B) and detailed sequence comparison permitted the deduction of the following overall structure for all RAD23 homologs: an ubiquitin-like N-terminus followed by a strongly conserved 50-amino-acid domain that is repeated at the C-terminus. We also found this domain as a specific C-terminal extension of one of the ubiquitin-conjugating enzymes, providing a second link with the ubiquitin pathway. By means of in situ hybridization, MHR23A was assigned to mouse chromosome 8C3 and MHR23B to 4B3. Because of the close chromosomal proximity of human XPC and HHR23B, the mouse XPC chromosomal location was determined (6D). Physical disconnection of the genes in mouse argues against a functional significance of the colocalization of these genes in human. Northern blot analysis revealed constitutive expression of both MHR23 genes in all tissues examined. Elevated RNA expression of both MHR23 genes was observed in testis. Although the RAD23 equivalents are well conserved during evolution, the mammalian genes did not express the UV-inducible phenotype of their yeast counterpart. This may point to a fundamental difference between the UV responses of yeast and human. No stage-specific mRNA expression during the cell cycle was observed for the mammalian RAD23 homologs. 38 refs., 5 figs.

  3. Cloning, functional expression, and chromosomal localization of the human and mouse gp180-carboxypeptidase D-like enzyme.

    PubMed

    Ishikawa, T; Murakami, K; Kido, Y; Ohnishi, S; Yazaki, Y; Harada, F; Kuroki, K

    1998-07-30

    We previously reported that a host cell glycoprotein, gp180, binds duck hepatitis B virus particles, and is encoded by a member of the carboxypeptidase gene family (Kuroki, K., Eng, F., Ishikawa, T., Turck, C., Harada, F., Ganem, D., 1995. gp180, a host cell glycoprotein that binds duck hepatitis B virus particles, is encoded by a member of the carboxypeptidase gene family. J. Biol. Chem. 270, 15022-15028). After that report, carboxypeptidase D (CPD) was subsequently purified from bovine pituitary and characterized as a novel carboxypeptidase E (CPE)-like enzyme, with many characteristics in common with duck gp180 (Song, L., Fricker, L.D., 1995. Purification and characterization of carboxypeptidase D, a novel carboxypeptidase E-like enzyme, from bovine pituitary. J. Biol. Chem. 270, 25007-25013). CPD is now supposed to play an important role in a secretory pathway. To clarify the function of gp180 further, we have isolated and analyzed human and mouse homologues of duck gp180. cDNA clones derived from human HepG2 cells and mouse livers have been isolated on the basis of homology to the duck gp180. The suggested open reading frames of the human and mouse cDNA encode 1380 and 1377 amino acid proteins, respectively and have three carboxypeptidase homologous domains (A, B, and C). Domains A and B have completely conserved the residues known to have the enzymatic activity of carboxypeptidase, but domain C in each cDNA does not. Northern blotting revealed a ubiquitous tissue distribution of human gp180 mRNA with several transcript species. Expression of human gp180 cDNA in transfected 293Tcells exhibited carboxypeptidase activity upon radiometric assay. The human and mouse homologues of duck gp180 have many characteristics in common with bovine CPD. Fluorescence in-situ hybridization reveals that the gene encoding human gp180 is located in region 17q11.2.

  4. High-level expression of a cloned HLA heavy chain gene introduced into mouse cells on a bovine papillomavirus vector.

    PubMed

    DiMaio, D; Corbin, V; Sibley, E; Maniatis, T

    1984-02-01

    A gene encoding the heavy chain of an HLA human histocompatibility antigen was isolated from a library of human DNA by recombination and selection in vivo. After insertion into a bovine papillomavirus (BPV) DNA expression vector, the gene was introduced into cultured mouse cells. Cells transformed with the HLA-BPV plasmids did not appear to contain extrachromosomal viral DNA, whereas BPV recombinants usually replicated as plasmids in transformed cell lines. Large amounts of HLA RNA were produced by the transformed cells, and the rate of synthesis of human heavy chain was several-fold higher than in the JY cell line, a well-characterized human lymphoblastoid cell line which expresses high levels of surface HLA antigen. Substantial amounts of human heavy chain accumulated in the transformed cells, and HLA antigen was present at the cell surface. These observations establish the feasibility of using BPV vectors to study the structure and function of HLA antigens and the expression of cloned HLA genes.

  5. Cloning of a hamster anti-mouse CD79B antibody sequences and identification of a new hamster immunoglobulin lambda constant IGLC gene region

    PubMed Central

    Haggart, Ryan; Perera, Jason; Huang, Haochu

    2013-01-01

    Anti-CD79 antibodies have been effective at targeting B cell lymphoma cells and depleting B cells in animal models. In order to engineer recombinant antibodies with additional effector functions in mice, we cloned and sequenced the full-length cDNAs of the heavy and light chain of a hamster anti-mouse CD79B antibody. Although hamster antibodies represent a unique source of monoclonal antibodies against mouse, rat, and human antigens, sequence information of hamster immunoglobulins (IG) is sparse. Here we report a new hamster (Cricetulus migratorius) IG lambda constant (IGLC) gene region that is most homologous to mouse IGLC2 and IGLC3. PMID:23558558

  6. MicroRNA (miRNA) cloning analysis reveals sex differences in miRNA expression profiles between adult mouse testis and ovary.

    PubMed

    Mishima, Takuya; Takizawa, Takami; Luo, Shan-Shun; Ishibashi, Osamu; Kawahigashi, Yutaka; Mizuguchi, Yoshiaki; Ishikawa, Tomoko; Mori, Miki; Kanda, Tomohiro; Goto, Tadashi; Takizawa, Toshihiro

    2008-12-01

    MicroRNAs (miRNAs) are endogenous non-coding small RNAs that can regulate the expression of complementary mRNA targets. Identifying tissue-specific miRNAs is the first step toward understanding the biological functions of miRNAs, which include the regulation of tissue differentiation and the maintenance of tissue identity. In this study, we performed small RNA library sequencing in adult mouse testis and ovary to reveal their characteristic organ- and gender-specific profiles and to elucidate the characteristics of the miRNAs expressed in the reproductive system. We obtained 10,852 and 11 744 small RNA clones from mouse testis and ovary respectively (greater than 10,000 clones per organ), which included 6630 (159 genes) and 10,192 (154 genes) known miRNAs. A high level of efficiency of miRNA library sequencing was achieved: 61% (6630 miRNA clones/10,852 small RNA clones) and 87% (10,192/11,744) for adult mouse testis and ovary respectively. We obtained characteristic miRNA signatures in testis and ovary; 55 miRNAs were detected highly, exclusively, or predominantly in adult mouse testis and ovary, and discovered two novel miRNAs. Male-biased expression of miRNAs occurred on the X-chromosome. Our data provide important information on sex differences in miRNA expression that should facilitate studies of the reproductive organ-specific roles of miRNAs.

  7. Nucleotide sequence of polypyrimidines from cloned mouse DNA as determined by base-specific blockage of exonuclease action

    SciTech Connect

    Deugau, K.V.; Mitchel, R.E.J.; Birnboim, H.C.

    1983-01-01

    Cloned fragments of mouse DNA have been screened for the presence of long polypyrimidine/polypurine segments. The polypyrimidine portion of one such segment (about 2000 nucleotides in length) has been isolated by acidic depurination of the entire cloned fragment and plasmid vector followed by selective precipitation and 5'-/sup 32/P labeling. This polypyrimidine has been used to demonstrate a new procedure for sequencing. Covalent modification of thymine with a water-soluble carbodiimide, or cytosine with glutaric anhydride, at low levels blocked in the action of snake venom exonuclease. After deblocking, separation of the products of digestion by polyacrylamide gel electrophoresis yields a sequence ladder which can be used to determine the position of C and T residues as in other sequencing methods. A sequence of 72 residues adjacent to the 5' end had been established, consisting principally of the repeating tetranucleotide (CCTT)n. A low ratio of endonuclease to exonuclease is essential for application of this method to sequences of this size. Accordingly, a very sensitive modification of a fluorometric endonuclease assay was developed and used to optimize pH and Mg/sup 2 +/ conditions to favor exonuclease activity over the accompanying endonuclease activity. The results clearly indicate that long polypyrimidine tracts can be efficiently prepared and their sequences determined with this method using commercially available exonuclease preparations without additional purification. 26 references, 5 figures.

  8. Molecular cloning, expression and purification of recombinant soluble mouse endostatin as an anti-angiogenic protein in Escherichia coli.

    PubMed

    Yari, Kheirollah; Afzali, Saboor; Mozafari, Hadi; Mansouri, Kamran; Mostafaie, Ali

    2013-02-01

    Inhibition of angiogenesis has become a particular interest for treatment of solid tumors. Endostatin, a C-terminal fragment of collagen XVIII, has been reported to exhibit potent inhibitory effect on endothelial cells proliferation, migration and tube formation. In this research, the cDNA library of endostatin was synthesized from mouse liver and inserted into the SacI and SalI enzyme-cutting sites of pUC18 cloning vector. The recombinant vector was transferred into Escherichia coli DH5a and the recombinant clone was selected on LB agar plate plus ampicillin. PCR analysis and DNA sequencing proved the presence of intact endostatin gene in pUC18. The endostatin gene subcloned into pET32a expression vector and the competent bacterial cells of E. coli BL21 were transformed by the vector harboring endostatin gene. In the optimum conditions, expression plasmid was induced with IPTG and recombinant soluble endostatin as a fusion with thioredoxin was purified with Ni-NTA (Ni(2+)-nitrilotriacetate) resin. The results showed that soluble recombinant endostatin as a fusion protein with thioredoxin is a homogenous polypeptide that inhibits angiogenesis (capillary tube formation) in human umbilical vein endothelial cells by 200 ng/ml.

  9. Hypoxia Increases Mouse Satellite Cell Clone Proliferation Maintaining both In Vitro and In Vivo Heterogeneity and Myogenic Potential

    PubMed Central

    Urbani, Luca; Piccoli, Martina; Franzin, Chiara

    2012-01-01

    Satellite cells (SCs) are essential for postnatal muscle growth and regeneration, however, their expansion potential in vitro is limited. Recently, hypoxia has been used to enhance proliferative abilities in vitro of various primary cultures. Here, by isolating SCs from single mouse hindlimb skeletal myofibers, we were able to distinguish two subpopulations of clonally cultured SCs (Low Proliferative Clones - LPC - and High Proliferative Clones - HPC), which, as shown in rat skeletal muscle, were present at a fixed proportion. In addition, culturing LPC and HPC at a low level of oxygen we observed a two fold increased proliferation both for LPC and HPC. LPC showed higher myogenic regulatory factor (MRF) expression than HPC, particularly under the hypoxic condition. Notably, a different myogenic potential between LPC and HPC was retained in vivo: green fluorescent protein (GFP)+LPC transplantation in cardiotoxin-injured Tibialis Anterior led to a higher number of new GFP+muscle fibers per transplanted cell than GFP+HPC. Interestingly, the in vivo myogenic potential of a single cell from an LPC is similar if cultured both in normoxia and hypoxia. Therefore, starting from a single satellite cell, hypoxia allows a larger expansion of LPC than normal O2 conditions, obtaining a consistent amount of cells for transplantation, but maintaining their myogenic regeneration potential. PMID:23166781

  10. Isolation and characterization of a cDNA clone for the complete protein coding region of the delta subunit of the mouse acetylcholine receptor.

    PubMed Central

    LaPolla, R J; Mayne, K M; Davidson, N

    1984-01-01

    A mouse cDNA clone has been isolated that contains the complete coding region of a protein highly homologous to the delta subunit of the Torpedo acetylcholine receptor (AcChoR). The cDNA library was constructed in the vector lambda 10 from membrane-associated poly(A)+ RNA from BC3H-1 mouse cells. Surprisingly, the delta clone was selected by hybridization with cDNA encoding the gamma subunit of the Torpedo AcChoR. The nucleotide sequence of the mouse cDNA clone contains an open reading frame of 520 amino acids. This amino acid sequence exhibits 59% and 50% sequence homology to the Torpedo AcChoR delta and gamma subunits, respectively. However, the mouse nucleotide sequence has several stretches of high homology with the Torpedo gamma subunit cDNA, but not with delta. The mouse protein has the same general structural features as do the Torpedo subunits. It is encoded by a 3.3-kilobase mRNA. There is probably only one, but at most two, chromosomal genes coding for this or closely related sequences. Images PMID:6096870

  11. X-linked gene transcription patterns in female and male in vivo, in vitro and cloned porcine individual blastocysts.

    PubMed

    Park, Chi-Hun; Jeong, Young Hee; Jeong, Yeun-Ik; Lee, Se-Yeong; Jeong, Yeon-Woo; Shin, Taeyoung; Kim, Nam-Hyung; Jeung, Eui-Bae; Hyun, Sang-Hwan; Lee, Chang-Kyu; Lee, Eunsong; Hwang, Woo Suk

    2012-01-01

    To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF), and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average) were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes) but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT) process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process, which may

  12. Molecular cloning and functional analysis of the FSH receptor gene promoter from the volcano mouse (Neotomodon alstoni alstoni).

    PubMed

    Pérez-Solis, Marco Allán; Macías, Héctor; Acosta-MontesdeOca, Adriana; Pasapera, Ana María; Fierro, Reyna; Ulloa-Aguirre, Alfredo; Gutiérrez-Sagal, Rubén

    2010-02-01

    To gain further insights on the genetic divergence and the species-specific characteristics of the follicle-stimulating hormone receptor (FSHR), we cloned 946 bp of the 5'-flanking region of the FSHR gene from the volcano mouse (Neotomodon alstoni alstoni), and compared its features with those from other mammalian species. The sequence of neotomodon FSHR (nFSHR) gene from the translation initiation site to -946 is 74, 71, 64, and 59% homologous to rat, mouse (129/J), human, and sheep, respectively. The nFSHR 5'-flanking region exhibits new interesting putative cis-regulatory elements including those for the SRY transcription factor, which had not been previously related to the FSHR gene. The transcriptional regulation properties of nFSHR gene were studied in mouse Sertoli (MSC-1) and non-Sertoli (H441) cell lines, and compared with those obtained with similar 129/J constructs. All constructs tested were more active in H441 than in MSC-1 cells. The low transcription levels detected in MSC-1 cells probably reflect the recruitment of Sertoli cells-specific nuclear factors that repress transcription of the FSHR gene. In H441 cells, 129/J constructs were more active than their neotomodon counterparts, indicating important species-specific differences in their transcription pattern. Functional analysis of a series of progressive 5'-deletion mutants identified regions involved in positive and negative transcriptional regulation as well as the strongest minimal promoter spanning 260 bp upstream the translation initiation site. The identification of inhibitory nuclear transcription factors, which are apparently expressed in MSC-1 cells, may contribute to a better understanding of the transcriptional regulation of the FSHR gene.

  13. 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

  14. 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.

  15. cDNA cloning and embryonic expression of mouse nuclear pore membrane glycoprotein 210 mRNA.

    PubMed

    Olsson, M; Ekblom, M; Fecker, L; Kurkinen, M; Ekblom, P

    1999-09-01

    In embryonic kidneys, mesenchymal cells convert into epithelium in response to an induction by the tip of the ureter bud. Metanephric mesenchyme can also be induced to convert into epithelium in vitro. It is a model system to identify genes that could be important for epithelial development. By differential screening of a cDNA library made from mesenchymes induced in transfilter cultures by embryonic spinal cord for 24 hours, we selected cDNA clones representing genes that were preferentially expressed in 24-hour-induced mesenchyme and not in uninduced mesenchyme. The sequence of one clone was determined and used to obtain the sequence of a complete open reading frame. By Northern blotting and in situ hybridization, the expression of the mRNA in embryonic kidneys was determined. We report the sequence and expression pattern of a marker for the 24-hour-induced state, mouse nuclear pore membrane glycoprotein 210 (mPOM210). The deduced 1886 amino acid sequence shows a 95% identity to the sequence of rat gp210. Northern blotting revealed a single 7.5 kb mRNA in 24-hour-induced mesenchyme, whereas message levels were fourfold to fivefold lower in uninduced mesenchyme. In situ hybridization of in vivo development confirmed the preferential expression of mPOM210 in epithelial cells. In the kidney, expression was seen in both the epithelium derived from the ureteric tree and the mesenchyme-derived epithelium. In other tissues of 13-day-old embryos, expression was also confined to the epithelium. In nervous tissues, the olfactory epithelium and walls of the lateral ventricle were the most prominently stained. Weak expression was seen in the heart. mPOM210 mRNA is an early marker for developing epithelial cells. Furthermore, our results suggest that nuclear pore membrane proteins could be more cell-type specific than previously anticipated.

  16. Characterizations of individual mouse red blood cells parasitized by Babesia microti using 3-D holographic microscopy.

    PubMed

    Park, HyunJoo; Hong, Sung-Hee; Kim, Kyoohyun; Cho, Shin-Hyeong; Lee, Won-Ja; Kim, Youngchan; Lee, Sang-Eun; Park, YongKeun

    2015-06-03

    Babesia microti causes "emergency" human babesiosis. However, little is known about the alterations in B. microti invaded red blood cells (Bm-RBCs) at the individual cell level. Through quantitative phase imaging techniques based on laser interferometry, we present the simultaneous measurements of structural, chemical, and mechanical modifications in individual mouse Bm-RBCs. 3-D refractive index maps of individual RBCs and in situ parasite vacuoles are imaged, from which total contents and concentration of dry mass are also precisely quantified. In addition, we examine the dynamic membrane fluctuation of Bm-RBCs, which provide information on cell membrane deformability.

  17. Characterizations of individual mouse red blood cells parasitized by Babesia microti using 3-D holographic microscopy

    PubMed Central

    Park, HyunJoo; Hong, Sung-Hee; Kim, Kyoohyun; Cho, Shin-Hyeong; Lee, Won-Ja; Kim, Youngchan; Lee, Sang-Eun; Park, YongKeun

    2015-01-01

    Babesia microti causes “emergency” human babesiosis. However, little is known about the alterations in B. microti invaded red blood cells (Bm-RBCs) at the individual cell level. Through quantitative phase imaging techniques based on laser interferometry, we present the simultaneous measurements of structural, chemical, and mechanical modifications in individual mouse Bm-RBCs. 3-D refractive index maps of individual RBCs and in situ parasite vacuoles are imaged, from which total contents and concentration of dry mass are also precisely quantified. In addition, we examine the dynamic membrane fluctuation of Bm-RBCs, which provide information on cell membrane deformability. PMID:26039793

  18. Cytoplasmic asters are required for progression past the first cell cycle in cloned mouse embryos.

    PubMed

    Miki, Hiromi; Inoue, Kimiko; Ogonuki, Narumi; Mochida, Keiji; Nagashima, Hiroshi; Baba, Tadashi; Ogura, Atsuo

    2004-12-01

    Unlike the oocytes of most other animal species, unfertilized murine oocytes contain cytoplasmic asters, which act as microtubule-organizing centers following fertilization. This study examined the role of asters during the first cell cycle of mouse nuclear transfer (NT) embryos. NT was performed by intracytoplasmic injection of cumulus cells. Cytoplasmic asters were localized by staining with an anti-alpha-tubulin antibody. Enucleation of MII oocytes caused no significant change in the number of cytoplasmic asters. The number of asters decreased after transfer of the donor nuclei into these enucleated oocytes, probably because some of the asters participated in the formation of the spindle that anchors the donor chromosomes. The cytoplasmic asters became undetectable within 2 h of oocyte activation, irrespective of the presence or absence of the donor chromosomes. After the standard NT protocol, a spindle-like structure persisted between the pseudopronuclei of these oocytes throughout the pronuclear stage. The asters reappeared shortly before the first mitosis and formed the mitotic spindle. When the donor nucleus was transferred into preactivated oocytes (delayed NT) that were devoid of free asters, the microtubules and microfilaments were distributed irregularly in the ooplasm and formed dense bundles within the cytoplasm. Thereafter, all of the delayed NT oocytes underwent fragmentation and arrested development. Treatment of these delayed NT oocytes with Taxol, which is a microtubule-assembling agent, resulted in the formation of several aster-like structures and reduced fragmentation. Some Taxol-treated oocytes completed the first cell cycle and developed further. This study demonstrates that cytoplasmic asters play a crucial role during the first cell cycle of murine NT embryos. Therefore, in mouse NT, the use of MII oocytes as recipients is essential, not only for chromatin reprogramming as previously reported, but also for normal cytoskeletal organization

  19. Selective Growth, In Vitro and In Vivo, of Individual T Cell Clones from Tumor-Infiltrating Lymphocytes Obtained from Patients with Melanoma

    PubMed Central

    Zhou, Juhua; Dudley, Mark E.; Rosenberg, Steven A.; Robbins, Paul F.

    2007-01-01

    In recent clinical trials in patients with metastatic melanoma, adoptive transfer of tumor-reactive lymphocytes mediated the regression of metastatic tumor deposits. To better understand the role of individual T cell clones in mediating tumor regression, a 5′ RACE technique was used to determine the distribution of TCR β-chain V region sequences expressed in the transferred cells as well as in tumor samples and circulating lymphocytes from melanoma patients following adoptive cell transfer. We found that dominant T cell clones were present in the in vitro-expanded and transferred tumor-infiltrating lymphocyte samples and certain T cell clones including the dominant T cell clones persisted at relatively high levels in the peripheral blood of the patients that demonstrated clinical responses to adoptive immunotherapy. However, these dominant clones were either undetected or present at a very low level in the resected tumor samples used for tumor-infiltrating lymphocyte generation. These data demonstrated that there was selective growth and survival, both in vitro and in vivo, of individual T cell clones from a relatively small number of T cells in the original tumor samples. These results suggest that the persistent T cell clones played an active role in mediating tumor regression and that 5′ RACE analysis may provide an important tool for the analysis of the role of individual T cell clones in mediating tumor regression. A similar analysis may also be useful for monitoring autoimmune responses. PMID:15585890

  20. Cloning, expression, purification and preliminary X-­ray crystallographic analysis of mouse protein arginine methyltransferase 7

    PubMed Central

    Cura, Vincent; Troffer-Charlier, Nathalie; Lambert, Marie-Annick; Bonnefond, Luc; Cavarelli, Jean

    2014-01-01

    Protein arginine methyltransferase 7 (PRMT7) is a unique but less characterized member of the family of protein arginine methyltransferases (PRMTs) that plays a role in male germline gene imprinting. PRMT7 is the only known PRMT member that catalyzes the monomethylation but not the dimethylation of the target arginine residues and harbours two catalytic domains in tandem. PRMT7 genes from five different species were cloned and expressed in Escherichia coli and Sf21 insect cells. Four gave soluble proteins from Sf21 cells, of which two were homogeneous and one gave crystals. The mouse PRMT7 structure was solved by the single anomalous dispersion method using a crystal soaked with thimerosal that diffracted to beyond 2.1 Å resolution. The crystal belonged to space group P43212, with unit-cell parameters a = b = 97.4, c = 168.1 Å and one PRMT7 monomer in the asymmetric unit. The structure of another crystal form belonging to space group I222 was solved by molecular replacement. PMID:24419624

  1. Cloning and expression of odorant-binding proteins Ia and Ib from mouse nasal tissue.

    PubMed

    Pes, D; Mameli, M; Andreini, I; Krieger, J; Weber, M; Breer, H; Pelosi, P

    1998-05-28

    We had previously reported the purification and partial characterisation of four distinct odorant-binding proteins from male mouse nasal epithelium. One of these, named OBP-I appeared to be a heterodimer, whose subunits, Ia and Ib showed significant similarity in their N-terminal amino acid sequences with hamster aphrodisin. In this paper, we report the complete amino acid sequences of these two polypeptide chains, as deduced from nucleotide sequences of their relative cDNA. These data confirm the high similarity of both proteins with hamster aphrodisin. A comparison with the sequences of other known OBPs indicate that they are more closely related to members of class I, including bovine OBP, rat OBP-I and pig OBP-I. A putative odorant-binding site is indicated by the presence of amino acid residues conserved with respect to the bovine protein, whose three-dimensional structure has been recently resolved. In-situ hybridisation has revealed identical expression patterns for the two proteins, further supporting the heterodimeric structure of these proteins in the nasal mucus.

  2. Cloning and characterization of mouse extracellular-signal-regulated protein kinase 3 as a unique gene product of 100 kDa.

    PubMed Central

    Turgeon, B; Saba-El-Leil, M K; Meloche, S

    2000-01-01

    MAP (mitogen-activated protein) kinases are a family of serine/threonine kinases that have a pivotal role in signal transduction. Here we report the cloning and characterization of a mouse homologue of extracellular-signal-regulated protein kinase (ERK)3. The mouse Erk3 cDNA encodes a predicted protein of 720 residues, which displays 94% identity with human ERK3. Transcription and translation of this cDNA in vitro generates a 100 kDa protein similar to the human gene product ERK3. Immunoblot analysis with an antibody raised against a unique sequence of ERK3 also recognizes a 100 kDa protein in mouse tissues. A single transcript of Erk3 was detected in every adult mouse tissue examined, with the highest expression being found in the brain. Interestingly, expression of Erk3 mRNA is acutely regulated during mouse development, with a peak of expression observed at embryonic day 11. The mouse Erk3 gene was mapped to a single locus on central mouse chromosome 9, adjacent to the dilute mutation locus and in a region syntenic to human chromosome 15q21. Finally, we provide several lines of evidence to support the existence of a unique Erk3 gene product of 100 kDa in mammalian cells. PMID:10657254

  3. Isolation of cDNA clones for the catalytic gamma subunit of mouse muscle phosphorylase kinase: expression of mRNA in normal and mutant Phk mice.

    PubMed Central

    Chamberlain, J S; VanTuinen, P; Reeves, A A; Philip, B A; Caskey, C T

    1987-01-01

    We have isolated and characterized cDNA clones for the gamma subunit of mouse muscle phosphorylase kinase (gamma-Phk). These clones were isolated from a lambda gt11 mouse muscle cDNA library via screening with a synthetic oligonucleotide probe corresponding to a portion of the rabbit gamma-Phk amino acid sequence. The gamma-Phk cDNA clones code for a 387-amino acid protein that shares 93% amino acid sequence identity with the corresponding rabbit amino acid sequence. RNA gel blot analysis reveals that the muscle gamma-Phk probe hybridizes to two mRNA species (2.4 and 1.6 kilobases) in skeletal muscle, cardiac muscle, and brain, but does not hybridize to liver RNA. Phk-deficient I-strain (Phk) mouse muscle contains reduced levels of gamma-Phk mRNA as compared with control mice. Although the Phk defect is an X-linked recessive trait, hybridization to a human-rodent somatic cell hybrid mapping panel shows that the gamma-Phk gene is not located on the X chromosome. Rather, the gamma-Phk cross-hybridizing human restriction fragments map to human chromosomes 7 (multiple) and 11 (single). Reduced gamma-Phk mRNA in I-strain mice, therefore, appears to be a consequence of the Phk-mutant trait and does not stem from a mutant gamma-subunit gene. Images PMID:3472241

  4. Characterization of a cDNA clone coding for a mouse 85 kDa heat shock protein from a 3-methylcholanthrene-induced tumor

    SciTech Connect

    Moore, S.K.; Robinson, E.A.; Ullrich, S.J.; Appella, E.

    1986-05-01

    Heat shock proteins (hsp) of approx. 85 kDa have been found associated with steroid hormone receptors and with the src oncogene product. Recently, the authors have shown that a mouse tumor-associated transplantation antigen from a 3-methylcholanthrene-induced tumor shares amino acid sequence homology with the 85 kDa hsp. Amino acid sequence of peptides from this antigen were used to synthesize oligonucleotide probes to screen a mouse cDNA library. A cDNA clone coding for a 85 kDa mouse hsp has been isolated and its sequence determined. Predicted amino acid sequences from this cDNA clone share significant homology to the published 90 kDa hsp of Saccaromyces cerevisiae and to the 83 kDa hsp of Drosophila melanogaster. In addition, the predicted amino acid sequence at the carboxyl terminus shares identity with that of the 70 kDa hsp from various species as well as that of the Escherichia coli dnaK gene product. Northern blot analysis indicates that the mouse 85 kDa hsp is coded for by a kb mRNA. The size of the mRNA is indistinguishable between normal and malignant cells.

  5. 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.

  6. Absence of MHC gene expression in lens and cloning of dbpB/YB-1, a DNA-binding protein expressed in mouse lens.

    PubMed

    Shaughnessy, M; Wistow, G

    1992-02-01

    The status of major histocompatibility complex (MHC) class I and II gene expression in the normal mouse lens was examined. No mRNA for either class I or II genes was detectable in mouse lens, while the expression of MHC genes in other tissues generally matched immunohistochemical data from human tissues. However it was observed that MHC class I mRNA is present in the mouse lens-derived cell line alpha TN4-1. From a new-born mouse lens cDNA library a clone was obtained for the murine homologue of the DNA-binding protein dbpB/YB-1, a protein originally identified in human lymphocytes and proposed to be a negative regulator of MHC class II gene expression. Northern blots detect dbpB/YB-1 mRNA in all mouse tissues and cells examined, including both mouse lens and alpha TN4-1 cells, suggesting that dbpB/YB-1 has a general and widespread role.

  7. 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.

  8. 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.

  9. Cloning and functional characterization of the mouse sodium-dependent organic anion transporter Soat (Slc10a6).

    PubMed

    Grosser, Gary; Fietz, Daniela; Günther, Sabine; Bakhaus, Katharina; Schweigmann, Helene; Ugele, Bernhard; Brehm, Ralph; Petzinger, Ernst; Bergmann, Martin; Geyer, Joachim

    2013-11-01

    The sodium-dependent organic anion transporter SOAT is a member of the Solute Carrier Family SLC10. In man, this carrier is predominantly expressed in the testis and has transport activity for sulfoconjugated steroid hormones. Here, we report on cloning, expression analysis and functional characterization of the mouse Soat (mSoat) and compare its characteristics with the human SOAT carrier. Quantitative mRNA expression analysis for mSoat in male mice revealed very high expression in lung and further high expression in testis and skin. Immunohistochemical studies showed expression of the mSoat protein in bronchial epithelial cells of the lung, in primary and secondary spermatocytes as well as round spermatids within the seminiferous tubules of the testis, in the epidermis of the skin, and in the urinary epithelium of the bladder. Stably transfected mSoat-HEK293 cells revealed sodium-dependent transport for dehydroepiandrosterone sulfate (DHEAS), estrone-3-sulfate, and pregnenolone sulfate (PREGS) with apparent Km values of 60.3μM, 2.1μM, and 2.5μM, respectively. In contrast to human SOAT, which has a preference for DHEAS as a substrate, mSoat exhibits the highest transport rate for PREGS, likely reflecting differences in the steroid pattern between both species. In conclusion, although certain differences between human SOAT and mSoat exist regarding quantitative gene expression in endocrine and non-endocrine tissues, as well as in the transport kinetics for steroid sulfates, in general, both can be regarded as homologous carriers. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. [Cloning: applications in humans 1. Technical aspects].

    PubMed

    Geraedts, J P; de Wert, G M

    2001-04-01

    The successful cloning experiments in mammals such as the sheep and mouse prompted speculations on clinical application in humans. Cloning is possible by nucleus transplantation and by embryo splitting. Nucleus transplantation does not result in a genetically completely identical individual because the mitochondrial DNA originates from the ovum donor. Embryo splitting may be regarded as the artificial production of a monozygotic multiplet. Possible applications of cloning in humans belong in the context of reproduction (treatment of couples with subfertility, with genetic problems or with a 'replica motive'), transplantation of genetically identical tissue, and scientific research.

  11. [Cloning: applications in humans. I. Technical aspects].

    PubMed

    Geraedts, J P; de Wert, G M

    2000-05-13

    The successful cloning experiments in mammals such as the sheep and mouse prompted speculations on clinical application in humans. Cloning is possible by nucleus transplantation and by embryo splitting. Nucleus transplantation does not result in a genetically completely identical individual because the mitochondrial DNA originates from the ovum donor. Embryo splitting may be regarded as the artificial production of a monozygotic multiplet. Possible applications of cloning in humans belong in the context of reproduction (treatment of couples with subfertility, with genetic problems or with a 'replica motive'), transplantation of genetically identical tissue, and scientific research.

  12. Sex-dependent expression of mouse testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha): cDNA cloning and pretranslational regulation.

    PubMed Central

    Harada, N; Negishi, M

    1985-01-01

    By using both double-colony hybridization and an in situ immunostaining assay for transformants, 39 cDNA clones (clone p-16 alpha) encoding mouse liver microsomal testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha) were isolated from a cDNA library constructed in the cloning vector pUC-9 with poly(A)+ RNA immunoenriched from total liver polysomes of male 129/J mice. mRNA selected by hybridization with clone p-16 alpha translated the P-450(16) alpha apoprotein in vitro. Total cellular proteins, which were prepared from immunopositive transformant Escherichia coli cells, were conjugated with Sepharose 4B. Antibody purified with the Sepharose 4B conjugate from mixed antiserum to P-450(16) alpha and P-450(15) alpha specifically inhibited testosterone 16 alpha-hydroxylase activity in microsomes. The cDNA insert of one recombinant plasmid (clone P-16 alpha-1) was 1.75 kilobases in size and contained one or more internal restriction sites for HindIII, BamHI, Bgl I, Pst I, Alu I, HinpI, and Rsa I. 32P-labeled clone p-16 alpha-1 hybridized with a single mRNA (2000 bases) that was 10 times more concentrated in liver cells from male 129/J mice than in female mice. This result was consistent with the finding that poly(A)+ RNA from male mice translated 10 times as much P-450(16) alpha in vitro as did the poly(A)+ RNA from females. Thus, the predominant expression of testosterone 16 alpha-hydroxylase in male 129/J mice is regulated pretranslationally, presumably at the transcriptional level of the P-450(16) alpha gene. Images PMID:3856880

  13. Combined effects of individual culture and atmospheric oxygen on preimplantation mouse embryos in vitro.

    PubMed

    Kelley, Rebecca L; Gardner, David K

    2016-11-01

    Embryos are routinely cultured individually, although this can reduce blastocyst development. Culture in atmospheric (20%) oxygen is also common, despite multiple detrimental effects on embryos. Although frequently occurring together, the consequences of this combination are unknown. Mouse embryos were cultured individually or grouped, under physiological (5%) or atmospheric (20%) oxygen. Embryos were assessed by time-lapse and blastocyst cell allocation. Compared with the control group (5% oxygen group culture), 5-cell cleavage (t5) was delayed in 5% oxygen individual culture and 20% oxygen group culture (59.91 ± 0.23, 60.70 ± 0.29, 63.06 ± 0.32 h post-HCG respectively, P < 0.05). Embryos in 20% oxygen individual culture were delayed earlier (3-cell cleavage), and at t5 cleaved later than embryos in other treatments (66.01 ± 0.40 h, P < 0.001), this delay persisting to blastocyst hatching. Compared with controls, hatching rate and cells per blastocyst were reduced in 5% oxygen single culture and 20% oxygen group culture (134.1 ± 3.4, 104.5 ± 3.2, 73.4 ± 2.2 cells, P < 0.001), and were further reduced in 20% oxygen individual culture (57.0 ± 2.8 cells, P < 0.001), as was percentage inner cell mass. These data indicate combining individual culture and 20% oxygen is detrimental to embryo development.

  14. 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.

  15. 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.

  16. Inhibition of hepatitis B virus (HBV) gene expression and replication by HBx gene silencing in a hydrodynamic injection mouse model with a new clone of HBV genotype B

    PubMed Central

    2013-01-01

    Background It has been suggested that different hepatitis B virus (HBV) genotypes may have distinct virological characteristics that correlate with clinical outcomes during antiviral therapy and the natural course of infection. Hydrodynamic injection (HI) of HBV in the mouse model is a useful tool for study of HBV replication in vivo. However, only HBV genotype A has been used for studies with HI. Methods We constructed 3 replication-competent clones containing 1.1, 1.2 and 1.3 fold overlength of a HBV genotype B genome and tested them both in vitro and in vivo. Moreover, A HBV genotype B clone based on the pAAV-MCS vector was constructed with the 1.3 fold HBV genome, resulting in the plasmid pAAV-HBV1.3B and tested by HI in C57BL/6 mice. Application of siRNA against HBx gene was tested in HBV genotype B HI mouse model. Results The 1.3 fold HBV clone showed higher replication and gene expression than the 1.1 and 1.2 fold HBV clones. Compared with pAAV-HBV1.2 (genotype A), the mice HI with pAAV-HBV1.3B showed higher HBsAg and HBeAg expression as well as HBV DNA replication level but a higher clearance rate. Application of two plasmids pSB-HBxi285 and pSR-HBxi285 expressing a small/short interfering RNA (siRNA) to the HBx gene in HBV genotype B HI mouse model, leading to an inhibition of HBV gene expression and replication. However, HBV gene expression may resume in some mice despite an initial delay, suggesting that transient suppression of HBV replication by siRNA may be insufficient to prevent viral spread, particularly if the gene silencing is not highly effective. Conclusions Taken together, the HI mouse model with a HBV genotype B genome was successfully established and showed different characteristics in vivo compared with the genotype A genome. The effectiveness of gene silencing against HBx gene determines whether HBV replication may be sustainably inhibited by siRNA in vivo. PMID:23805945

  17. cDNA cloning reveals two mouse beta5 integrin transcripts distinct in cytoplasmic domains as a result of alternative splicing.

    PubMed Central

    Zhang, H; Tan, S M; Lu, J

    1998-01-01

    The integrin beta5 subunit has only been found to form a heterodimer with subunit alphav which acts as a vitronectin receptor. Integrin alphavbeta5 has been implicated in cell migration and growth factor-induced angiogenesis. In the present study, a mouse liver cDNA library was screened using a human beta5 cDNA fragment obtained by reverse transcriptase PCR (RT-PCR). Three of the clones (MB5, MB15 and MB17) overlapped to give an open reading frame, called beta5A, which is homologous to the human beta5 subunit. The sequence of another clone (MB26), called beta5B, was identical with beta5A, except for a deletion of 29 bp near the 3' end of the open reading frame. The 29 bp deletion resulted in an open-reading-frame shift and a completely different C-terminal sequence in beta5B. beta5A and beta5B were shown, by RT-PCR, to be co-expressed in most mouse tissues tested, although beta5B mRNA was detected at much lower levels than beta5A. beta5A and beta5B mRNAs were also detected in the mouse monocytic cell line, J774, and in isolated mouse peritoneal macrophages. Adhesion of peritoneal macrophages has been shown to up-regulate the expression of both beta5A and beta5B mRNAs. The 29 bp sequence begins with a putative intron-splicing donor site (GTGAT...). A 3' fragment of the mouse integrin beta5 gene was cloned by PCR and sequenced showing that the 29 bp sequence was also immediately followed by an intron. Therefore, the 29 bp sequence was apparently expressed as part of the beta5A mRNA but was spliced out as part of the downstream intron in beta5B. Since the cytoplasmic domains of the integrin beta subunits are important in cytoskeleton attachment and signalling, the two alternatively spliced beta5 isoforms may have distinct roles in cell adhesion and other cellular functions. PMID:9531507

  18. 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.

  19. Molecular cloning, gene structure, expression profile and functional characterization of the mouse glutamate transporter (EAAT3) interacting protein GTRAP3-18.

    PubMed

    Butchbach, Matthew E R; Lai, Liching; Lin, Chien-liang Glenn

    2002-06-12

    Glutamate is an important amino acid implicated in energy metabolism, protein biosynthesis and neurotransmission. The Na(+)-dependent high-affinity excitatory amino acid transporter EAAT3 (EAAC1) facilitates glutamate uptake into most cells. Recently, a novel rat EAAT3-interacting protein called GTRAP3-18 has been identified by a yeast two-hybrid screening. GTRAP3-18 functions as a negative modulator of EAAT3-mediated glutamate transport. In order to further understand the function and regulation of GTRAP3-18, we cloned the mouse orthologue to GTRAP3-18 and determined its gene structure and its expression pattern. GTRAP3-18 encodes a 188-residue hydrophobic protein whose sequence is highly conserved amongst vertebrates. Mouse and human GTRAP3-18 genes contain three exons separated by two introns. The GTRAP3-18 gene is found on mouse chromosome 6D3 and on human chromosome 3p14, a susceptibility locus for cancer and epilepsy. GTRAP3-18 protein and RNA were found both in neuronal rich regions of the brain and in non-neuronal tissues such as the kidney, heart and skeletal muscle. Mouse GTRAP3-18 inhibited EAAT3-mediated glutamate transport in a dose-dependent manner. These studies show that GTRAP3-18 is a ubiquitously expressed protein that functions as a negative regulator of EAAT3 function.

  20. 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

  1. Single body parts are processed by individual neurons in the mouse dorsolateral striatum.

    PubMed

    Coffey, Kevin R; Nader, Miles; West, Mark O

    2016-04-01

    Interest in the dorsolateral striatum (DLS) has generated numerous scientific studies of its neuropathologies, as well as its roles in normal sensorimotor integration and learning. Studies are informed by knowledge of DLS functional organization, the guiding principle being its somatotopic afferent projections from primary somatosensory (S1) and motor (M1) cortices. The potential to connect behaviorally relevant function to detailed structure is elevated by mouse models, which have access to extensive genetic neuroscience tool kits. Remaining to be demonstrated, however, is whether the correspondence between S1/M1 corticostriatal terminal distributions and the physiological properties of DLS neurons demonstrated in rats and non-human primates exists in mice. Given that the terminal distribution of S1/M1 projections to the DLS in mice is similar to that in rats, we studied whether firing rates (FRs) of DLS neurons in awake, behaving mice are related to activity of individual body parts. MSNs exhibited robust, selective increases in FR during movement or somatosensory stimulation of single body parts. Properties of MSNs, including baseline FRs, locations, responsiveness to stimulation, and proportions of responsive neurons were similar to properties observed in rats. Future studies can be informed by the present demonstration that the mouse lateral striatum functions as a somatic sensorimotor sector of the striatum and appears to be a homolog of the primate putamen, as demonstrated in rats (Carelli and West, 1991). Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Single Body Parts are Processed by Individual Neurons in the Mouse Dorsolateral Striatum

    PubMed Central

    Coffey, Kevin R.; Nader, Miles; West, Mark O.

    2016-01-01

    Interest in the dorsolateral striatum (DLS) has generated numerous scientific studies of its neuropathologies, as well as its roles in normal sensorimotor integration and learning. Studies are informed by knowledge of DLS functional organization, the guiding principle being its somatotopic afferent projections from primary somatosensory (S1) and motor (M1) cortices. The potential to connect behaviorally relevant function to detailed structure is elevated by mouse models, which have access to extensive genetic neuroscience tool kits. Remaining to be demonstrated, however, is whether the correspondence between S1/M1 corticostriatal terminal distributions and the physiological properties of DLS neurons demonstrated in rats and non-human primates exists in mice. Given that the terminal distribution of S1/M1 projections to the DLS in mice is similar to that in rats, we studied whether firing rates (FRs) of DLS neurons in awake, behaving mice are related to activity of individual body parts. MSNs exhibited robust, selective increases in FR during movement or somatosensory stimulation of single body parts. Properties of MSNs, including baseline FRs, locations, responsiveness to stimulation, and proportions of responsive neurons were similar to properties observed in rats. Future studies can be informed by the present demonstration that the mouse lateral striatum functions as a somatic sensorimotor sector of the striatum and appears to be a homolog of the primate putamen, as demonstrated in rats (Carelli and West, 1991). PMID:26827625

  3. [Cloning and characterization of a novel mouse short-chain dehydrogenase/reductases cDNA mHsdl2#, encoding a protein with a SDR domaid and a SCP2 domain].

    PubMed

    Dai, J; Li, P; Ji, Ch; Feng, C; Gui, M; Sun, Y; Zhang, J; Zhu, J; Dou, Ch; Gu, Sh

    2005-01-01

    The short-chain dehydrogenases/reductases (SDRs) play important roles in body's metabolism. We cloned a novel mouse SDR cDNA which encodes a deduced HSD-like protein with a conserved SDR domain and a SCP2 domain. The 1.8 kb cDNA consists of 11 exons and is mapped to mouse chromosome 4B3. The corresponding gene is widely expressed in normal mouse tissues and its expression level in liver increases after inducement with cholesterol food. The predicted mouse HSDL2 protein, which has a peroxisomal target signal, is localized in the cytoplasm of NIH 3T3 cells.

  4. 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

  5. Russell body inducing threshold depends on the variable domain sequences of individual human IgG clones and the cellular protein homeostasis.

    PubMed

    Stoops, Janelle; Byrd, Samantha; Hasegawa, Haruki

    2012-10-01

    Russell bodies are intracellular aggregates of immunoglobulins. Although the mechanism of Russell body biogenesis has been extensively studied by using truncated mutant heavy chains, the importance of the variable domain sequences in this process and in immunoglobulin biosynthesis remains largely unknown. Using a panel of structurally and functionally normal human immunoglobulin Gs, we show that individual immunoglobulin G clones possess distinctive Russell body inducing propensities that can surface differently under normal and abnormal cellular conditions. Russell body inducing predisposition unique to each immunoglobulin G clone was corroborated by the intrinsic physicochemical properties encoded in the heavy chain variable domain/light chain variable domain sequence combinations that define each immunoglobulin G clone. While the sequence based intrinsic factors predispose certain immunoglobulin G clones to be more prone to induce Russell bodies, extrinsic factors such as stressful cell culture conditions also play roles in unmasking Russell body propensity from immunoglobulin G clones that are normally refractory to developing Russell bodies. By taking advantage of heterologous expression systems, we dissected the roles of individual subunit chains in Russell body formation and examined the effect of non-cognate subunit chain pair co-expression on Russell body forming propensity. The results suggest that the properties embedded in the variable domain of individual light chain clones and their compatibility with the partnering heavy chain variable domain sequences underscore the efficiency of immunoglobulin G biosynthesis, the threshold for Russell body induction, and the level of immunoglobulin G secretion. We propose that an interplay between the unique properties encoded in variable domain sequences and the state of protein homeostasis determines whether an immunoglobulin G expressing cell will develop the Russell body phenotype in a dynamic cellular setting.

  6. 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.

  7. 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.

    1990-01-01

    Three inbred mouse strains, C57BL/6 (H-2/b/), A/J (H-2/a/), and BALB/C (H-2/d/), and one outbred strain, CD-1, demonstrated differences in susceptibility to challenge intravenously with the ANKA clone of Plasmodium berghei. Mice were challenged with 100, 1000, or 10000 sporozoites, then evaluated daily beginning on day 4 for patency. CD-1 mice were further evaluated at challenge doses of 125000, 25000, and 50000 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 25000 sporozoites per 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 1000 sporozoites. In contrast BALB/C mice, immunized with a single dose of 1000 irradiated sporozoites, are protected against a 10000 sporozoite challenge. These data suggest that both infectivity and protection are genetically restricted and that susceptibility to infection may be inversely related to protection.

  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. A novel mouse running wheel that senses individual limb forces: biomechanical validation and in vivo testing.

    PubMed

    Roach, Grahm C; Edke, Mangesh; Griffin, Timothy M

    2012-08-15

    Biomechanical data provide fundamental information about changes in musculoskeletal function during development, adaptation, and disease. To facilitate the study of mouse locomotor biomechanics, we modified a standard mouse running wheel to include a force-sensitive rung capable of measuring the normal and tangential forces applied by individual paws. Force data were collected throughout the night using an automated threshold trigger algorithm that synchronized force data with wheel-angle data and a high-speed infrared video file. During the first night of wheel running, mice reached consistent running speeds within the first 40 force events, indicating a rapid habituation to wheel running, given that mice generated >2,000 force-event files/night. Average running speeds and peak normal and tangential forces were consistent throughout the first four nights of running, indicating that one night of running is sufficient to characterize the locomotor biomechanics of healthy mice. Twelve weeks of wheel running significantly increased spontaneous wheel-running speeds (16 vs. 37 m/min), lowered duty factors (ratio of foot-ground contact time to stride time; 0.71 vs. 0.58), and raised hindlimb peak normal forces (93 vs. 115% body wt) compared with inexperienced mice. Peak normal hindlimb-force magnitudes were the primary force component, which were nearly tenfold greater than peak tangential forces. Peak normal hindlimb forces exceed the vertical forces generated during overground running (50-60% body wt), suggesting that wheel running shifts weight support toward the hindlimbs. This force-instrumented running-wheel system provides a comprehensive, noninvasive screening method for monitoring gait biomechanics in mice during spontaneous locomotion.

  10. RNAi-mediated knockdown of Xist does not rescue the impaired development of female cloned mouse embryos.

    PubMed

    Oikawa, Mami; Matoba, Shogo; Inoue, Kimiko; Kamimura, Satoshi; Hirose, Michiko; Ogonuki, Narumi; Shiura, Hirosuke; Sugimoto, Michihiko; Abe, Kuniya; Ishino, Fumitoshi; Ogura, Atsuo

    2013-01-01

    In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of Xist during the preimplantation period in both sexes. We found that this aberrant Xist expression could be impeded by deletion of Xist from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of Xist-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of Xist at the morula stage. In this study, we examined whether the same knockdown strategy could work as well in female SCNT-derived embryos. Embryos were reconstructed with cumulus cell nuclei and injected with Xist-specific siRNA at 6-7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed Xist RNA at the morula stage, as shown by the significant decrease in the number of cloud-type Xist signals in the blastomere nuclei. However, blastomeres with different sizes (from "pinpoint" to "cloud") and numbers of Xist RNA signals remained within single embryos. After implantation, the dysregulated Xist expression was normalized autonomously, as in male clones, to a state of monoallelic expression in both embryonic and extraembryonic tissues. However, at term there was no significant improvement in the survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective in repressing the Xist overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic Xist expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.

  11. Functional correction of renal defects in a mouse model for ARPKD through expression of the cloned wild-type Tg737 cDNA.

    PubMed

    Yoder, B K; Richards, W G; Sommardahl, C; Sweeney, W E; Michaud, E J; Wilkinson, J E; Avner, E D; Woychik, R P

    1996-10-01

    Autosomal recessive polycystic kidney disease (ARPKD) is characterized by the formation of large collecting tubule and ductular cysts that often result in renal insufficiency within the first decade of life. Understanding the process leading to cyst formation will require the identification and characterization of genes involved in the etiology of this disease. In this regard, we previously described the generation of a mouse model (TgN737Rpw) for ARPKD and the cloning of a candidate gene. Here we show direct involvement of the Tg737 gene in collecting duct cyst formation by expressing the wild-type Tg737 cDNA as a transgene in TgN737Rpw mutants. In contrast to TgN737Rpw mutants, the "rescued" animals survive longer, have normal renal function and normal localization of the EGFr to the basolateral surfaces of collecting duct epithelium.

  12. 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.

  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. 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.

  15. Molecular cloning and expression of a cDNA encoding an olfactory-specific mouse phenol sulphotransferase.

    PubMed Central

    Tamura, H O; Harada, Y; Miyawaki, A; Mikoshiba, K; Matsui, M

    1998-01-01

    Previously we demonstrated the presence of phenol sulphotransferase (P-ST) in mouse nasal cytosols and identified its zonal location in mouse nasal cavity by staining with an antiserum raised against a rat liver P-ST isoenzyme, PSTg. In the present study a cDNA was isolated from a mouse olfactory cDNA library by immunological screening with the antiserum. The isolated cDNA consisted of 1347 bp with a 912 bp open reading frame encoding a 304-residue polypeptide. Both the nucleotide and deduced amino acid sequences of the cDNA were 94% identical with those of a rat liver P-ST isoenzyme, ST1C1. The expressed enzyme in Escherichia coli displayed high P-ST activity towards phenolic odorants such as eugenol and guaiacol, and it showed a high N-hydroxy-2-acetylaminofluorene sulphation activity in comparison with the rat ST1C1 enzyme. These results indicate that the olfactory P-ST encoded by the cDNA is a mouse orthologue of rat ST1C1; however, expression of the olfactory P-ST mRNA is specific for nasal tissues as revealed by reverse transcriptase-mediated PCR (RT-PCR). PMID:9560327

  16. Expression and distribution of NBCn2 (Slc4a10) splice variants in mouse brain: cloning of novel variant NBCn2-D.

    PubMed

    Liu, Ying; Xu, Jiu-Ying; Wang, Deng-Ke; Boron, Walter F; Chen, Li-Ming

    2011-05-16

    The SLC4A10 gene, which is highly expressed in the mammalian brain, contains two known alternative splicing units, inserts A and B, and is theoretically capable of producing four NBCn2 splice variants: NBCn2-A, -B, -C, and -D. By immunoprecipitation and western blotting, a previous study showed the putative NBCn2-D to be expressed predominantly in the subcortex (SCX) and medulla (MD) of mouse brain. However, no evidence has been provided, in any species, for the existence of a full-length transcript encoding NBCn2-D. In the present study, we report for the first time the cloning of the full-length cDNAs encoding NBCn2-D from mouse SCX and MD. Based on the frequency of bacterial colonies obtained after PCR, we conclude that in SCX, the NBCn2-A transcript is dominant, whereas in MD, NBCn2-B is dominant. NBCn2-D is the least abundant transcript in each of these two brain regions. An analysis based upon the present PCR data as well as the previous immunoprecipitation/western-blot data suggests the following prevalence of NBCn2 variants in total mouse brain: NBCn2-A (~83%), NBCn2-B (~10%), NBCn2-C (~5%), and NBCn2-D (~2%). We also estimate the prevalence of each variant in each of the five brain regions (i.e., cerebral cortex, SCX, cerebellum, hippocampus, and MD). We hypothesize that the expression of different NBCn2 splice variants is characteristic of specific tissue/cells. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. The mouse insulin-like growth factor II/cation-independent mannose 6-phosphate (IGF-II/MPR) receptor gene: Molecular cloning and genomic organization

    SciTech Connect

    Szebenyi, G.; Rotwein, P. )

    1994-01-01

    The mammalian insulin-like growth factor III/cation-independent mannose 6-phosphate receptor (IGF-II/MPR) is a multifunctional protein that binds both IGF-II and ligands containing a mannose 6-phosphate recognition marker through distinct high-affinity sites. This receptor plays an integral part in lysosomal enzyme transport, has a potential role in growth factor maturation and clearance, and may mediate IGF-II-activated signal transduction through a G-protein-coupled mechanism. Recent studies have shown that production of IGF-II/MPR mRNA and protein begins in the mouse embryo soon after fertilization and have demonstrated that the receptor gene is on mouse chromosome 17 and is maternally imprinted. In this paper, the authors report the cloning and characterization of the mouse IGF-II/MPR gene. The gene is 93 kb long, is composed of 48 exons, and codes for a predicted protein of 2482 amino acids. The extracellular part of the receptor is encoded by exons 1-46, with each of 15 related repeating motifs being determined by parts of 3-5 exons. A single fibronectin type II-like element is found in exon 39. The transmembrane portion of the receptor also is encoded by exon 46, and the cytoplasmic region by exons 46-48. The positions of exon-intron splice junctions are conserved between several of the repeats in the IGF-II/MPR and the homologous extracellular region of the gene for the other known lysosomal sorting receptor, the cation-dependent mannose 6-phosphate receptor. The gene duplications that gave rise to the modern IGF-II/MPR probably occurred before the divergence of mammals, since there is more extensive protein sequence conservation between receptors from different species than between any pair of repeating motifs within a single receptor. 55 refs., 7 figs., 1 tab.

  18. Cloning and characterization of a novel human homolog* of mouse U26, a putative PQQ-dependent AAS dehydrogenase.

    PubMed

    Wang, Liu; Jil, Chaoneng; Xu, Yiren; Xu, Jian; Dai, Jianfeng; Wu, Qihan; Wu, Maoqing; Zou, Xianqiong; Sun, Liyun; Gu, Shaohua; Xie, Yi; Mao, Yumin

    2005-03-01

    Mouse U26 has been defined as a 2-aminoadipic 6-semialdehyde dehydrogenase. It was speculated to be a PQQ-dependent AAS dehydrogenase due to the research of demonstrating PQQ as a new B vitamin. We isolated a novel human cDNA from the human fetal brain cDNA library we constructed. Its deduced protein was most related to mouse U26. Thus, we termed it human U26. This putative protein contains an AMP-binding domain, a Phosphopantetheine-binding domain and six PQQ-binding motifs. Human U26 mRNA is ubiquitously expressed in adult tissues and is highly expressed in colon adenocarcinoma (CX-1) and colon adenocarcinoma (GI-112) cell lines. Further study should be made to clarify the precise function of human U26.

  19. 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.

  20. Cloning of the VASP (Vasodilator-Stimulated Phosphoprotein) genes in human and mouse: Structure, sequence, and chromosomal localization

    SciTech Connect

    Zimmer, M.; Fischer, L.; Hauser, W.

    1996-09-01

    The genes encoding the vasodilator-stimulated phosphoprotein (VASP) in human and mouse were isolated, and major parts were sequenced. In both species the gene is composed of 13 exons with conserved exon-intron positions. The mouse VASP cDNA sequence was deduced from the genomic sequence. The predicted amino acid sequence is 89% identical to the human protein. The high nucleotide sequence homology extends not only over the coding regions but also into the 3{prime}-UTRs, indicating a possible function in mRNA targeting or regulation of translation. Prominent 5{prime} CpG islands including multiple SP1 sites indicate a housekeeping function of VASP. Using cosmid DNA as a probe for fluorescence in situ hybridization, the human VASP gene was assigned to chromosome 19q13.2-q13.3, an extended region with homology to mouse chromosome 7. A sequence overlap of the VASP 5{prime}-region with the telomeric end of a cosmid contig physically links the VASP gene with ERCC1. VASP is located about 92 kb distal to ERCC1 and about 300 kb proximal to the myotonic dystrophy protein kinase gene. 43 refs., 6 figs.

  1. Molecular cloning of mouse amino acid transport system B0, a neutral amino acid transporter related to Hartnup disorder.

    PubMed

    Bröer, Angelika; Klingel, Karin; Kowalczuk, Sonja; Rasko, John E J; Cavanaugh, Juleen; Bröer, Stefan

    2004-06-04

    Resorption of amino acids in kidney and intestine is mediated by transporters, which prefer groups of amino acids with similar physico-chemical properties. It is generally assumed that most neutral amino acids are transported across the apical membrane of epithelial cells by system B(0). Here we have characterized a novel member of the Na(+)-dependent neurotransmitter transporter family (B(0)AT1) isolated from mouse kidney, which shows all properties of system B(0). Flux experiments showed that the transporter is Na(+)-dependent, electrogenic, and actively transports most neutral amino acids but not anionic or cationic amino acids. Superfusion of mB(0)AT1-expressing oocytes with neutral amino acids generated inward currents, which were proportional to the fluxes observed with labeled amino acids. In situ hybridization showed strong expression in intestinal microvilli and in the proximal tubule of the kidney. Expression of mouse B(0)AT1 was restricted to kidney, intestine, and skin. It is generally assumed that mutations of the system B(0) transporter underlie autosomal recessive Hartnup disorder. In support of this notion mB(0)AT1 is located on mouse chromosome 13 in a region syntenic to human chromosome 5p15, the locus of Hartnup disorder. Thus, the human homologue of this transporter is an excellent functional and positional candidate for Hartnup disorder.

  2. Epigenetic regulation of genetic integrity is reprogrammed during cloning.

    PubMed

    Murphey, Patricia; Yamazaki, Yukiko; McMahan, C Alex; Walter, Christi A; Yanagimachi, Ryuzo; McCarrey, John R

    2009-03-24

    Cloning by somatic cell nuclear transfer (SCNT) circumvents processes that normally function during gametogenesis to prepare the gamete genomes to support development of new progeny following fertilization. One such process is enhanced maintenance of genetic integrity in germ cells, such that germ cells typically carry fewer spontaneously acquired mutations than somatic cells in the same individual. Thus, embryos produced from somatic cells by SCNT could directly inherit more mutations than naturally conceived embryos. Alternatively, they could inherit epigenetic programming that predisposes more rapid accumulation of de novo mutations during development. We used a transgenic mouse system to test these possibilities by producing cloned midgestation mouse fetuses from three different donor somatic cell types carrying significantly different initial frequencies of spontaneous mutations. We found that on an individual locus basis, mutations acquired spontaneously in a population of donor somatic cells are not likely to be propagated to cloned embryos by SCNT. In addition, we found that the rate of accumulation of spontaneous mutations was similar in fetuses produced by either natural conception or cloning, indicating that cloned fetuses do not acquire mutations more rapidly than naturally conceived fetuses. These results represent the first direct demonstration that the process of cloning by SCNT does not lead to an increase in the frequency of point mutations. These results also demonstrate that epigenetic mechanisms normally contribute to the regulation of genetic integrity in a tissue-specific manner, and that these mechanisms are subject to reprogramming during cloning.

  3. Relationship between individual neuron and network spontaneous activity in developing mouse cortex.

    PubMed

    Barnett, Heather M; Gjorgjieva, Julijana; Weir, Keiko; Comfort, Cara; Fairhall, Adrienne L; Moody, William J

    2014-12-15

    Spontaneous synchronous activity (SSA) that propagates as electrical waves is found in numerous central nervous system structures and is critical for normal development, but the mechanisms of generation of such activity are not clear. In previous work, we showed that the ventrolateral piriform cortex is uniquely able to initiate SSA in contrast to the dorsal neocortex, which participates in, but does not initiate, SSA (Lischalk JW, Easton CR, Moody WJ. Dev Neurobiol 69: 407-414, 2009). In this study, we used Ca(2+) imaging of cultured embryonic day 18 to postnatal day 2 coronal slices (embryonic day 17 + 1-4 days in culture) of the mouse cortex to investigate the different activity patterns of individual neurons in these regions. In the piriform cortex where SSA is initiated, a higher proportion of neurons was active asynchronously between waves, and a larger number of groups of coactive cells was present compared with the dorsal cortex. When we applied GABA and glutamate synaptic antagonists, asynchronous activity and cellular clusters remained, while synchronous activity was eliminated, indicating that asynchronous activity is a result of cell-intrinsic properties that differ between these regions. To test the hypothesis that higher levels of cell-autonomous activity in the piriform cortex underlie its ability to initiate waves, we constructed a conductance-based network model in which three layers differed only in the proportion of neurons able to intrinsically generate bursting behavior. Simulations using this model demonstrated that a gradient of intrinsic excitability was sufficient to produce directionally propagating waves that replicated key experimental features, indicating that the higher level of cell-intrinsic activity in the piriform cortex may provide a substrate for SSA generation.

  4. Relationship between individual neuron and network spontaneous activity in developing mouse cortex

    PubMed Central

    Barnett, Heather M.; Gjorgjieva, Julijana; Weir, Keiko; Comfort, Cara; Fairhall, Adrienne L.

    2014-01-01

    Spontaneous synchronous activity (SSA) that propagates as electrical waves is found in numerous central nervous system structures and is critical for normal development, but the mechanisms of generation of such activity are not clear. In previous work, we showed that the ventrolateral piriform cortex is uniquely able to initiate SSA in contrast to the dorsal neocortex, which participates in, but does not initiate, SSA (Lischalk JW, Easton CR, Moody WJ. Dev Neurobiol 69: 407–414, 2009). In this study, we used Ca2+ imaging of cultured embryonic day 18 to postnatal day 2 coronal slices (embryonic day 17 + 1–4 days in culture) of the mouse cortex to investigate the different activity patterns of individual neurons in these regions. In the piriform cortex where SSA is initiated, a higher proportion of neurons was active asynchronously between waves, and a larger number of groups of coactive cells was present compared with the dorsal cortex. When we applied GABA and glutamate synaptic antagonists, asynchronous activity and cellular clusters remained, while synchronous activity was eliminated, indicating that asynchronous activity is a result of cell-intrinsic properties that differ between these regions. To test the hypothesis that higher levels of cell-autonomous activity in the piriform cortex underlie its ability to initiate waves, we constructed a conductance-based network model in which three layers differed only in the proportion of neurons able to intrinsically generate bursting behavior. Simulations using this model demonstrated that a gradient of intrinsic excitability was sufficient to produce directionally propagating waves that replicated key experimental features, indicating that the higher level of cell-intrinsic activity in the piriform cortex may provide a substrate for SSA generation. PMID:25185811

  5. The Memory Cytotoxic T-Lymphocyte (CTL) Response to Human Cytomegalovirus Infection Contains Individual Peptide-Specific CTL Clones That Have Undergone Extensive Expansion In Vivo

    PubMed Central

    Weekes, Michael P.; Wills, Mark R.; Mynard, Kim; Carmichael, Andrew J.; Sissons, J. G. Patrick

    1999-01-01

    Human cytomegalovirus (HCMV)-specific CD8+ cytotoxic T lymphocytes (CTL) appear to play an important role in the control of virus replication and in protection against HCMV-related disease. We have previously reported high frequencies of memory CTL precursors (CTLp) specific to the HCMV tegument protein pp65 in the peripheral blood of healthy virus carriers. In some individuals, the CTL response to this protein is focused on only a single epitope, whereas in other virus carriers CTL recognized multiple epitopes which we identified by using synthetic peptides. We have analyzed the clonal composition of the memory CTL response to four of these pp65 epitopes by sequencing the T-cell receptors (TCR) of multiple independently derived epitope-specific CTL clones, which were derived by formal single-cell cloning or from clonal CTL microcultures. In all cases, we have observed a high degree of clonal focusing: the majority of CTL clones specific to a defined pp65 peptide from any one virus carrier use only one or two different TCRs at the level of the nucleotide sequence. Among virus carriers who have the same major histocompatibility complex (MHC) class I allele, we observed that CTL from different donors that recognize the same peptide-MHC complex often used the same Vβ segment, although other TCR gene segments and CDR3 length were not in general conserved. We have also examined the clonal composition of CTL specific to pp65 peptides in asymptomatic human immunodeficiency virus-infected individuals. We have observed a similarly focused peptide-specific CTL response. Thus, the large population of circulating HCMV peptide-specific memory CTLp in virus carriers in fact contains individual CTL clones that have undergone extensive clonal expansion in vivo. PMID:9971792

  6. 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.

  7. Isolation of a cDNA Clone for Mouse Urinary Proteins: Age- And Sex-Related Expression of Mouse Urinary Protein Genes is Transcriptionally Controlled

    NASA Astrophysics Data System (ADS)

    Derman, Eva

    1981-09-01

    A recombinant cDNA plasmid derived from mouse urinary protein (MUP) mRNA was isolated and used to determine the level of control of the developmentally regulated and the sex-linked expression of MUP genes by monitoring the transcription of MUP mRNA sequences in isolated liver nuclei. No transcription of MUP genes could be detected in liver nuclei of prepubescent animals whose livers do not contain measurable MUP mRNA. Transcription of MUP genes in the livers of adult male mice was 6-fold higher than in the livers of adult female mice, proportional to the difference in MUP mRNA concentrations. Transcriptional control mechanisms are therefore implicated as responsible for both the developmentally and the sex-linked changes in the expression of MUP genes.

  8. 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.

  9. [Molecular cloning, prokaryotic expression and double-antibody sandwich ELISA development of 17β-hsd10 in mouse].

    PubMed

    Liu, Chuanzhi; Niu, Yingying; Chen, Yuan'an; Wu, Cheng; Yu, Yuanhua

    2014-11-01

    We expressed 17-hydroxysteroid dehydrogenase10 (17β-hsd10) recombinant protein, prepared anti-17β- hsd10 polyclonal antibodies and established sandwich enzyme linked immunosorbent assay (ELISA) test for detection of 17β-hsd10. RT-PCR was used to get the gene of 17β-hsd10 of mouse liver, and a prokaryotic protein expression system pET 15b-17β-hsd10/Escherichia coli BL21 (DE3) which induced with isopropyl-1-thio-β-galactopyranoside (IPTG) for recombinant protein expression was constructed subsequently. The target protein purified using His-Binding-resin column was used to immunize BALB/c mice and rabbits, serum total IgGs from immunized animals were purified by ammonium sulfate precipitation method. We established a Double-antibody Sandwich enzyme linked immunosorbent assay about 17β-hsd10 using the two antibodies we prepared. We got the concentration of 1.5 mg/mL of 17β-hsd10 protein with molecular weight of 29.5 kDa, and polyclonal antibodies from mouse and rabbit with the tite 1.25 x 10(4) and 2.5 x 10(4) respectively. The concentration of 0.1 g/mL of 17β-hsd10 can be detected by the Double-antibody Sandwich ELISA we established, and the assay was sensitive and specific. It can be widely used in clinical and experimental study.

  10. Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase.

    PubMed Central

    Thelander, L; Berg, P

    1986-01-01

    Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins M1 and M2, which are differentially regulated during the cell cycle. We have isolated expressible cDNA clones of both subunits from an Okayama-Berg cDNA library made with mRNA from hydroxyurea-resistant, M2 protein-overproducing mouse TA3 cells. Expression of M2 protein could be demonstrated by electron paramagnetic resonance spectroscopy after transfection of COS-7 monkey cells with the plasmid. Electrophoresis and blot analyses of the parent and hydroxyurea-resistant TA3 mRNA revealed two M2 transcripts, a major one of 2.1 kilobases and a minor one of about 1.6 kilobases. Restriction endonuclease mapping of the corresponding cDNAs indicated that the two mRNAs differed only in the length of the 3' untranslated ends. By contrast, there was only one mRNA corresponding to the M1 protein, and its mobility corresponded to about 3.1 kilobases. The hydroxyurea-resistant TA3 cells contained a 50- to 100-fold excess of the M2 mRNAs over that of the parent cells and a 10-fold excess of the M1 mRNA. However, a Southern blot analysis of the corresponding genomic DNA sequences showed that the M2 gene was amplified fivefold but the M1 gene was still single copy. The complete nucleotide sequence of the 2,111-base-pair-long M2 cDNA revealed an open reading frame coding for 390 amino acids, which corresponds to a molecular weight of 45,100. The mouse M2 protein sequence was quite homologous to the equivalent protein in the clam Spisula solidissima, while the homology to the smaller subunits of Epstein-Barr virus, herpes simplex virus type 2, and Escherichia coli ribonucleotide reductases were less pronounced. Images PMID:3025593

  11. Cloning and characterization of GRIPE, a novel interacting partner of the transcription factor E12 in developing mouse forebrain.

    PubMed

    Heng, Julian Ik Tsen; Tan, Seong-Seng

    2002-11-08

    The helix-loop-helix (HLH) family of transcription factors are key contributors to a wide array of developmental processes, including neurogenesis and hematopoiesis. These factors are thought to exert their regulatory influences by binding to cognate promoter-DNA sequences as dimers. Although studies in mice have convincingly demonstrated that neurogenic HLH proteins such as NeuroD are intimately involved in neuronal fate determination, the role of the ubiquitously expressed HLH protein, E12, in mammalian neurogenesis remains ambiguous. To address this, a yeast two-hybrid interaction screen was employed to identify dimerization partners to E12. Screening of an embryonic day 11.5 forebrain library resulted in the cloning of GRIPE, a novel GAP-related interacting protein to E12. GRIPE binds to the HLH region of E12 and may require E12 for nuclear import. Furthermore, GRIPE may negatively regulate E12-dependent target gene transcription. High levels of GRIPE and E12 mRNA were coincidentally detected during embryogenesis, but only GRIPE mRNA levels remained high in adult brain, particularly in neurons of the cortex and hippocampus. These observations were recapitulated through an in vitro model of neurogenesis. Taken together, these results indicate that GRIPE is a novel protein dimerization of which with E12 has important consequences for cells undergoing neuronal differentiation.

  12. Dual bidirectional promoters at the mouse dhfr locus: cloning and characterization of two mRNA classes of the divergently transcribed Rep-1 gene.

    PubMed Central

    Linton, J P; Yen, J Y; Selby, E; Chen, Z; Chinsky, J M; Liu, K; Kellems, R E; Crouse, G F

    1989-01-01

    The mouse dihydrofolate reductase gene (dhfr) is a housekeeping gene expressed under the control of a promoter region embedded in a CpG island--a region rich in unmethylated CpG dinucleotides. A divergent transcription unit exists immediately upstream of the dhfr gene which is coamplified with dhfr in some but not all methotrexate-resistant cell lines. We show that the promoter region for this gene pair consists of two bidirectional promoters, a major and minor promoter, which are situated within a 660-base-pair region upstream of the dhfr ATG translation initiation codon. The major promoter controls over 90% of dhfr transcription, while the minor promoter directs the transcription of the remaining dhfr mRNAs. The major promoter functions bidirectionally, transcribing a divergent 4.0-kilobase poly(A) mRNA (class A) in the direction opposite that of dhfr transcription. The predicted protein product of this mRNA is 105 kilodaltons. The minor promoter also functions bidirectionally, directing the transcription of at least two divergent RNAs (class B). These RNAs, present in quantities approximately 1/10 to 1/50 that of the class A mRNAs, are 4.4- and 1.6-kilobase poly(A) mRNAs. cDNAs representing both class A and class B mRNAs have been cloned from a mouse fibroblast cell line which has amplified the dhfr locus (3T3R500). DNA sequence analysis of these cDNAs reveals that the class A and class B mRNAs share, for the most part, the same exons. On the basis of S1 nuclease protection analysis of RNA preparations from several mouse tissues, both dhfr and divergent genes showed similar levels of expression but did show some specificity in start site utilization. Computer homology searches have revealed sequence similarity of the divergent transcripts with bacterial genes involved in DNA mismatch repair, and we therefore have named the divergently transcribed gene Rep-1. Images PMID:2674679

  13. Cloning of wrinkle-free, a previously uncharacterized mouse mutation, reveals crucial roles for fatty acid transport protein 4 in skin and hair development.

    PubMed

    Moulson, Casey L; Martin, Daniel R; Lugus, Jesse J; Schaffer, Jean E; Lind, Anne C; Miner, Jeffrey H

    2003-04-29

    Wrinkle-free (wrfr) is a previously uncharacterized, spontaneous, autosomal recessive mouse mutation resulting in very tight, thick skin. wrfr mutant mice exhibit severe breathing difficulties secondary to their tight skin and die shortly after birth. This phenotype is strikingly similar to a very rare human genetic disorder, restrictive dermopathy. wrfr mutant mice display a defective skin barrier, which is normally imparted by the cornified envelope, a composite of protein and lipid that prevents loss of water from within and entry of potentially harmful substances from without. In addition, hair growth from grafted wrfr skin is impaired. Positional cloning of the wrfr mutation revealed a retrotransposon insertion into a coding exon of Slc27a4, the gene encoding fatty acid transport protein (FATP)4. FATP4 is the primary intestinal FATP and is thought to play a major role in dietary fatty acid uptake; it therefore is viewed as a target to prevent or reverse obesity. However, its function in vivo had not been determined. Our results demonstrate an unexpected yet critical role for FATP4 in skin and hair development and suggest Slc27a4 to be a candidate gene for restrictive dermopathy.

  14. Cloning of the mouse BTG3 gene and definition of a new gene family (the BTG family) involved in the negative control of the cell cycle.

    PubMed

    Guéhenneux, F; Duret, L; Callanan, M B; Bouhas, R; Hayette, S; Berthet, C; Samarut, C; Rimokh, R; Birot, A M; Wang, Q; Magaud, J P; Rouault, J P

    1997-03-01

    It is well known that loss of tumor suppressor genes and more generally of antiproliferative genes plays a key role in the development of most tumors. We report here the cloning of the mouse BTG3 gene and show that its human counterpart maps on chromosome 21. This evolutionarily conserved gene codes for a 30 kDa protein and is expressed in most adult murine and human tissues analyzed. However, we demonstrate that its expression is cell cycle dependent and peaks at the end of the G1 phase. This gene is homologous to the human BTG1, BTG2 and TOB genes which were demonstrated to act as inhibitors of cell proliferation. Its description allowed us to define better this seven gene family (the BTG gene family) at the structural level and to speculate about its physiological role in normal and tumoral cells. This family is mainly characterized by the presence of two conserved domains (BTG boxes A and B) of as yet undetermined function which are separated by a non-conserved 20-25 amino acid sequence.

  15. Molecular cloning and mapping of the ecotropic leukemia provirus Emv-23 provides molecular access to the albino-deletion complex in mouse chromosome 7.

    PubMed

    Rinchik, E M; Machanoff, R; Cummings, C C; Johnson, D K

    1989-04-01

    Genetic analysis of radiation-induced deletion mutations involving the chromosome 7 albino (c) locus has expanded the functional map of this 6 to 11-cM region of the mouse genome. To generate one of many points of molecular access necessary for intensifying the analysis of the genes and phenotypes associated with this particular complex of deletions, we have cloned an endogenous ecotropic leukemia provirus (Emv-23), known to be closely linked to c, along with its flanking chromosome 7 sequences. A unique-sequence probe (23.3), derived from a region immediately 5' to the proviral integration site, was found to map less than 0.5 cM from c in a standard backcross analysis. Southern blot analysis of DNAs from animals carrying homozygous or overlapping albino deletions demonstrated that the 23.3 probe was deleted in several relatively small c-region deletions. The deletion mapping of the 23.3 probe places the Emv-23 locus between c and Mod-2, just proximal to a region important for male fertility and juvenile fitness. Mapping of this locus also provides a refinement of the genetic/deletion map for several mutations within this deletion complex.

  16. The effect of molecular weights of microencapsulating polymers on viability of mouse-cloned pancreatic β-cells: biomaterials, osmotic forces and potential applications in diabetes treatment.

    PubMed

    Mooranian, Armin; Tackechi, Ryu; Jamieson, Emma; Morahan, Grant; Al-Salami, Hani

    2017-05-09

    Ideal cell-containing microcapsules should be capable of maintaining cell viability and exhibit significant structural stability to support cellular functionality. To date, such microcapsules remain unavailable; thus, this study used our well-established microencapsulating methods to examine a total of 32 different microencapsulating formulations and correlate polymers' molecular weights (Mwt) and UDCA addition, with cell viability and microcapsules' stability, postmicroencapsulation. MIN6 mouse-cloned pancreatic β-cells were microencapsulated using control (n = 16; without UDCA) and test (n = 16; with UDCA) different polymers. Confocal microscopic imaging, cell viability, and microcapsules' stability were assessed. Best cell viability (>50%) was obtained at average Mwt of 50,000 g/mol (poly-l-ornithine), followed by 110,000 g/mol (poly-l-lysine). There was no linear correlation between Mwt and viability. Confocal imagining showed similar microcapsules' shape and cell distribution among all different polymers' molecular weights, which suggests that the microencapsulating method was efficient and maintained microcapsules' uniformity. UDCA addition resulted in enhanced osmotic stability of the microcapsules and improved cell viability, when the formulation contained 1% polylornithine, 1% polyethylene glycol, 20% Eudragit(®) NM30D, 1% polytetrafluoroethylene, or 5% pentamethylcyclopentasiloxane. UDCA addition improved microenvironmental conditions within the microcapsules but this effect was largely dependent on the polymer systems used.

  17. 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.

  18. 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

  19. 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

  20. Molecular genetic analysis of the V kappa Ser group associated with two mouse light chain genetic markers. Complementary DNA cloning and southern hybridization analysis

    PubMed Central

    1985-01-01

    Previous studies (21) have shown that two mouse kappa light (L) chain variable (V) region polymorphisms, the IB-peptide and Efla markers, reflect expression of a characteristic group of V kappa regions, called V kappa Ser, by some inbred strains and not others. Expression of V kappa Ser is controlled by a locus on chromosome 6, the chromosome that contains the kappa locus. To further characterize this V kappa group and begin to analyze the basis for its strain-specific expression, full- length complementary DNA (cDNA) copies were produced of L chain mRNA from the M75 myeloma that had been induced in the C.C58 strain of mice, and which produces a V kappa Ser L chain. The C.C58 strain is congenic with BALB/cAn, differing in the region of chromosome 6 that controls expression of the V kappa polymorphisms and the Lyt-2 and Lyt-3 T cell alloantigens. The complete nucleotide sequence of this cloned cDNA was determined and compared with the nucleotide sequences the most closely related BALB/c myeloma L chains known. Results indicated significant differences throughout the variable region, but particularly toward the 5' portion of the sequence. A probe corresponding to 200 bp of the 5' end of the cloned V kappa Ser cDNA was used in Southern hybridizations of restriction digests of liver DNA from a number of inbred, recombinant, and recombinant inbred strains. Under stringent hybridization conditions, one strongly-hybridizing fragment was observed in Bam HI, Hind III, and Eco RI digests, and based on the size of the fragments, strains could be organized into two groups. The presence of strongly hybridizing Bam HI, Hind III, and Eco RI fragments of 3.2, 2.8, and 2.1 kb, respectively, was found to correlate completely with expression by the strain of the IB-peptide and Efla markers. All nonexpressor strains yielded hybridizing fragments of 7.8, 8.4, and 2.8 kb, respectively. Possible explanations for strain- specific expression of V kappa Ser-associated phenotypic markers are

  1. Pharmacology of a cloned potassium channel from mouse brain (MK-1) expressed in CHO cells: effects of blockers and an 'inactivation peptide'.

    PubMed Central

    Robertson, B.; Owen, D. G.

    1993-01-01

    1. Chinese hamster ovary cells (CHO), maintained in cell culture, were stably transfected with DNA for the MK-1 voltage-activated potassium channel, previously cloned from a mouse brain library. 2. Voltage-activated currents were recorded by the whole cell patch clamp method. In CHO cells transfected with the vector only, there were no significant outward voltage activated currents. However, large outward voltage-activated potassium currents were always observed in those cells which had been transfected with the vector containing the DNA encoding for MK-1. 3. These potassium currents activated from -40 mV, and reversed at the potassium equilibrium potential. The half-maximal conductance of MK-1 was at -10 mV and had a slope factor of 11 mV when fitted with a Boltzmann function. There was only very slight (< 10%) inactivation of MK-1 even at very large positive voltages. 4. MK-1 was reversibly blocked by: 4-aminopyridine (4-AP, 0.1-4 mM), Toxin I 10-100 nM), mast cell degranulating peptide (1 microM), tetraethylammonium (TEA, 4-10 mM), tedisamil (100 microM), quinine (100 microM) and ciclazindol (100 microM); all applied to the outside of the cell from a 'U tube' rapid perfusion system. 4-AP may block closed as well as open MK-1 potassium channels. 5. A synthetic 20 amino acid peptide derived from the N-terminus sequence of the Shaker B potassium channel (the 'inactivation peptide') produced dramatic inactivation of MK-1 when applied to the inside, but not the outside of the cell. Reducing peptide concentration or 'degrading' the peptide produced less inactivation. 6. The block of MK-1 by the synthetic inactivation peptide was quite different in time dependence from block by internal TEA (0.4-4 mM), which probably blocks much more quickly but less potently than the peptide. PMID:8358568

  2. 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.

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

    PubMed Central

    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

  4. Developmental phases of individual mouse preimplantation embryos characterized by lipid signatures using desorption electrospray ionization mass spectrometry.

    PubMed

    Ferreira, Christina R; Pirro, Valentina; Eberlin, Livia S; Hallett, Judy E; Cooks, R Graham

    2012-12-01

    Knowledge of the lipids present in individual preimplantation embryos is of interest in fundamental studies of embryology, in attempts to understand cellular pluripotency and in optimization of in vitro culture conditions necessary for the application and development of biotechnologies such as in vitro fertilization and transgenesis. In this work, the profiles of fatty acids and phospholipids (PL) in individual mouse preimplantation embryos and oocytes were acquired using an analytical strategy based on desorption electrospray ionization mass spectrometry (DESI-MS). The methodology avoids sample preparation and provides information on the lipids present in these microscopic structures. Differences in the lipid profiles observed for unfertilized oocytes, two- and four-cell embryos, and blastocysts were characterized. For a representative set of embryos (N = 114) using multivariate analysis (specifically principal component analysis) unfertilized oocytes showed a narrower range of PL species than did blastocysts. Two- and four-cell embryos showed a wide range of PLs compared with unfertilized oocytes and high abundances of fatty acids, indicating pronounced synthetic activity. The data suggest that the lipid changes observed in mouse preimplantation development reflect acquisition of a degree of cellular membrane functional and structural specialization by the blastocyst stage. It is also noteworthy that embryos cultured in vitro from the two-cell through the blastocyst stage have a more homogeneous lipid profile as compared with their in vivo-derived counterparts, which is ascribed to the restricted diversity of nutrients present in synthetic culture media. The DESI-MS data are interpreted from lipid biochemistry and previous reports on gene expression of diverse lipids known to be vital to early embryonic development.

  5. Molecular cloning of the mouse grb2 gene: differential interaction of the Grb2 adaptor protein with epidermal growth factor and nerve growth factor receptors.

    PubMed Central

    Suen, K L; Bustelo, X R; Pawson, T; Barbacid, M

    1993-01-01

    We report the isolation and molecular characterization of the mouse grb2 gene. The product of this gene, the Grb2 protein, is highly related to the Caenorhabditis elegans sem-5 gene product and the human GRB2 protein and displays the same SH3-SH2-SH3 structural motifs. In situ hybridization studies revealed that the mouse grb2 gene is widely expressed throughout embryonic development (E9.5 to P0). However, grb2 transcripts are not uniformly distributed, and in certain tissues (e.g., thymus) they appear to be regulated during development. Recent genetic and biochemical evidence has implicated the Grb2 protein in the signaling pathways that link cell surface tyrosine kinase receptors with Ras. We have investigated the association of the Grb2 protein with epidermal growth factor (EGF) and nerve growth factor (NGF) receptors in PC12 pheochromocytoma cells. EGF treatment of PC12 cells results in the rapid association of Grb2 with the activated EGF receptors, an interaction mediated by the Grb2 SH2 domain. However, Grb2 does not bind to NGF-activated Trk receptors. Mitogenic signaling of NGF in NIH 3T3 cells ectopically expressing Trk receptors also takes place without detectable association between Grb2 and Trk. These results suggest that whereas EGF and NGF can activate the Ras signaling pathway in PC12 cells, only the EGF receptor is likely to do so through a direct interaction with Grb2. Finally, binding studies with glutathione S-transferase fusion proteins indicate that Grb2 binds two distinct subsets of proteins which are individually recognized by its SH2 and SH3 domains. These observations add further support to the concept that Grb2 is a modular adaptor protein. Images PMID:7689150

  6. 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

  7. Molecular cloning and heterologous expression of a cDNA encoding a mouse glutathione S-transferase Yc subunit possessing high catalytic activity for aflatoxin B1-8,9-epoxide.

    PubMed Central

    Hayes, J D; Judah, D J; Neal, G E; Nguyen, T

    1992-01-01

    Resistance to the carcinogenic effects of aflatoxin B1 (AFB1) in the mouse is due to the constitutive expression of an Alpha-class glutathione S-transferase (GST), YcYc, with high detoxification activity towards AFB1-8,9-epoxide. A cDNA clone (pmusGST Yc) for a murine GST Yc polypeptide has been isolated. Sequencing has shown the cDNA insert of pmusGST Yc to be 922 bp in length, with an open reading frame of 663 bp that encodes a polypeptide of M(r) 25358. The primary structure of the murine GST Yc subunit predicted by pmusGST Yc is in complete agreement with the partial amino acid sequence of the aflatoxin-metabolizing mouse liver GST described previously [McLellan, Kerr, Cronshaw & Hayes (1991) Biochem. J. 276, 461-469]. A plasmid, termed pKK-musGST Yc, which permits the expression of the murine Yc subunit in Escherichia coli, has been constructed. The murine GST expressed in E. coli was purified and found to be catalytically active towards several GST substrates, including AFB1-8,9-epoxide. This enzyme was also found to possess electrophoretic and immunochemical properties closely similar to those of the GST Yc subunit from mouse liver. However, the GST synthesized in E. coli and the constitutive mouse liver Alpha-class GST exhibited small differences in their chromatographic behaviour during reverse-phase h.p.l.c. Automated Edman degradation revealed alanine to be the N-terminal amino acid in the GST Yc subunit expressed in E. coli, whereas the enzyme in mouse liver possesses a blocked N-terminus. Although sequencing showed that the purified Yc subunit from E. coli lacked the initiator methionine, the amino acid sequence obtained over the first eleven N-terminal residues agreed with that predicted from the cDNA clone, pmusGST Yc. Comparison of the deduced amino acid sequence of the mouse Yc polypeptide with the primary structures of the rat Alpha-class GST enzymes revealed that it is more closely related to the ethoxyquin-induced rat liver Yc2 subunit than to

  8. 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.

  9. Complementation of a defect in the asparagine-linked glycosylation of a mouse FM3A mutant G258 cell line by spheroplast fusion of a human mega YAC clone 923f5.

    PubMed

    Masuda, Takahisa; Moriya, Masayuki; Kataoka, Kensuke; Nishikawa, Yoshihisa

    2012-01-01

    Mouse G258 mutant stopped both cell growth and the synthesis of lipid-linked oligosaccharide at the Man(3)GlcNAc(2)-P-P-Dolichol at a restricted temperature with a single gene mutation. To clarify the lesion in the G258 mutant, we isolated human genomic DNA transformants of the G258 mutant, which recovered from both defects by way of cell hybridization with X-ray irradiated HeLa cells. We detected a common 1.3-kb product by inter-human specific sequence in the L1 (L1Hs) PCR in the transformants (Kataoka et al., Somat. Cell Mol. Genet., 24, 235-243 (1998)). In the present study, we screened a human mega yeast artificial chromosome (YAC) library by PCR with primers designed according to the 1.3-kb DNA, and selected YAC clone 923f5. Moreover, we found by spheroplast fusion that YAC clone 923f5 complemented both defects of the G258 mutant. Since the human counterpart of the yeast ALG11 gene is localized in the region, the G258 mutant might have a defect in the mouse ALG11 gene.

  10. [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.

  11. Effect of culture medium volume and embryo density on early mouse embryonic development: tracking the development of the individual embryo.

    PubMed

    Dai, Shan-Jun; Xu, Chang-Long; Wang, Jeffrey; Sun, Ying-Pu; Chian, Ri-Cheng

    2012-07-01

    To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly. (1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining. The blastocyst development was significantly higher (P < 0.05) when nine embryos were cultured in 50 μl of droplet of culture medium compared with other volumes. The blastocyst development was significantly reduced (P < 0.05) in single embryo culture compared to group embryo culture with or without the WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P < 0.05) in single embryo culture with the WOW

  12. 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.

  13. 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

  14. Individual USH2 proteins make distinct contributions to the ankle link complex during development of the mouse cochlear stereociliary bundle

    PubMed Central

    Zou, Junhuang; Mathur, Pranav D.; Zheng, Tihua; Wang, Yong; Almishaal, Ali; Park, Albert H.; Yang, Jun

    2015-01-01

    Usher syndrome (USH) is the leading cause of inherited deaf-blindness, with type 2 (USH2) being the most common clinical form. Studies suggest that proteins encoded by USH2 causative genes assemble into the ankle link complex (ALC) at the hair cell stereociliary bundle; however, little is known about the in vivo assembly and function of this complex. Using various USH2 mutant mice, we showed by immunofluorescence that USH2 proteins play different roles in cochlear ALC assembly, with G protein-coupled receptor 98 being the most important protein. Complex assembly likely occurs at the stereociliary bundle but not along the protein transport route in the cell body. Stereociliary morphological defects in USH2 mutant mice suggest roles for the ALC in regulating inner hair cell stereociliary growth and differentiation as well as outer hair cell stereociliary rigidity and organization during development. These roles are unique from the bundle cohesion role of Usher syndrome type 1 protein complexes. Loss of individual USH2 gene expressions leads to variable morphological and functional consequences, correlating with the severity of ALC disruption. This finding suggests a potential genotype–phenotype correlation in USH2 patients. In summary, this study provides novel insights into the molecular mechanism underlying cochlear stereociliary bundle development and hearing loss pathogenesis of various USH2 subtypes. Our thorough phenotypical characterization of USH2 mouse models is essential for future use of these animal models in therapeutic development. PMID:26401052

  15. Nuclear reprogramming to produce cloned mice and embryonic stem cells from somatic cells.

    PubMed

    Wakayama, Sayaka; Cummins, James M; Wakayama, Teruhiko

    2008-04-01

    Cloning methods in mice are now well described and are becoming routine. However, the frequency at which cloned mice are produced remains below 5%, irrespective of the nucleus donor species or cell type. Only a few laboratories have made clones from adult mouse somatic cells and most strains have never produced cloned mice. On the other hand, nuclear transfer can be used to generate human embryonic stem (ntES) cell lines from a patient's own somatic cells. It has been shown that such cells can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. This technique could be used in regenerative medicine and, in theory, in infertility clinics to treat completely infertile individuals. However, these results suggest that the reprogramming integrity of each cloned embryo differs: some cloned embryos can be converted to ntES cells, but these embryos cannot achieve full term development. This review outlines the nature of genomic reprogramming potential and its application, and suggests new approaches to avoid the ethical problems of creating embryos by nuclear transfer.

  16. Population dynamics of natural antibodies in normal and autoimmune individuals.

    PubMed Central

    Varela, F; Andersson, A; Dietrich, G; Sundblad, A; Holmberg, D; Kazatchkine, M; Coutinho, A

    1991-01-01

    We have measured the quantities of naturally occurring autoantibodies in the serum of normal, unmanipulated individuals. These changes over time following broad-band complex dynamical patterns that are similar in mouse and man. The patterns more likely reflect the network architecture of the natural antibody repertoire, regulating the activation and decay of individual clones. The temporal changes of both disease-specific and nonspecific autoantibodies are consistently modified in autoimmune individuals. PMID:2062870

  17. 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.

  18. Cloning of a functional mannose-6-phosphate reductase (M6PR) gene homolog from Egyptian celery plants (Apium graveolens): overexpression in non-mannitol producing plants resulted in mannitol accumulation in transgenic individuals.

    PubMed

    Khalil, Shaimaa R M; Ibrahim, Amr S; Hussien, Basita A; Hussien, Ebtissam A; Tawfik, Mohamed S

    2017-10-01

    Salinity is a major limiting factor affecting crops production, survival and distribution worldwide. Engineering dehydration stress tolerance in commercial crops is a trait of economic importance, especially in saline-affected areas. In this work, we are reporting the cloning of the M6PR gene homolog (encoding a key enzyme, mannose-6-phosphate reductase, for mannitol biosynthesis in celery) from Egyptian celery plants. Using RACE technique, the full-length Egyptian-M6PR gene (1333 bp) was cloned into pRI-201AN plant expression vector. Analysis of the cloned gene revealed that both American and Egyptian clones had both start and stop codons in frame and was found to be 930 base long. The newly cloned EM6PR gene was found to be 126 base longer than its American counterpart at the non-coding region. Six differences at nucleotide level between the Egyptian and American sequences were observed, three of which in the coding region resulting in three polymorphic amino acids differences (tryptophan vs. leucine, glutamine vs. histidine and isoleucine vs. leucine). The newly cloned gene was introduced to tobacco via Agrobacterium and PCR analysis of T0 plants indicated the presence of the EM6PR gene into 10 out of 38 tobacco individuals. Moreover, RT-PCR analysis confirmed the presence of EM6PR transcripts in 9 out of the 10 PCR positive plants. GC/MS analysis of some RT positive individuals indicated the accumulation of mannitol in transgenics tobacco, while mannitol was absent in non-transgenic controls.

  19. Human Cloning

    DTIC Science & Technology

    2006-07-20

    not believe that noncoital, asexual reproduction , such as cloning, would be considered a fundamental right by the Supreme Court. A ban on human...society by “crossing the boundary from sexual to asexual reproduction , thus approving in principle the genetic manipulation and control of nascent human... reproductive cloning and, by a vote of 10 to 7, a four-year moratorium on cloning for medical research purposes. The ethical issues surrounding reproductive

  20. Clone clustering by hybridization

    SciTech Connect

    Milosavijevic, A.; Zeremski, M.; Paunesku, T.

    1995-05-01

    DNA sequencing by hybridization (SBH) Format 1 technique is based on experiments in which thousands of short oligomers are consecutively hybridized with dense arrays of clones. In this paper the authors present the description of a method for obtaining hybridization signatures for individual clones that guarantees reproducibility despite a wide range of variations in experimental circumstances, a sensitive method for signature comparison at prespecified significance levels, and a clustering algorithm that correctly identifies clusters of significantly similar signatures. The methods and the algorithm have been verified experimentally on a control set of 422 signatures that originate from 9 distinct clones of known sequence. Experiments indicate that only 30 to 50 oligomer probes suffice for correct clustering. This information about the identity of clones can be used to guide both genomic and cDNA sequencing by SBH or by standard gel-based methods. 12 refs., 5 figs., 2 tabs.

  1. 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…

  2. 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…

  3. 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.

  4. 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.

  5. 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.

  6. Equine cloning: applications and outcomes.

    PubMed

    Vanderwall, Dirk K; Woods, Gordon L; Roser, Janet F; Schlafer, Donald H; Sellon, Debra C; Tester, David F; White, Kenneth L

    2006-01-01

    Cloning is one of several new assisted reproductive techniques being developed for clinical use in the equine industry. Potential uses of equine cloning include: (1) the preservation of genetics from individual animals that would otherwise not be able to reproduce, such as geldings; (2) the preservation of genetic material of endangered and/or exotic species, such as the Mongolian wild horse (Przewalski's horse); and (3) because of the companion animal role that horses fill for some individuals, it is likely that some horse owners will have individual animals cloned for emotional fulfillment. Although equine cloning has been successful, like other species, it remains a very inefficient process (<3% success). In most species, the inefficiency of cloning results from a high incidence of embryonic, fetal and/or placental developmental abnormalities that contribute to extremely high rates of embryonic loss, abortion and stillbirths throughout gestation and compromised neonatal health after birth. The present review describes some of the ultrasonographic, endocrinological and histopathological characteristics of successful (produced viable offspring) and unsuccessful (resulted in pregnancy failure) cloned equine (mule and horse) pregnancies we have produced. A total of 21 cloned mule pregnancies were established using fetal fibroblast cells, whereas a total of seven cloned horse pregnancies were established using adult cumulus cells. Three of the cloned mule conceptuses were carried to term, resulting in the birth of three healthy clones. This information adds to an accumulating body of knowledge about the outcome of cloned equine pregnancies, which will help to establish when, and perhaps why, many cloned equine pregnancies fail.

  7. cDNA cloning and characterization of mouse nifS-like protein, m-Nfs1: mitochondrial localization of eukaryotic NifS-like proteins.

    PubMed

    Nakai, Y; Yoshihara, Y; Hayashi, H; Kagamiyama, H

    1998-08-14

    We have isolated a mouse cDNA which shows significant sequence similarity to the yeast nifS-like gene (y-NFS1), and termed it m-Nfs1. The deduced protein sequence (459 amino acids long) has several characteristic features common to those of bacterial NifS proteins, but distinct from them by its amino-terminal extension which contains a typical mitochondrial targeting presequence. m-Nfs1 was found to be a soluble 47-kDa protein in the matrix fraction of mouse liver mitochondria. The m-Nfs1 gene was ubiquitously expressed in most tissues, suggesting its housekeeping function in vivo. We also found that the gamma-NFS1 protein was localized in the mitochondrial matrix in yeast cells. These results suggest that both eukaryotic NifS-like proteins may play some roles in mitochondrial functions.

  8. Why Clone?

    MedlinePlus

    ... disease. Find out more about Stem Cells . Reviving Endangered or Extinct Species You might have seen the Jurassic Park movies. ... related goat species to make a male. Cloning endangered species is much easier, mainly because the surviving animals ...

  9. 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.

  10. 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. © 2012 Blackwell Verlag GmbH.

  11. 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

  12. 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.

  13. 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

  14. Alloreactive T cell clones.

    PubMed

    Fitch, F W

    1984-01-01

    T cell clones are useful models for studying lymphocyte function both at the level of the individual cell and in interacting systems. Murine cytolytic and non- cytolyic T cell clones have been obtained with relative ease, and the particular procedure used to derive and maintain T cell clones may influence profoundly the characteristics of the resulting cells. The method of choice depends on the specific question to be asked. Although some clones have characteristics that would have been expected on the basis of results observed with bulk cell populations, other clones have rather unexpected properties. Although most T cell clones appear to be either cytolytic or non-cytolytic, this distinction is not always absolute. A high proportion of both cytolytic and non-cytolytic T cell clones have dual reactivity. This is true for cells which by other criteria appear to be true clones. The frequency of such cells is high enough to suggest that most if not all T cells may have reactivity for more than one antigenic determinant or that antigenic determinants recognized by T cells are shared widely and unexpectedly. It is not clear whether one or two different antigen receptors account for such dual reactivity. The nature of the T cell receptor for antigen remains obscure. T cell clones, because of their homogeneous nature, should make it easier to answer these important immunological questions. Although it remains to be determined how many distinct molecules account for the numerous biological activities found in the culture supernatants from antigen-stimulated T cell clones, it is clear that these factors influence several different types of cells that are involved directly and indirectly in immune responses. IL-2 stimulates both cytolytic and non-cytolytic T cells to proliferate. BCSF causes polyclonal activation of B cells, and there may be other factors which influence B cell responses to antigenic stimulation. IL-3 apparently stimulates maturation of immature T cells

  15. Differential Gene Expression in Chemically Induced Mouse Lung Adenomas1

    PubMed Central

    Yao, Ruisheng; Wang, Yian; Lubet, Ronald A; You, Ming

    2003-01-01

    Abstract Because of similarities in histopathology and tumor progression stages between mouse and human lung adenocarcinomas, the mouse lung tumor model with lung adenomas as the endpoint has been used extensively to evaluate the efficacy of putative lung cancer chemopreventive agents. In this study, a competitive cDNA library screening (CCLS) was employed to determine changes in the expression of mRNA in chemically induced lung adenomas compared with paired normal lung tissues. A total of 2555 clones having altered expression in tumors were observed following competitive hybridization between normal lung and lung adenomas after primary screening of over 160,000 clones from a mouse lung cDNA library. Among the 755 clones confirmed by dot blot hybridization, 240 clones were underexpressed, whereas 515 clones were overexpressed in tumors. Sixty-five clones with the most frequently altered expression in six individual tumors were confirmed by semiquantitative RT-PCR. When examining the 58 known genes, 39 clones had increased expression and 19 had decreased expression, whereas the 7 novel genes showed overexpression. A high percentage (>60%) of overexpressed or underexpressed genes was observed in at least two or three of the lesions. Reproducibly overexpressed genes included ERK-1, JAK-1, surfactant proteins A, B, and C, NFAT1, α-1 protease inhibitor, helix-loop-helix ubiquitous kinase (CHUK), α-adaptin, α-1 PI2, thioether S-methyltransferase, and CYP2C40. Reproducibly underexpressed genes included paroxanase, ALDH II, CC10, von Ebner salivary gland protein, and α- and β-globin. In addition, CCLS identified several novel genes or genes not previously associated with lung carcinogenesis, including a hypothetical protein (FLJ11240) and a guanine nucleotide exchange factor homologue. This study shows the efficacy of this methodology for identifying genes with altered expression. These genes may prove to be helpful in our understanding of the genetic basis of lung

  16. Highly Variable Genomic Landscape of Endogenous Retroviruses in the C57BL/6J Inbred Strain, Depending on Individual Mouse, Gender, Organ Type, and Organ Location

    PubMed Central

    Lee, Kang-Hoon; Lim, Debora; Greenhalgh, David

    2017-01-01

    Transposable repetitive elements, named the “TREome,” represent ~40% of the mouse genome. We postulate that the germ line genome undergoes temporal and spatial diversification into somatic genomes in conjunction with the TREome activity. C57BL/6J inbred mice were subjected to genomic landscape analyses using a TREome probe from murine leukemia virus-type endogenous retroviruses (MLV-ERVs). None shared the same MLV-ERV landscape within each comparison group: (1) sperm and 18 tissues from one mouse, (2) six brain compartments from two females, (3) spleen and thymus samples from four age groups, (4) three spatial tissue sets from two females, and (5) kidney and liver samples from three females and three males. Interestingly, males had more genomic MLV-ERV copies than females; moreover, only in the males, the kidneys had higher MLV-ERV copies than the livers. Perhaps, the mouse-, gender-, and tissue/cell-dependent MLV-ERV landscapes are linked to the individual-specific and dynamic phenotypes of the C57BL/6J inbred population. PMID:28951865

  17. Valproic acid treatment from the 4-cell stage improves Oct4 expression and nuclear distribution of histone H3K27me3 in mouse cloned blastocysts.

    PubMed

    Isaji, Yuuki; Murata, Moeko; Takaguchi, Naoya; Mukai, Toshita; Tajima, Yosuke; Imai, Hiroshi; Yamada, Masayasu

    2013-01-01

    We examined effects of treatment with valproic acid (0, 0.2, 1 or 2 mM, VPA), an inhibitor of class I and IIa histone deacetylases (HDACs), of mouse somatic cell nuclear transfer (SCNT) embryos for 24 h from 48 h (4-cell stage), 24 h (2-cell stage) or immediately after oocyte activation on blastocyst formation rates and qualities of the resultant blastocysts. Blastocyst formation rates (33.4-37.0%) were not improved by VPA treatments compared with the untreated control (35.1-36.4%). However, immunofluorescence staining revealed that Oct4 expression levels, evaluated from percentages of embryos expressing Oct4 strongly and having more than 10 Oct4-positive cells, in blastocysts from SCNT embryos treated with 1 mM VPA for 24 h from the 4-cell stage (VPA-4C) were highest among all the groups and that the proportion of cells with a normal nuclear distribution of histone H3 trimethylated at lysine 27 (H3K27me3), a marker of the state of X-chromosome inactivation, significantly increased in the VPA-4C group (36.6%) compared with the control group (12.4%, P<0.05). Treatments with scriptaid and sodium butyrate, inhibitors of class I and IIa/b HDACs, for 24 h from the 4-cell stage also had beneficial effects on SCNT blastocysts. These findings indicate that treatment with 1 mM VPA from the 4-cell stage improves the Oct4 expression and nuclear distribution of H3K27me3 in mouse SCNT blastocysts and suggest that the inhibition of class I and IIa HDACs from the 4-cell stage plays an important role in these effects.

  18. Positional cloning of zinc finger domain transcription factor Zfp69, a candidate gene for obesity-associated diabetes contributed by mouse locus Nidd/SJL.

    PubMed

    Scherneck, Stephan; Nestler, Matthias; Vogel, Heike; Blüher, Matthias; Block, Marcel-Dominique; Berriel Diaz, Mauricio; Herzig, Stephan; Schulz, Nadja; Teichert, Marko; Tischer, Sina; Al-Hasani, Hadi; Kluge, Reinhart; Schürmann, Annette; Joost, Hans-Georg

    2009-07-01

    Polygenic type 2 diabetes in mouse models is associated with obesity and results from a combination of adipogenic and diabetogenic alleles. Here we report the identification of a candidate gene for the diabetogenic effect of a QTL (Nidd/SJL, Nidd1) contributed by the SJL, NON, and NZB strains in outcross populations with New Zealand Obese (NZO) mice. A critical interval of distal chromosome 4 (2.1 Mbp) conferring the diabetic phenotype was identified by interval-specific congenic introgression of SJL into diabetes-resistant C57BL/6J, and subsequent reporter cross with NZO. Analysis of the 10 genes in the critical interval by sequencing, qRT-PCR, and RACE-PCR revealed a striking allelic variance of Zfp69 encoding zinc finger domain transcription factor 69. In NZO and C57BL/6J, a retrotransposon (IAPLTR1a) in intron 3 disrupted the gene by formation of a truncated mRNA that lacked the coding sequence for the KRAB (Krüppel-associated box) and Znf-C2H2 domains of Zfp69, whereas the diabetogenic SJL, NON, and NZB alleles generated a normal mRNA. When combined with the B6.V-Lep(ob) background, the diabetogenic Zfp69(SJL) allele produced hyperglycaemia, reduced gonadal fat, and increased plasma and liver triglycerides. mRNA levels of the human orthologue of Zfp69, ZNF642, were significantly increased in adipose tissue from patients with type 2 diabetes. We conclude that Zfp69 is the most likely candidate for the diabetogenic effect of Nidd/SJL, and that retrotransposon IAPLTR1a contributes substantially to the genetic heterogeneity of mouse strains. Expression of the transcription factor in adipose tissue may play a role in the pathogenesis of type 2 diabetes.

  19. Cloning and characterization of the 5'-upstream sequence governing the cell cycle-dependent transcription of mouse DNA polymerase alpha 68 kDa subunit gene.

    PubMed

    Nishikawa, N S; Izumi, M; Uchida, H; Yokoi, M; Miyazawa, H; Hanaoka, F

    2000-04-01

    We have isolated the genomic DNA fragment spanning the 5-end and the first four exons encoding the 68 kDa subunit (p68) of the mouse DNA polymerase alpha-primase complex [corrected]. The p68 promoter region lacks TATA and CAAT boxes, but contains a GC-rich sequence, two palindrome sequences and two putative E2F-binding sites [corrected]. A series of transient expression assays using a luciferase reporter gene indicated that a region from nucleotide position -89 to -30 (-89/-30) with respect to the transcription initiation site is crucial for basal transcription of the p68 gene in proliferating NIH 3T3 cells. In particular, part of the GC-rich sequence (-57/-46) and the palindrome (-81/-62) elements were necessary for promoter activity, both of which share homology with the E-box sequence. Gel mobility shift assays using NIH 3T3 nuclear extracts revealed that the upstream stimulatory factor, known as an E-box-binding protein, binds to these sites. Moreover, we observed binding of E2F to two sites near the transcription initiation site (-11/-3 and +9/+16). A transient luciferase expression assay using synchronized NIH 3T3 cells in G(0)phase revealed that these E2F sites are essential for transcription induction of the p68 gene after serum stimulation, but are dispensable for basal transcription. These results indicate that growth-dependent regulation of transcription of the mouse p68 and p180 genes is mediated by a common factor, E2F; however, basal transcription of the genes, interestingly, is regulated by different transcription factors.

  20. Cloning and characterization of the 5′-upstream sequence governing the cell cycle-dependent transcription of mouse DNA polymerase α 68 kDa subunit gene

    PubMed Central

    Nishikawa, Naoko S.; Izumi, Masako; Uchida, Hiroshi; Yokoi, Masayuki; Miyazawa, Hiroshi; Hanaoka, Fumio

    2000-01-01

    We have isolated and determined the structure of the gene encoding the 68 kDa subunit (p68) of the mouse DNA polymerase α–primase complex. The p68 gene consists of four exons and the p68 promoter region lacks TATA and CAAT boxes, but contains a GC-rich sequence, two palindrome sequences and two putative E2F-binding sites. A series of transient expression assays using a luciferase reporter gene indicated that a region from nucleotide position –89 to –30 (–89/–30) with respect to the transcription initiation site is crucial for basal transcription of the p68 gene in proliferating NIH 3T3 cells. In particular, part of the GC-rich sequence (–57/–46) and the palindrome (–81/–62) elements were necessary for promoter activity, both of which share homology with the E-box sequence. Gel mobility shift assays using NIH 3T3 nuclear extracts revealed that the upstream stimulatory factor, known as an E-box-binding protein, binds to these sites. Moreover, we observed binding of E2F to two sites near the transcription initiation site (–11/–3 and +9/+16). A transient luciferase expression assay using synchronized NIH 3T3 cells in G0 phase revealed that these E2F sites are essential for transcription induction of the p68 gene after serum stimulation, but are dispensable for basal transcription. These results indicate that growth-dependent regulation of transcription of the mouse p68 and p180 genes is mediated by a common factor, E2F; however, basal transcription of the genes, interestingly, is regulated by different transcription factors. PMID:10710418

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

    PubMed

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

    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-tetra-decanoylphorbol-13-acetate treatment decreasing D52 and D53 transcript levels in these cell lines. The presence of a coiled-coil domain, combined with observed co- or independent expression of the D52 and D53 genes, suggests that D52 and D53 proteins may be capable of hetero- and/or homodimer formation.

  2. 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.

  3. Molecular cloning.

    PubMed

    Lessard, Juliane C

    2013-01-01

    This protocol describes the basic steps involved in conventional plasmid-based cloning. The goals are to insert a DNA fragment of interest into a receiving vector plasmid, transform the plasmid into E. coli, recover the plasmid DNA, and check for correct insertion events.

  4. Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing.

    PubMed

    Veres, Adrian; Gosis, Bridget S; Ding, Qiurong; Collins, Ryan; Ragavendran, Ashok; Brand, Harrison; Erdin, Serkan; Cowan, Chad A; Talkowski, Michael E; Musunuru, Kiran

    2014-07-03

    Genome editing has attracted wide interest for the generation of cellular models of disease using human pluripotent stem cells and other cell types. CRISPR-Cas systems and TALENs can target desired genomic sites with high efficiency in human cells, but recent publications have led to concern about the extent to which these tools may cause off-target mutagenic effects that could potentially confound disease-modeling studies. Using CRISPR-Cas9 and TALEN targeted human pluripotent stem cell clones, we performed whole-genome sequencing at high coverage in order to assess the degree of mutagenesis across the entire genome. In both types of clones, we found that off-target mutations attributable to the nucleases were very rare. From this analysis, we suggest that, although some cell types may be at risk for off-target mutations, the incidence of such effects in human pluripotent stem cells may be sufficiently low and thus not a significant concern for disease modeling and other applications.

  5. MicroRNA-Attenuated Clone of Virulent Semliki Forest Virus Overcomes Antiviral Type I Interferon in Resistant Mouse CT-2A Glioma.

    PubMed

    Martikainen, Miika; Niittykoski, Minna; von und zu Fraunberg, Mikael; Immonen, Arto; Koponen, Susanna; van Geenen, Maartje; Vähä-Koskela, Markus; Ylösmäki, Erkko; Jääskeläinen, Juha E; Saksela, Kalle; Hinkkanen, Ari

    2015-10-01

    Glioblastoma is a terminal disease with no effective treatment currently available. Among the new therapy candidates are oncolytic viruses capable of selectively replicating in cancer cells, causing tumor lysis and inducing adaptive immune responses against the tumor. However, tumor antiviral responses, primarily mediated by type I interferon (IFN-I), remain a key problem that severely restricts viral replication and oncolysis. We show here that the Semliki Forest virus (SFV) strain SFV4, which causes lethal encephalitis in mice, is able to infect and replicate independent of the IFN-I defense in mouse glioblastoma cells and cell lines originating from primary human glioblastoma patient samples. The ability to tolerate IFN-I was retained in SFV4-miRT124 cells, a derivative cell line of strain SFV4 with a restricted capacity to replicate in neurons due to insertion of target sites for neuronal microRNA 124. The IFN-I tolerance was associated with the viral nsp3-nsp4 gene region and distinct from the genetic loci responsible for SFV neurovirulence. In contrast to the naturally attenuated strain SFV A7(74) and its derivatives, SFV4-miRT124 displayed increased oncolytic potency in CT-2A murine astrocytoma cells and in the human glioblastoma cell lines pretreated with IFN-I. Following a single intraperitoneal injection of SFV4-miRT124 into C57BL/6 mice bearing CT-2A orthotopic gliomas, the virus homed to the brain and was amplified in the tumor, resulting in significant tumor growth inhibition and improved survival. Although progress has been made in development of replicative oncolytic viruses, information regarding their overall therapeutic potency in a clinical setting is still lacking. This could be at least partially dependent on the IFN-I sensitivity of the viruses used. Here, we show that the conditionally replicating SFV4-miRT124 virus shares the IFN-I tolerance of the pathogenic wild-type SFV, thereby allowing efficient targeting of a glioma that is refractory

  6. 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.

  7. Expression of cloned herpesvirus genes. I. Detection of nuclear antigens from herpes simplex virus type 2 inverted repeat regions in transfected mouse cells.

    PubMed Central

    Middleton, M H; Reyes, G R; Ciufo, D M; Buchan, A; Macnab, J C; Hayward, G S

    1982-01-01

    Three different recombinant plasmids containing the entire 15-kilobase L and S inverted repeat sequence of herpes simplex virus type 2 DNA have been introduced into cultured Ltk- or BSC cells by both the calcium and DEAE-dextran transfection procedures. In each case, after 24 h approximately 1% of the cells gave strongly positive nuclear staining when assayed by immunofluorescence with hyperimmune antisera made against early and immediate-early infected-cell polypeptides. The nuclear fluorescence pattern and intensity mimicked that observed within 2 to 3 h after infection of Ltk- cells with either herpes simplex virus type 1 or type 2 wild-type virus. Herpes simplex virus type 1 (KOStsB2)-infected Ltk- cells under nonpermissive conditions did not express these antigens in the nucleus. Therefore, we conclude that either one or both of the 185,000- and 110,000-molecular-weight immediate early proteins, or some other as yet unknown gene product encoded entirely within the inverted repeats, can be transiently expressed in large amounts in transfected cells in the absence of other viral genes or accompanying virion components. Permanent mouse cell lines derived from transfection with these plasmids by using the thymidine kinase coselection procedure did not express sufficient nuclear antigen to be detectable by immunofluorescence. Images PMID:6292452

  8. Cloning and molecular characterization of cDNA encoding a mouse male-enhanced antigen-2 (Mea-2): a putative family of the Golgi autoantigen.

    PubMed

    Kondo, M; Sutou, S

    1997-01-01

    The male-enhanced antigen-2 (Mea-2) gene was originally identified with a monoclonal histocompatibility Y (H-Y) antibody (mAb4VII). There is no report of the full length cDNA encode for Mea-2 product until this report. In this study, we isolated the full length mouse Mea-2 cDNA by screening a testis cDNA library with a PCR-amplified Mea-2 product, and direct PCR amplification of its upstream sequences from the cDNA library. The primary structure of the Mea-2 peptide, deduced from this nucleotide sequence, shows that it encode a 150 kDa protein, of 1325 amino acid residues, which contained five putative N-glycosylation sites and four leucine zipper motifs. A data bank search indicated that it has high homology with a human Golgi autoantigen (golgin-160) both in its nucleotides (78%) and amino acids sequence (83%). This suggests that Mea-2 gene product may encode a golgi structural protein. In situ hybridization analysis suggested that the Mea-2 gene is expressed in spermatids during spermatogenesis as already shown by Mea-1, suggesting that Mea-2 gene product as well as Mea-1 have also some role for spermatogenesis.

  9. Cloning and Immunocytochemical Localization of a Cyclic Nucleotide–Gated Channel α-Subunit to All Cone Photoreceptors in the Mouse Retina

    PubMed Central

    HIRANO, ARLENE A.; HACK, IRIS; WÄSSLE, HEINZ; DUVOISIN, ROBERT M.

    2010-01-01

    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 α-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. PMID:10813773

  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, bacterial expression, and unique structure of adenosylhomocysteine hydrolase-like protein 1, or inositol 1,4,5-triphosphate receptor-binding protein from mouse kidney.

    PubMed

    Gomi, Tomoharu; Takusagawa, Fusao; Nishizawa, Mikio; Agussalim, Bukhari; Usui, Isao; Sugiyama, Eiji; Taki, Hirofumi; Shinoda, Kouichiro; Hounoki, Hiroyuki; Miwa, Toshiro; Tobe, Kazuyuki; Kobayashi, Masashi; Ishimoto, Tetsuya; Ogawa, Hirofumi; Mori, Hisashi

    2008-11-01

    Adenosylhomocysteine hydrolase (SAHase)-like protein 1 (SAH-L), also called inositol 1,4,5-triphosphate receptor-binding protein (IRBIT) is a novel protein involved in fish embryo development and calcium release in mammalian cells through protein-protein interactions. To better understand its reaction mechanism, purified protein is indispensable. Here we describe a simple purification procedure and the unique properties of SAH-L. The cDNA was isolated from mouse kidney by RT-PCR and inserted into various pETtrade mark vectors. Escherichia coli harboring a plasmid coding for SAH-L with a C-terminal His-tag could solely produce a soluble protein. SAH-L purified through a Ni(2+) column gave M(r)s of 59,000 and 190,000 by SDS-PAGE and gel filtration, respectively, which is suggestive of a trimer, but chemical cross-linking experiments demonstrated a dimer. The incompatible M(r) values implicate an irregular structure of SAH-L. In fact, SAH-L was partially purified in a form lacking the 31 N-terminal residues, and was found to be extremely susceptible to proteases in the region around residue 70. The N-terminal polypeptide (residues 1-98) was also expressed as a soluble form and was trypsin-sensitive. Circular dichroism revealed a low alpha-helix content but not a randomly extended structure. Interestingly, SAH-L contained tightly bound NAD(+) despite showing no SAHase activity. The characterized properties of SAH-L and its N-terminal fragment present the notion that the structure of the protease-sensitive N-terminal region is relatively loose and flexible rather than compact, and which protrudes from the major SAHase-like domain. This structure is supposed to be favorable to interact with the IP(3) receptor.

  12. A Japanese encephalitis virus genotype 5 molecular clone is highly neuropathogenic in a mouse model: impact of the structural protein region on virulence.

    PubMed

    de Wispelaere, Mélissanne; Frenkiel, Marie-Pascale; Desprès, Philippe

    2015-06-01

    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. 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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. 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.

  14. 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

  15. 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.

  16. Structure of cloned delta-globin genes from a normal subject and a patient with delta-thalassemia; sequence polymorphisms found in the delta-globin gene region of Japanese individuals.

    PubMed

    Kimura, A; Matsunaga, E; Ohta, Y; Fujiyoshi, T; Matsuo, T; Nakamura, T; Imamura, T; Yanase, T; Takagi, Y

    1982-10-11

    The delta-globin genes of a normal Japanese and a Japanese patient with homozygous delta-thalassemia were cloned, and the nucleotide sequence of a region including the gene was determined. Comparison of the nucleotide sequences of these two individuals with that of pH delta 1, delta-globin clone from the gene library constructed by Maniatis et al., showed differences in the large intervening sequence (IVS 2), at positions 137, 151, 186, 188, 291, 292 and 540 as one base substitutions, at 339 and 823 as one base additions, at 548 as a one base deletion, and a 9 bp duplication between positions 651 and 659, and differences in the 3'-flanking sequence at 51 and 98 nucleotides 3' to the AATAAA sequence. However, in the region studied, no differences was observed in the nucleotide sequences of the normal subject and the patient with delta-thalassemia. Therefore, these differences may represent polymorphisms of the delta-globin gene present in Japanese individuals. These data suggest that IVS 2 is more divergent than other regions, and that a DNA region(s) other than the globin gene may affect expression of the gene.

  17. Cloning of rat homeobox genes

    SciTech Connect

    Sakoyama, Yasuhiko; Mizuta, Ikuko; Ogasawara, Naotake

    1994-10-01

    We report the isolation of nine rat cognates of mouse homeoboxes within the four Hox gene clusters and a rat homologue of mouse IPF1 homeobox, RHbox No. 13A. The sequences of nine cloned homeoboxes are highly similar to those of the mouse and human homeoboxes in the Hox clusters. The restriction enzyme sites and map distances between each of the homeoboxes on the rat genome are nearly identical to those of mouse and human. Thus, we conclude that the isolated homeoboxes are the rat homologues of mouse homeoboxes within the four Hox clusters. A novel homeobox RHbox No. 13A is different from the Drosophila Antennapedia (Antp) sequence but is highly similar to the XlHbox8 (Xenopus laevis) and HtrA2 (Helobdella triserialis) homeoboxes. Forty-two amino acids of the last two-thirds of the RHbox No. 13A, XlHbox8, and mouse IPF1 homeodomains completely matched. In addition, these four homeodomains contain a unique His residue in the recognition helix of a helix-turn-helix DNA-binding motif. This His residue is not found in any of the previously published mammalian homeodomain sequences except mouse IPF1. 24 refs., 4 figs.

  18. Biomimetic Cloning of Quantum Observables

    NASA Astrophysics Data System (ADS)

    Alvarez-Rodriguez, U.; Sanz, M.; Lamata, L.; Solano, E.

    2014-05-01

    We propose a bio-inspired sequential quantum protocol for the cloning and preservation of the statistics associated to quantum observables of a given system. It combines the cloning of a set of commuting observables, permitted by the no-cloning and no-broadcasting theorems, with a controllable propagation of the initial state coherences to the subsequent generations. The protocol mimics the scenario in which an individual in an unknown quantum state copies and propagates its quantum information into an environment of blank qubits. Finally, we propose a realistic experimental implementation of this protocol in trapped ions.

  19. Biomimetic Cloning of Quantum Observables

    PubMed Central

    Alvarez-Rodriguez, U.; Sanz, M.; Lamata, L.; Solano, E.

    2014-01-01

    We propose a bio-inspired sequential quantum protocol for the cloning and preservation of the statistics associated to quantum observables of a given system. It combines the cloning of a set of commuting observables, permitted by the no-cloning and no-broadcasting theorems, with a controllable propagation of the initial state coherences to the subsequent generations. The protocol mimics the scenario in which an individual in an unknown quantum state copies and propagates its quantum information into an environment of blank qubits. Finally, we propose a realistic experimental implementation of this protocol in trapped ions. PMID:24809937

  20. 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.

  1. 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

  2. Mouse reproductive fitness is maintained up to an ambient temperature of 28℃ when housed in individually-ventilated cages.

    PubMed

    Helppi, J; Schreier, D; Naumann, R; Zierau, O

    2016-08-01

    Production of genetically-modified mice is strongly dependent on environmental conditions. Mice are commonly housed at 22℃, which is significantly lower than their thermoneutral zone. But, when given a choice, mice often seem to prefer higher ambient temperatures. In the current study we investigated the effect of higher ambient temperature on the production of transgenic mice, with emphasis on embryo and sperm yield and quality. Mice (C57BL/6JOlaHsd) were housed under four different ambient temperatures (22, 25, 28 and 30℃). Female mice were superovulated, and mated with males. As indicators for reproductive fitness, the success of the mating was observed, including embryo yield and quality, as well as sperm count, motility and progressivity. Female mice were found to produce high amounts of high quality embryos from 22 to 28℃. Sperm count dropped continuously from 22 to 30℃, but sperm motility and progressivity remained high from 22 to 28℃. We conclude that mice can be housed at significantly higher temperatures than is commonly recommended without compromising embryo production and quality, or sperm quality. These results could lead to fundamental changes in how mouse facilities are built and operated - especially in warmer climates whereby energy consumption and therefore costs could be significantly reduced.

  3. The spectral karyotype of L5178Y TK⁺/⁻ mouse lymphoma cells clone 3.7.2C and factors affecting mutant frequency at the thymidine kinase (tk) locus in the microtitre mouse lymphoma assay.

    PubMed

    Fellows, Mick D; McDermott, Angela; Clare, Katie R; Doherty, Ann; Aardema, Marilyn J

    2014-01-01

    There has been much discussion on acceptable spontaneous mutant frequencies in the mouse lymphoma assay (MLA). This culminated in the International Workshop on Genotoxicity Testing (IWGT) recommended control limits for the microtitre version of 50-170 mutants/10(6) viable cells, which has now been included in the draft Organization for Economic Co-Operation and Development guideline for assays investigating mammalian cell gene mutation at the tk locus. Some of the factors affecting mutant frequency have been investigated. It was shown that when culturing methotrexate cleansed TK⁺/⁻ cells, a spontaneous mutant frequency of ∼100 mutants/10⁶ viable cells was achieved after only 26 doublings. However, after further culturing for ∼6 months the spontaneous mutant frequency only gradually increased. Culturing for this time did not affect the karyotype of the cell in so much as the modal chromosome number remained stable. The spontaneous mutant frequency could effectively be manipulated by cleansing with various concentrations of methotrexate. The necessity for using appropriately heat-inactivated horse serum was confirmed. Finally, following treatment with 4-nitroquinoline-N-oxide, cells did not preferentially survive when plated at high cell densities (1.6 cells plus 2,000 feeder cells/well) versus cells at low density (1.6 cells/well). It was considered that these findings confirm that the dynamics of spontaneous mutant formation in the MLA are complex. However, the karyotype of L5178Y cells is remarkably stable and assuming investigators are using cells with appropriate provenance and good culturing technique, it is clear that the IWGT recommendations are achievable.

  4. Expression profiling in mouse fetal thymus reveals clusters of coordinately expressed genes that mark individual stages of T-cell ontogeny.

    PubMed

    Ramialison, Mirana; Mohr, Elodie; Nal, Béatrice; Saboul, Thierry; Carrier, Alice; Tagett, Rebecca; Granjeaud, Samuel; Nguyen, Catherine; Gautheret, Daniel; Jordan, Bertrand R; Ferrier, Pierre

    2002-10-01

    To search for genes that participate in regulatory networks sustaining mouse embryonic T-cell development, we have performed expression profiling using nylon macroarrays. Labeled samples representative of individual developmental stages were utilized, taking advantage of cell homogeneity during early thymus ontogeny. cDNAs revealing differential expression were further selected using labeled samples derived from lymphoid versus non-lymphoid tissues, and from mutant thymi exhibiting T-cell developmental defects. We thus identified clusters of coexpressed genes during T-cell embryogenesis and characterized their sequences through bioinformatics. We compare our results with those from other profiling analyses in the immune system, and discuss their implications for the definition of genes whose products are involved in T-cell development.

  5. Group and Individual Regulation of Physiology and Behavior: A Behavioral, Thermographic, and Acoustic Study of Mouse Development

    PubMed Central

    Harshaw, Christopher; Alberts, Jeffrey R.

    2012-01-01

    The traditional approach to the study of thermoregulation in young animals focuses on the regulatory capacities of individuals, which, for multiparous species, risks ignoring critical aspects of the early developmental niche. Here, we examined the ontogeny of regulatory behavior in C57BL/6 mice, employing simultaneous behavioral, thermographic, and acoustic measures of groups and individual pups. Litters of mice were placed in a chamber on Postnatal Day (PND) 2, 4, or 8, in which the ambient temperature (Ta) gradually cycled (over 50 min) from warm (36.5°C) to cool (20°C) and back (to 36.5°C). Litters of all three ages displayed “group regulatory behavior,” whereby group size varied with changes in Ta. This coupling, moreover, improved with age. Infrared thermography was used to monitor skin temperature of pups’ interscapular (TIS) and rump (Trump) areas, and to estimate brown adipose tissue (BAT) thermogenesis (TIS − Trump) in PND4 and PND8 individuals and huddles. Huddling was found to significantly reduce heat loss in pups subject to thermal challenge as groups, compared to pups challenged as individuals. Additionally, females were found to display significantly warmer TIS and Trump values than male huddlemates. Huddling did not have a consistent effect on emissions of ultrasonic vocalizations, which were generally correlated with ambient temperature and BAT activation. Our results indicate that simultaneous measures of behavioral and physiological response to cooling may prove useful for a variety of applications, including the phenotyping of social dysfunction. PMID:22580514

  6. Group and individual regulation of physiology and behavior: a behavioral, thermographic, and acoustic study of mouse development.

    PubMed

    Harshaw, Christopher; Alberts, Jeffrey R

    2012-07-16

    The traditional approach to the study of thermoregulation in young animals focuses on the regulatory capacities of individuals, which, for multiparous species, risks ignoring critical aspects of the early developmental niche. Here, we examined the ontogeny of regulatory behavior in C57BL/6 mice, employing simultaneous behavioral, thermographic, and acoustic measures of groups and individual pups. Litters of mice were placed in a chamber on Postnatal Day (PND) 2, 4, or 8, in which the ambient temperature (T(a)) gradually cycled (over 50 min) from warm (36.5°C) to cool (20°C) and back (to 36.5°C). Litters of all three ages displayed "group regulatory behavior," whereby group size varied with changes in T(a). This coupling, moreover, improved with age. Infrared thermography was used to monitor skin temperature of pups' interscapular (T(IS)) and rump (T(rump)) areas, and to estimate brown adipose tissue (BAT) thermogenesis (T(IS)-T(rump)) in PND4 and PND8 individuals and huddles. Huddling was found to significantly reduce heat loss in pups subject to thermal challenge as groups, compared to pups challenged as individuals. Additionally, females were found to display significantly warmer T(IS) and T(rump) values than male huddlemates. Huddling did not have a consistent effect on emissions of ultrasonic vocalizations, which were generally correlated with ambient temperature and BAT activation. Our results indicate that simultaneous measures of behavioral and physiological response to cooling may prove useful for a variety of applications, including the phenotyping of social dysfunction. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Context-Dependent Sensitivity to Mutations Disrupting the Structural Integrity of Individual EGF Repeats in the Mouse Notch Ligand DLL1.

    PubMed

    Schuster-Gossler, Karin; Cordes, Ralf; Müller, Julia; Geffers, Insa; Delany-Heiken, Patricia; Taft, Manuel; Preller, Matthias; Gossler, Achim

    2016-03-01

    The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans.

  8. Context-Dependent Sensitivity to Mutations Disrupting the Structural Integrity of Individual EGF Repeats in the Mouse Notch Ligand DLL1

    PubMed Central

    Schuster-Gossler, Karin; Cordes, Ralf; Müller, Julia; Geffers, Insa; Delany-Heiken, Patricia; Taft, Manuel; Preller, Matthias; Gossler, Achim

    2016-01-01

    The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans. PMID:26801181

  9. 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

  10. 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.

  11. [Eugenics and human cloning].

    PubMed

    Boloz, W

    2001-01-01

    Because of legislative bans there are still no reports of human cloning. However eager public debate is currently running, concerning medical, legal, social and ethical aspects of human cloning. Arguments for and against human cloning are presented. An important argument against cloning is the danger of eugenic tendencies connected with cloning, which could lead to genetic discrimination.

  12. Cloning and expression analysis in mature individuals of two chicken type-II GnRH (cGnRH-II) genes in common carp (Cyprinus carpio).

    PubMed

    Li, Shuangfei; Hu, Wei; Wang, Yaping; Zhu, Zuoyan

    2004-08-01

    Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide family, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II precursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplication hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the expression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.

  13. 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.

  14. Sham Surgery and Inter-Individual Heterogeneity Are Major Determinants of Monocyte Subset Kinetics in a Mouse Model of Myocardial Infarction

    PubMed Central

    Hoffmann, Jedrzej; Ospelt, Manuel; Troidl, Christian; Voss, Sandra; Liebetrau, Christoph; Kim, Won-Keun; Rolf, Andreas; Wietelmann, Astrid; Braun, Thomas; Troidl, Kerstin; Sadayappan, Sakthivel; Barefield, David; Hamm, Christian; Nef, Holger; Möllmann, Helge

    2014-01-01

    Aims Mouse models of myocardial infarction (MI) are commonly used to explore the pathophysiological role of the monocytic response in myocardial injury and to develop translational strategies. However, no study thus far has examined the potential impact of inter-individual variability and sham surgical procedures on monocyte subset kinetics after experimental MI in mice. Our goal was to investigate determinants of systemic myeloid cell subset shifts in C57BL/6 mice following MI by developing a protocol for sequential extensive flow cytometry (FCM). Methods and Results Following cross-sectional multiplex FCM analysis we provide for the first time a detailed description of absolute quantities, relative subset composition, and biological variability of circulating classical, intermediate, and non-classical monocyte subsets in C57BL/6 mice. By using intra-individual longitudinal measurements after MI induction, a time course of classical and non-classical monocytosis was recorded. This approach disclosed a significant reduction of monocyte subset dispersion across all investigated time points following MI. We found that in the current invasive model of chronic MI the global pattern of systemic monocyte kinetics is mainly determined by a nonspecific inflammatory response to sham surgery and not by the extent of myocardial injury. Conclusions Application of sequential multiplexed FCM may help to reduce the impact of biological variability in C57BL/6 mice. Furthermore, the confounding influence of sham surgical procedures should always be considered when measuring monocyte subset kinetics in a murine model of MI. PMID:24893162

  15. 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

  16. 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.

  17. [Cloning: applications in humans 2. Ethical considerations].

    PubMed

    de Wert, G M; Geraedts, J P

    2001-05-01

    Reproductive cloning in adults/children evokes unfavourable reactions. Direct objections are that cloning is unnatural, that it affects human dignity and violates the individual's right to genetic uniqueness. Consequential objections concern unjustified health risks for the progeny, unjustified psychosocial risks for the clone child and the risk of cloning for eugenetic purposes. There is consensus that reproductive cloning of existing persons is unjustify as yet because of the health risks for the offspring. Reproductive cloning of embryos is possible by means of nucleus transplantation and of embryo splitting. The ethical analysis of reproductive cloning of embryos depends on the purposes and applications. At least some of the moral objections against cloning of adults/children are not or not completely applicable to reproductive cloning of embryos. Conditions to be put to reproductive cloning of embryos are efficacy, safety and, at least for the time being, avoidance of asynchrony in transferring identical embryos. The ethical aspects of its application in the context of genetical reproductive techniques must be evaluated separately. Therapeutic cloning may be acceptable if alternatives are lacking.

  18. [Cloning: applications in humans. II. Ethical considerations].

    PubMed

    de Wert, G M; Geraedts, J P

    2000-05-13

    Reproductive cloning in adults/children evokes unfavourable reactions. Direct objections are that cloning is unnatural, that it affects human dignity and violates the individual's right to genetic uniqueness. Consequential objections concern unjustified health risks for the progeny, unjustified psychosocial risks for the clone child and the risk of cloning for eugenetic purposes. There is consensus that reproductive cloning of existing persons is unjustifiable as yet because of the health risks for the offspring. Reproductive cloning of embryos is possible by means of nucleus transplantation and of embryo splitting. The ethical analysis of reproductive cloning of embryos depends on the purposes and applications. At least some of the moral objections against cloning of adults/children are not or not completely applicable to reproductive cloning of embryos. Conditions to be put to reproductive cloning of embryos are efficacy, safety and, at least for the time being, avoidance of asynchrony in transferring identical embryos. The ethical aspects of its application in the context of genetical reproductive techniques must be evaluated separately. Therapeutic cloning may be acceptable if alternatives are lacking.

  19. 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.

  20. Exocytosis elicited by action potentials and voltage-clamp calcium currents in individual mouse pancreatic B-cells.

    PubMed Central

    Ammälä, C; Eliasson, L; Bokvist, K; Larsson, O; Ashcroft, F M; Rorsman, P

    1993-01-01

    1. Measurements of membrane capacitance, as an indicator of exocytosis, and intracellular Ca2+ concentration ([Ca2+]i) were used to determine the Ca2+ dependence of secretion in single pancreatic B-cells. 2. Exocytosis was dependent on a rise in [Ca2+]i and could be evoked by activation of voltage-dependent Ca2+ currents. The threshold for depolarization-induced release was 0.5 microM [Ca2+]i. Once the [Ca2+]i threshold was exceeded, exocytosis was rapidly (< 50 ms) initiated. When individual pulses were applied, exocytosis stopped immediately upon repolarization and the Ca2+ channels closed, although [Ca2+]i remained elevated for several seconds. 3. During repetitive stimulation (1 Hz), when [Ca2+]i attained micromolar levels, exocytosis also took place during the interpulse intervals albeit at a slower rate than during the depolarizations. 4. Exocytosis could be initiated by simulated action potentials. Whereas a single action potential only produced a small capacitance increase, and in some cells even failed to stimulate release, larger and more consistent responses were obtained with > or = four action potentials. 5. Comparison of the rates of exocytosis measured in response to depolarization, mobilization of Ca2+ from intracellular stores or infusion of Ca2+ through the patch pipette suggests that [Ca2+]i at the secretory sites attains a concentration of several micromolar. This is much higher than the average [Ca2+]i detected by microfluorimetry suggesting the existence of steep spatial gradients of [Ca2+]i within the B-cell. 6. Inclusion of inhibitors of Ca2+/calmodulin-dependent protein kinase II in the intracellular solution reduced the depolarization-induced exocytotic responses suggesting this enzyme may be involved in the coupling between elevation of [Ca2+]i to stimulation of the secretory machinery. 7. The size of the unitary exocytotic event was 2 fF, corresponding to a secretory granule diameter of 250 nm. 8. Over short periods, exocytosis may be

  1. Differential regulation by agonist and phorbol ester of cloned m1 and m2 muscarinic acetylcholine receptors in mouse Y1 adrenal cells and in Y1 cells deficient in cAMP-dependent protein kinase

    SciTech Connect

    Scherer, N.M.; Nathanson, N.M. )

    1990-09-11

    Cloned muscarinic acetylcholine m1 and m2 receptors were expressed in stably transfected mouse Y1 adrenal cells and in a variant Y1 line, Kin-8, which is deficient in cAMP-dependent protein kinase activity (PKA{sup {minus}}). m1 and m2 receptors were rapidly internalized following exposure of transfected PKA{sup +} or PKA{sup {minus}} cells to the muscarinic agonist carbachol. Thus, agonist-dependent internalization of m1 and m2 did not require PKA activity. A differential effect of PKA on regulation by agonist of the m2 receptor, but not the m1 receptor, was unmasked in PKA{sup {minus}} cells. These data indicate that the basal activity of PKA may modulate the agonist-dependent internalization of the m2 receptor, but not the m1 receptor. The internalization of the m1 and m2 receptors in both PKA{sup +} and PKA{sup {minus}} cells was accompanied by desensitization of functional responses. Exposure of PKA{sup +} cells to 10{sup {minus}7} M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, resulted in a 30 {plus minus} 9% decrease in the number of m1 receptors on the cell surface. The m2 receptor was not internalized following treatment of either PKA{sup +} or PKA{sup {minus}} cells with PMA. Thus, the m1 and m2 receptors show differential sensitivity to internalization by PMA. Agonist-dependent internalization of the m1 receptor appeared to be independent of activation of PKC because (1) agonist-dependent internalization of m1 was not attenuated in PKA{sup {minus}} cells, (2) the rate and extent of internalization of m1 in cells exposed to PMA were less than those in cells exposed to agonist, and (3) treatment of cells with concanavalin A selectivity blocked internalization of m1 in cells exposed to PMA, but not to agonist. The effects of agonist and PMA on receptor internalization were not additive. Exposure of PKA{sup +} or PKA{sup {minus}} cells to PMA reduced the magnitude of pilocarpine-stimulated PI hydrolysis by about 25%.

  2. 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.

  3. 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.

  4. 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/.

  5. 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…

  6. 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…

  7. 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

  8. 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.

  9. A new mouse model for infantile neuroaxonal dystrophy, inad mouse, maps to mouse chromosome 1.

    PubMed

    Matsushima, Yoshibumi; Kikuchi, Tateki; Kikuchi, Hisae; Ichihara, Nobutsune; Ishikawa, Akira; Ishijima, Yasushi; Tachibana, Masayoshi

    2005-02-01

    Infantile neuroaxonal dystrophy (INAD) is a rare autosomal recessive hereditary neurodegenerative disease of humans. So far, no responsible gene has been cloned or mapped to any chromosome. For chromosome mapping and positional cloning of the responsible gene, establishment of an animal model would be useful. Here we describe a new mouse model for INAD, named inad mouse. In this mouse, the phenotype is inherited in an autosomal recessive manner, symptoms occur in the infantile period, and the mouse dies before sexual maturity. Axonal dystrophic change appearing as spheroid bodies in central and peripheral nervous system was observed. These features more closely resembled human INAD than did those of the gad mouse, the traditional mouse model for INAD. Linkage analysis linked the inad gene to mouse Chromosome 1, with the highest LOD score (=128.6) at the D1Mit45 marker, and haplotype study localized the inad gene to a 7.5-Mb region between D1Mit84 and D1Mit25. In this linkage area some 60 genes exist: Mutation of one of these 60 genes is likely responsible for the inad mouse phenotype. Our preliminary mutation analysis in 15 genes examining the nucleotide sequence of exons of these genes did not find any sequence difference between inad mouse and C57BL/6 mouse.

  10. Developing a framework for predicting upper extremity muscle activities, postures, velocities, and accelerations during computer use: the effect of keyboard use, mouse use, and individual factors on physical exposures.

    PubMed

    Bruno Garza, Jennifer L; Catalano, Paul J; Katz, Jeffrey N; Huysmans, Maaike A; Dennerlein, Jack T

    2012-01-01

    Prediction models were developed based on keyboard and mouse use in combination with individual factors that could be used to predict median upper extremity muscle activities, postures, velocities, and accelerations experienced during computer use. In the laboratory, 25 participants performed five simulated computer trials with different amounts of keyboard and mouse use ranging from a highly keyboard-intensive trial to a highly mouse-intensive trial. During each trial, muscle activity and postures of the shoulder and wrist and velocities and accelerations of the wrists, along with percentage keyboard and mouse use, were measured. Four individual factors (hand length, shoulder width, age, and gender) were also measured on the day of data collection. Percentage keyboard and mouse use explained a large amount of the variability in wrist velocities and accelerations. Although hand length, shoulder width, and age were each significant predictors of at least one median muscle activity, posture, velocity, or acceleration exposure, these individual factors explained very little variability in addition to percentage keyboard and mouse use in any of the physical exposures investigated. The amounts of variability explained for models predicting median wrist velocities and accelerations ranged from 75 to 84% but were much lower for median muscle activities and postures (0-50%). RMS errors ranged between 8 to 13% of the range observed. While the predictions for wrist velocities and accelerations may be able to be used to improve exposure assessment for future epidemiologic studies, more research is needed to identify other factors that may improve the predictions for muscle activities and postures.

  11. Metabolomic phenotyping of a cloned pig model

    PubMed Central

    2011-01-01

    Background Pigs are widely used as models for human physiological changes in intervention studies, because of the close resemblance between human and porcine physiology and the high degree of experimental control when using an animal model. Cloned animals have, in principle, identical genotypes and possibly also phenotypes and this offer an extra level of experimental control which could possibly make them a desirable tool for intervention studies. Therefore, in the present study, we address how phenotype and phenotypic variation is affected by cloning, through comparison of cloned pigs and normal outbred pigs. Results The metabolic phenotype of cloned pigs (n = 5) was for the first time elucidated by nuclear magnetic resonance (NMR)-based metabolomic analysis of multiple bio-fluids including plasma, bile and urine. The metabolic phenotype of the cloned pigs was compared with normal outbred pigs (n = 6) by multivariate data analysis, which revealed differences in the metabolic phenotypes. Plasma lactate was higher for cloned vs control pigs, while multiple metabolites were altered in the bile. However a lower inter-individual variability for cloned pigs compared with control pigs could not be established. Conclusions From the present study we conclude that cloned and normal outbred pigs are phenotypically different. However, it cannot be concluded that the use of cloned animals will reduce the inter-individual variation in intervention studies, though this is based on a limited number of animals. PMID:21859467

  12. A physical map of the mouse genome.

    PubMed

    Gregory, Simon G; Sekhon, Mandeep; Schein, Jacqueline; Zhao, Shaying; Osoegawa, Kazutoyo; Scott, Carol E; Evans, Richard S; Burridge, Paul W; Cox, Tony V; Fox, Christopher A; Hutton, Richard D; Mullenger, Ian R; Phillips, Kimbly J; Smith, James; Stalker, Jim; Threadgold, Glen J; Birney, Ewan; Wylie, Kristine; Chinwalla, Asif; Wallis, John; Hillier, LaDeana; Carter, Jason; Gaige, Tony; Jaeger, Sara; Kremitzki, Colin; Layman, Dan; Maas, Jason; McGrane, Rebecca; Mead, Kelly; Walker, Rebecca; Jones, Steven; Smith, Michael; Asano, Jennifer; Bosdet, Ian; Chan, Susanna; Chittaranjan, Suganthi; Chiu, Readman; Fjell, Chris; Fuhrmann, Dan; Girn, Noreen; Gray, Catharine; Guin, Ran; Hsiao, Letticia; Krzywinski, Martin; Kutsche, Reta; Lee, Soo Sen; Mathewson, Carrie; McLeavy, Candice; Messervier, Steve; Ness, Steven; Pandoh, Pawan; Prabhu, Anna-Liisa; Saeedi, Parvaneh; Smailus, Duane; Spence, Lorraine; Stott, Jeff; Taylor, Sheryl; Terpstra, Wesley; Tsai, Miranda; Vardy, Jill; Wye, Natasja; Yang, George; Shatsman, Sofiya; Ayodeji, Bola; Geer, Keita; Tsegaye, Getahun; Shvartsbeyn, Alla; Gebregeorgis, Elizabeth; Krol, Margaret; Russell, Daniel; Overton, Larry; Malek, Joel A; Holmes, Mike; Heaney, Michael; Shetty, Jyoti; Feldblyum, Tamara; Nierman, William C; Catanese, Joseph J; Hubbard, Tim; Waterston, Robert H; Rogers, Jane; de Jong, Pieter J; Fraser, Claire M; Marra, Marco; McPherson, John D; Bentley, David R

    2002-08-15

    A physical map of a genome is an essential guide for navigation, allowing the location of any gene or other landmark in the chromosomal DNA. We have constructed a physical map of the mouse genome that contains 296 contigs of overlapping bacterial clones and 16,992 unique markers. The mouse contigs were aligned to the human genome sequence on the basis of 51,486 homology matches, thus enabling use of the conserved synteny (correspondence between chromosome blocks) of the two genomes to accelerate construction of the mouse map. The map provides a framework for assembly of whole-genome shotgun sequence data, and a tile path of clones for generation of the reference sequence. Definition of the human-mouse alignment at this level of resolution enables identification of a mouse clone that corresponds to almost any position in the human genome. The human sequence may be used to facilitate construction of other mammalian genome maps using the same strategy.

  13. The effect of superovulation on the contributions of individual blastomeres from 2-cell stage CF1 mouse embryos to the blastocyst.

    PubMed

    Katayama, Mika; Roberts, R Michael

    2010-01-01

    It remains controversial whether blastomeres of 2-cell stage mouse embryos show bias in their contribution to the blastocyst and whether there is any effect of superovulation. Two-cell stage embryos from CF1 mice were derived by either natural breeding (N) or superovulation (S) and cultured in vitro. At blastocyst, inner cell mass and trophectoderm were distinguished by Cdx2 and Oct4 immunostaining. A fluorescent dye (CM-Dil) was also used to tag individual blastomeres at the 2-cell stage, and the descendant cells identified by their red fluorescence. S and N embryos developed to blastocyst at the same rate and contained a similar number of cells. However, with S embryos, the descendants of the blastomere labeled with CM-DiI contributed predominantly to either the embryonic or abembryonic pole about 70% of the time, whereas most N embryos displayed random patterning, with no restriction to one or other of the poles. In S-embryos, but not N-embryos, the leading blastomere at second cleavage contributed preferentially to the embryonic pole of the blastocyst and the lagging blastomere to the abembryonic pole and hence mural trophectoderm. In addition, a tetrahedral rather than a flat morphology at the 4-cell stage of S-embryos was strongly biased to displaying the embryonic/abembryonic pattern at blastocyst. In contrast, S-embryos lacking a zona pellucida resembled N embryos in their patterning. In CF1 mice, superovulation has little effect on development to blastocyst, but enforces a greater degree of lineage restriction than natural breeding, most likely through constraints imposed by the zona pellucida.

  14. Immune-modulating effects in mouse dendritic cells of lactobacilli and bifidobacteria isolated from individuals following omnivorous, vegetarian and vegan diets.

    PubMed

    Luongo, Diomira; Treppiccione, Lucia; Sorrentino, Alida; Ferrocino, Ilario; Turroni, Silvia; Gatti, Monica; Di Cagno, Raffaella; Sanz, Yolanda; Rossi, Mauro

    2017-09-01

    Lactobacilli and bifidobacteria play a primary role in modulation of gut immunity. By considering that microbiota composition depends on various factors, including diet, we asked whether functional differences could characterize faecal populations of lactobacilli and bifidobacteria isolated from individuals with different dietary habits. 155 healthy volunteers who followed omnivorous, ovo-lacto-vegetarian or vegan diets were recruited at four Italian centres (Turin, Parma, Bologna and Bari). Faecal samples were collected; lactobacilli and bifidobacteria were isolated on selective media and their immunomodulatory activity was tested in mouse dendritic cells (DCs). Pre-incubation with lactobacilli increased LPS-induced expression of the maturation markers CD80 and CD86, whereas pre-incubation with bifidobacteria decreased such expression. Analysis of the cytokine profile indicated that strains of both genera induced down-regulation of IL-12 and up-regulation of IL-10, whereas expression of TNF-α was not modulated. Notably, analysis of anti-inflammatory potential (IL-10/IL-12 ratio) showed that lactobacilli evoked a greater anti-inflammatory effect than did bifidobacteria in the omnivorous group (P<0.05). We also found significantly reduced anti-inflammatory potential in the bacterial strains isolated from Bari's volunteers in comparison with those from the cognate groups from the other centres. In conclusion, lactobacilli and bifidobacteria showed a genus-specific ability of modulating in vitro innate immunity associated with a specific dietary habit. Furthermore, the geographical area had a significant impact on the anti-inflammatory potential of some components of faecal microbiota. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Dioxin-like activity of brominated dioxins as individual compounds or mixtures in in vitro reporter gene assays with rat and mouse hepatoma cell lines.

    PubMed

    Suzuki, G; Nakamura, M; Michinaka, C; Tue, N M; Handa, H; Takigami, H

    2017-10-01

    In vitro reporter gene assays detecting dioxin-like compounds have been developed and validated since the middle 1990's, and applied to the determination of dioxin-like activities in various samples for their risk management. Data on characterizing the potency of individual brominated dioxins and their activity in mixture with chlorinated dioxins are still limited on the cell-based assay. This study characterized the dioxin-like activities of the 32 brominated dioxins, such as polybrominated dibenzo-p-dioxins, polybrominated dibenzofurans (PBDFs), coplanar polybrominated biphenyls, mixed halogenated dibenzo-p-dioxins and dibenzofurans (PXDFs), as a sole component or in a mixture by DR-CALUX (dioxin-responsive chemically activated luciferase expression) using the rat hepatoma H4IIE cell line and XDS-CALUX (xenobiotic detection systems-chemically activated luciferase expression) assays using the mouse hepatoma H1L6.1 cell line. The 2,3,7,8-TCDD-relative potencies (REPs) of most of the brominated dioxins were within a factor of 10 of the WHO toxicity equivalency factor (WHO-TEF) for the chlorinated analogues. The REPs of a few PXDFs were an order of magnitude higher than the corresponding WHO-TEFs, indicating their toxicological importance. Results with reconstituted mixtures suggest that the activity of brominated and chlorinated dioxins in both CALUX assays was dose-additive. Thus, obtained results indicated the applicability of the CALUX assays as screening tools of brominated dioxins together with their chlorinated analogues. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. 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.

  17. [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.

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. Cloning in reproductive medicine.

    PubMed

    Illmensee, K

    2001-08-01

    This review article summarizes the historical development of mammalian cloning, presents current advances and presumed risk factors in the field of reproductive cloning, discusses possible clinical applications of therapeutic and diagnostic cloning and outlines prospective commercial trends in pharmaceutical cloning. Predictable progress in biotechnology and stem cell engineering should prove to be advantageous for patients' health and for novel benefits in reproductive and regenerative medicine.

  6. 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.

  7. 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.

  8. 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.

  9. Quick and clean cloning.

    PubMed

    Thieme, Frank; Marillonnet, Sylvestre

    2014-01-01

    Identification of unknown sequences that flank known sequences of interest requires PCR amplification of DNA fragments that contain the junction between the known and unknown flanking sequences. Since amplified products often contain a mixture of specific and nonspecific products, the quick and clean (QC) cloning procedure was developed to clone specific products only. QC cloning is a ligation-independent cloning procedure that relies on the exonuclease activity of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. A specific feature of QC cloning is the use of vectors that contain a sequence called catching sequence that allows cloning specific products only. QC cloning is performed by a one-pot incubation of insert and vector in the presence of T4 DNA polymerase at room temperature for 10 min followed by direct transformation of the incubation mix in chemo-competent Escherichia coli cells.

  10. 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

  11. 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.

  12. Nuclear transfer and cloning.

    PubMed

    Wolf, D P

    2001-10-01

    The use of nuclear transfer in human reproductive and therapeutic cloning is reviewed with attention on the origins of this technology from its evolution to the present. The successes and limitations of mammalian reproductive cloning are itemized. A case is made against the use of human reproductive cloning to reproduce an existing person, based on the unacceptable risks to the embryo, fetus, or newborn. However, support is extended for human therapeutic cloning involving the derivation and use of embryonic stem cells to treat human disease.

  13. [Cloning--ethical aspects].

    PubMed

    Munzarová, M

    2004-01-01

    Ethical problems related to cloning are discussed on three model situations: cloning of human beings (for example by utilizing the techniques of embryo splitting or nuclear transfer), use of embryonic cells in cloning techniques and cloning of nonembryonic cells. The first situation is strictly condemned, the second has been examined up present (it should be condemned as well) and the third is--under certain conditions--fully acceptable. The issue is discussed from the point of view of relevant Council of Europe documents as well.

  14. 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.

  15. 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.

  16. Tracking the Evolution of Code Clones

    NASA Astrophysics Data System (ADS)

    Bakota, Tibor

    It is believed by many academic and industrial experts, that source code cloning (copy&paste programming) represents a significant threat to maintainability in an evolving software system. The real threat does not lie in the existence of duplications, but the fears are in connection with their evolution. There exist an abundance of algorithms for finding code clones in one particular version of a software system, but eliminating or even evaluating these clones often seems hopeless, as there may exist several thousands of them. Tracking the evolution of individual clones might solve the problem, as it could help identifying the inconsistently changing duplications: the clones which are really dangerous at a particular moment. In this paper we present an approach for mapping code duplications from one particular version of the software to another one, based on a similarity distance function. For the suspicious evolution patterns we introduce the term of "clone smells". By defining the relevant categories of the possible evolution patterns, the proposed method also gives a clue about why the reported code fragments might be dangerous. In the case study, clone smells were extracted, evaluated, and manually categorized throughout many versions of the jEdit system. The findings suggest that roughly half of the reported smells refer to inconsistent changes in the code.

  17. [Worldviews and philosophical basis of human cloning].

    PubMed

    Lukowska, A T

    2001-01-01

    The article presents three standpoints on the question of moral permissibility of human cloning and shows the philosophical principles of it. 1. The moral consent to human cloning with the purposes of reproduction and therapy. The followers of human cloning refer to materialistic anthropology also to subjectivistic, relativistic and utilitarian ethics. 2. Those, who are adverse to human cloning with the purpose of reproduction, but they acquiesce to the so-called therapeutic cloning. They reject that human embryos and foetuses are individuals who come under protection of law. 3. Those, who reject human cloning for the purposes of reproduction and therapy alike. They assent to a personalistic anthropology and Christian ethics. A human being was created by God and human life begins at the moment of insemination. All three groups use various argumentation. The arguments for and against cloning are extracted from biology as well as psychology, philosophy, law and religion. The author of the article takes the last standpoint, but she does not see such arguments, that might convince the opposite parties to a suit.

  18. [On the problem of human cloning].

    PubMed

    Smorag, Z

    2001-01-01

    Somatic cell cloning technique in mammals is still not very efficient, but intensive efforts have been made to improve it. Considering the great biological affinity of humans and animals, the cloning technique can in the not too distant future be applied in human cloning and improved to the point of becoming safe. Even when we make such an assumption, I consider it irrational and dangerous to clone the human in order to make their copies (with human cloning for therapeutic purposes being another problem). Life, which is generated by the union of egg cell and spermatozoon is an unforeseeable combination of genetic possibilities, but at the same time it offers a unique chance for the human being, both as an individual and a species. The creation of life by genetic duplication of an already formed individual means a great reduction not only in the biological sense. Action like this is evidence of extreme egocentrism and totalitarian thinking, and its proponents should first answer the question whether they would consider cloning themselves. An answer in the affirmative would help to establish the underlying reasons for their approval.

  19. Cloning, killing, and identity.

    PubMed Central

    McMahan, J

    1999-01-01

    One potentially valuable use of cloning is to provide a source of tissues or organs for transplantation. The most important objection to this use of cloning is that a human clone would be the sort of entity that it would be seriously wrong to kill. I argue that entities of the sort that you and I essentially are do not begin to exist until around the seventh month of fetal gestation. Therefore to kill a clone prior to that would not be to kill someone like you or me but would be only to prevent one of us from existing. And even after one of us begins to exist, the objections to killing it remain comparatively weak until its psychological capacities reach a certain level of maturation. These claims support the permissibility of killing a clone during the early stages of its development in order to use its organs for transplantation. PMID:10226909

  20. A Combinatory Antibody–Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries

    PubMed Central

    Jansson, Bo; Stuhr-Hansen, Nicolai; Kovács, András; Welinder, Charlotte

    2016-01-01

    We have developed a combinatory antibody–antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a “spot-on-spot” print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation. PMID:28002485

  1. 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.

  2. 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.

  3. [Human cloning or cannibalism].

    PubMed

    Sokolowski, L M

    2001-01-01

    In this article I develop the idea presented in my previous work that human cloning would be of little practical use since almost any aim that one would like to attain by multiple cloning of a concrete man or a group of people, are unattainable or it might be achieved by easier, cheaper and more efficient traditional methods. For this reason cloning of a man is unlikely to occur on a larger scale and only few people will decide to clone themselves. In this sense no social effects of human cloning will be disastrous for the human population. Yet investigations in human genetics are very important since they may provide medical applications far more important than human cloning. It is argued that the main trend of modern medicine: organ transplantation from an alien donor, will become socially dangerous in near future since the number of donors will be drastically smaller than the number of potential patients waiting for transplantations. This in turn may cause social conflicts and a form of medical cannibalism may arise. These problems and conflicts will be avoided if organ transplantation from an alien donor is replaced by organ cloning, i.e. by transplanting an organ developed from the patient.

  4. Evaluation and Optimization of Blood Micro-Sampling Methods: Serial Sampling in a Cross-Over Design from an Individual Mouse.

    PubMed

    Patel, Nita J; Wickremsinhe, Enaksha; Hui, Yu-Hua; Barr, Alexandar; Masterson, Nicholas; Ruterbories, Kenneth; Weller, Jennifer; Hanes, Jennifer; Kern, Tom; Perkins, Everett

    Current practices applied to mouse pharmacokinetic (PK) studies often use large numbers of animals with sporadic or composite sampling that inadequately describe PK profiles.  The purpose of this work was to evaluate and optimize blood microsampling techniques coupled with dried blood spot (DBS) and LC-MS/MS analysis to generate reliable PK data in mice.  In addition, the feasibility of cross-over designs was assessed and recommendations are presented. The work describes a comprehensive evaluation of five blood microsampling techniques (tail clip, tail vein with needle hub, submandibular, retro-orbital, and saphenous bleeding) in CD-1 mice.  The feasibility of blood sampling was evaluated based on animal observations, ease of bleeding, and ability to collect serial samples.  Methotrexate, gemfibrozil and glipizide were used as test compounds and were dosed either orally or intravenously, followed by DBS collection and LC-MS/MS analysis to compare PK with various bleeding methods. Submandibular and retro-orbital methods that required non-serial blood collections did not allow for inter-animal variability assessments and resulted in poorly described absorption and distribution kinetics.  The submandibular and tail vein with needle-hub methods were the least favorable from a technical feasibility perspective.  Serial bleeding was possible with cannulated animals or saphenous bleeding in non-cannulated animals.   Of the methods that allowed serial sampling, the saphenous method when executed as described in this report, was most practical, reproducible and provided for assessment of inter-animal variability.  It enabled the collection of complete exposure profiles from a single mouse and the conduct of an intravenous/oral cross-over study design.  This methodology can be used routinely, it promotes the 3Rs principles by achieving reductions in the number of animals used, decreased restraints and animal stress, and improved the quality of data obtained in mouse

  5. Initial steps for antibody fragment cloning from hybridoma cells

    SciTech Connect

    Schlager, J.J.; Clark, J.H.; Legere, R.H.; Courtney, B.C.; Brecht, K.M.

    1993-05-13

    Our present research has focused on the production, isolation and cloning of analytically active mouse antibody fragments (Fab) which are capable of accelerating organophosphorus acid fluoride hydrolysis. As an initial part of this effort, mouse hybridoma cells were produced to isolate a catalytic immunoglobulin for incorporation as a positive control in Fab cloning experiments. Mice were inoculated biweekly with the organophosphorus transition state analog (TSA) conjugated to porcine thyroglobulin (PTG), N-4(PTG-succinyl) 2(4-amino-3,3-dimethyl-2-butoxy)2-menthoxy(l,3,2-dioxa-4,5(di-tert- butyl)) benzophosphol, until exhibiting hyperimmune polyclonal binding sera. A 50% polyethylene glycol fusion was performed between splenocytes from a PTG-menthoxy inoculated Balb/c mouse and op2 myeloma cells. From the most successful fusion to date, 850/1344 wells produced visible cell clones and supernatants from 88 wells exhibited binding activity in ELISA to bovine albumin-menthoxy TSA. Supernatants from 10 of these same parental wells exhibited catalytic activity toward the soman derivative 4-nitrophenyl (1,2,2-trimethyl)propyl methyl-phosphonate (4-NPMP). The expressed catalytic immunoglobulin mRNA will be cloned into either the lambda or M13 vector systems for optimizing the binding and catalytic activity screening of mouse splenic Fab immunoglobulin libraries.

  6. Murine T-cell clones against Entamoeba histolytica: in vivo and in vitro characterization.

    PubMed Central

    Denis, M; Chadee, K

    1989-01-01

    Eleven T-cell clones were raised from the spleens of BALB/c mice hyperimmunized against a crude soluble extract of Entamoeba histolytica trophozoites. Seven clones were of the Lyt-1+, and four of the Lyt-23+ phenotype. All clones proliferated in the presence of E. histolytica antigens but not to a purified protein derivative; five clones proliferated to a crude extract of the E. histolytica-like Laredo amoebae. Ten clones secreted T-cell growth factors in response to E. histolytica antigens. Two clones (Lyt-23+) mediated direct lymphocytotoxicity (73% and 86%) against amoebic trophozoites that was inhibited with rabbit anti-mouse TNF-alpha. Supernatants of five of the clones (all Lyt-1+) activated mouse peritoneal macrophages (Mphi) to kill E. histolytica trophozoites in vitro, seemingly independent of secreted reactive oxygen intermediates (O2- and H2O2) in the case of three clones supernatants. All of the clones that were activating Mphi to kill amoeba in vitro also mediated a local DTH reaction in mouse footpad. Our results demonstrate direct lymphocyte cytotoxicity via a cytolytic molecule antigenically related to TNF-alpha and lymphokines activating Mphi for amoebic killing by oxidative and non-oxidative mechanisms, the latter process mediated by a macrophage-activating factor (MAF) distinct from interferon-gamma (IFN-gamma). PMID:15493266

  7. Targeted Genomic Disruption of H-ras and N-ras, Individually or in Combination, Reveals the Dispensability of Both Loci for Mouse Growth and Development

    PubMed Central

    Esteban, Luis M.; Vicario-Abejón, Carlos; Fernández-Salguero, Pedro; Fernández-Medarde, Alberto; Swaminathan, Nalini; Yienger, Kate; Lopez, Eva; Malumbres, Marcos; McKay, Ron; Ward, Jerrold M.; Pellicer, Angel; Santos, Eugenio

    2001-01-01

    Mammalian cells harbor three highly homologous and widely expressed members of the ras family (H-ras, N-ras, and K-ras), but it remains unclear whether they play specific or overlapping cellular roles. To gain insight into such functional roles, here we generated and analyzed H-ras null mutant mice, which were then also bred with N-ras knockout animals to ascertain the viability and properties of potential double null mutations in both loci. Mating among heterozygous H-ras+/− mice produced H-ras−/− offspring with a normal Mendelian pattern of inheritance, indicating that the loss of H-ras did not interfere with embryonic and fetal viability in the uterus. Homozygous mutant H-ras−/− mice reached sexual maturity at the same age as their littermates, and both males and females were fertile. Characterization of lymphocyte subsets in the spleen and thymus showed no significant differences between wild-type and H-ras−/− mice. Analysis of neuronal markers in the brains of knockout and wild-type H-ras mice showed that disruption of this locus did not impair or alter neuronal development. Breeding between our H-ras mutant animals and previously available N-ras null mutants gave rise to viable double knockout (H-ras−/−/N-ras−/−) offspring expressing only K-ras genes which grew normally, were fertile, and did not show any obvious phenotype. Interestingly, however, lower-than-expected numbers of adult, double knockout animals were consistently obtained in Mendelian crosses between heterozygous N-ras/H-ras mice. Our results indicate that, as for N-ras, H-ras gene function is dispensable for normal mouse development, growth, fertility, and neuronal development. Additionally, of the three ras genes, K-ras appears to be not only essential but also sufficient for normal mouse development. PMID:11238881

  8. 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

  9. Effects of Donor Fibroblast Cell Type and Transferred Cloned Embryo Number on the Efficiency of Pig Cloning

    PubMed Central

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

    2013-01-01

    Abstract 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

  10. 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

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

    PubMed

    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.

  12. Cloning the human SUMO1 promoter.

    PubMed

    Nanos-Webb, Angela; Deyrieux, Adeline; Bian, Xue-lin; Rosas-Acosta, Germán; Wilson, Van G

    2010-03-01

    Regulation of the sumoylation system at the level of gene expression has not yet been explored. To begin to define transcriptional regulatory features, the promoter region for the SUMO1 gene was cloned from human genomic DNA and characterized. Initially, a 532 base pair fragment upstream of and including the predicted SUMO1 transcription start site (TSS) was cloned and shown to possess promoter activity. Subsequent deletion analysis showed that a smaller fragment containing 158 bp upstream of the TSS region exhibited basal promoter activity in both human and rodent cell lines. Within this basal promoter fragment, there were predicted binding sites for numerous transcription factors, including the nude mouse gene product, Whn (FoxN1). Electrophoretic mobility shift assays showed that Whn could bind to an ACGC motif adjacent to the TSR, and in transfection studies Whn stimulated a 3-fold increase in transcription from this cloned promoter in keratinocytes (HaCaT cells). Mutation of the ACGC motif abrogated both Whn binding and transcriptional activation, indicating that the Whn effect is likely due to direct interaction with this promoter element. Consistent with these observations on the cloned promoter region, Whn also modestly stimulated transcription from the endogenous, genomic SUMO1 promoter in HaCaT cells, consistent with Whn potentially playing a regulatory role for SUMO1 transcription in keratinocytes.

  13. 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)

  14. 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)

  15. Statement on Human Cloning

    MedlinePlus

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

  16. Statement on Human Cloning

    MedlinePlus

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

  17. Much ado about cloning in the public square.

    PubMed

    McLean, M R

    2001-01-01

    The dawnings of the age of human cloning and genetics is shaping lives, ideologies, and social structures. How will we--as individuals and as communities--respond to the possibilities, challenges, and changes of the clone age? This essay invites engagement in communal moral deliberation through broadening conversations about serious matters, including human cloning. A framework that includes important moral markers for significant "kitchen table talk" is offered. Clone age justice is also discussed. Attention is paid to the renewed role of religious voices in the public square.

  18. 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.

  19. 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.

  20. Development of an in vitro cloning method for Cowdria ruminantium.

    PubMed Central

    Perez, J M; Martinez, D; Debus, A; Sheikboudou, C; Bensaid, A

    1997-01-01

    Cowdria ruminantium is a tick-borne rickettsia which causes severe disease in ruminants. All studies with C. ruminantium reported so far were carried out with stocks consisting of infective blood collected from reacting animals or from the same stocks propagated in vitro. Cloned isolates are needed to conduct studies on immune response of the host, on genetic diversity of the parasite, and on mechanisms of attenuation and the development of vaccines. A method of cloning based on the particular chlamydia life cycle of Cowdria was developed. Instead of cloning extracellular elementary bodies, it appeared more convenient to clone endothelial cells infected by one morula resulting from the infection of the cell by one elementary body of Cowdria. Two hundred and sixteen clones were obtained by limiting dilution of infected cells. The method was experimentally validated by comparing randomly amplified polymorphic DNA fingerprints from individual clones obtained from endothelial cell cultures coinfected with two different stocks of C. ruminantium. PMID:9302217

  1. [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.

  2. 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

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

    PubMed

    Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

    2013-01-05

    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.

  4. 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.

  5. A comparative analysis of shotgun-cloning and tagged-random amplification-cloning of chromatin immunoprecipitation-isolated genome fragments.

    PubMed

    White, Robert B; Ziman, Melanie R

    2006-07-28

    The cloning of transcription factor antibody-immunoprecipitated genomic fragments from chromatin immunoprecipitation (ChIP) experiments is a technically challenging procedure, especially when the input genomic DNA is isolated from whole tissues (in vivo) rather than cultured cells. Here we adapt a technique known as Tagged-Random PCR (T-PCR) to amplify ChIP-immunoprecipitated DNA from mouse embryonic tissue prior to cloning. Importantly, we then compare this technique with tandem shotgun-cloning experiments in terms of its capacity to identify target genes. We find that T-PCR dramatically increases the efficiency of cloning ChIP fragments without distortion of the relative location of cloned fragments to putative target genes. Thus, T-PCR is a simple procedure which greatly enhances the efficiency of cloning tissue-derived ChIP fragments.

  6. 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

  7. 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.

  8. Molecular characterization of individual D3 dopamine receptor-expressing cells isolated from multiple brain regions of a novel mouse model

    PubMed Central

    Li, Ying

    2012-01-01

    Among dopamine receptors, the expression and function of the D3 receptor subtype is not well understood. The receptor has the highest affinity for dopamine and many drugs that target dopamine receptors. In this paper, we examined, at the single cell level, the characteristics of D3 receptor-expressing cells isolated from different brain regions of male and female mice that were either 35 or 70 days old. The brain regions included nucleus accumbens, Islands of Calleja, olfactory tubercle, retrosplenial cortex, dorsal subiculum, mammillary body, amygdala and septum. The expression analysis was done in the drd3-enhanced green fluorescent protein transgenic mice that report the endogenous expression of D3 receptor mRNA. Using single cell reverse transcriptase PCR, we determined if the D3 receptor-expressing fluorescent cells in these mice were neurons or glia and if they were glutamatergic, GABAergic or catecholaminergic. Next, we determined if the fluorescent cells co-expressed the four other dopamine receptor subtypes, adenylate cyclase V (ACV) isoform, and three different isoforms of G protein-coupled inward rectifier potassium (GIRK) channels. The results suggest that D3 receptor is expressed in neurons, with region-specific expression in glutamatergic and GABAergic neurons. The D3 receptor primarily co-expressed with D1 and D2 dopamine receptors with regional, sex and age-dependent differences in the co-expression pattern. The percentage of cells co-expressing D3 receptor and ACV or GIRK channels varied significantly by brain region, sex and age. The molecular characterization of D3 receptor-expressing cells in mouse brain reported here will facilitate the characterization of D3 receptor function in physiology and pathophysiology. PMID:22286951

  9. Potential for cloning dogs.

    PubMed

    Westhusin, M E; Burghardt, R C; Ruglia, J N; Willingham, L A; Liu, L; Shin, T; Howe, L M; Kraemer, D C

    2001-01-01

    The aim of this study was to determine whether nuclear transplantation could be used to clone a dog using donor nucleus cells collected from an adult female. Fibroblasts obtained from skin biopsies were fused with enucleated bovine or canine oocytes. The resulting cloned embryos were cultured in vitro to monitor embryonic development. A proportion of the resulting embryos was transferred into surrogate bitches for development to term. When canine oocytes were used as recipient ova for canine fibroblasts, 23% of the resulting embryos cleaved at least once after culture in vitro. Five cloned embryos were transferred into three synchronized recipient bitches, but no pregnancies resulted. When bovine oocytes were used as recipinets for canine fibroblasts, 38% cleaved to the two- to four-cell stage and 43% cleaved to the eight- to 16-cell stage. Forty-seven of these embryos were transferred into four recipient females, resulting in a single conceptus that ceased development at about day 20 of gestation. The desire for cloned dogs is considerable and will undoubtedly incite the development of successful methods for cloning companion animals. However, significant investment into additional research is required, especially in the areas of in vitro maturation of oocytes and control of the oestrous cycle of bitches.

  10. 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.

  11. 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.

  12. Use of BAC clones as standardized reagents for Marek’s disease virus research

    USDA-ARS?s Scientific Manuscript database

    The cloning of the Marek’s disease virus (MDV) genome as an infectious bacterial artificial chromosome (BAC) clone have led to major advances through our ability to study individual gene function by making precise insertions and deletions in the viral genome. We believe that MDV BAC clones will repl...

  13. Extremal quantum cloning machines

    NASA Astrophysics Data System (ADS)

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

    2005-10-01

    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 displacements.

  14. 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.

  15. A screen for RAS mutations in individuals at risk of secondary leukaemia due to occupational exposure to petrochemicals.

    PubMed

    Taylor, C; Hughes, D C; Zappone, E; Cazzola, M; Carter, G; Jacobs, A; Padua, R A

    1995-05-01

    Occupational exposure to petrochemicals, in particular benzene, has been identified as a risk factor in the development of acute leukaemia. A cohort of exposed (n = 44) and non-exposed individuals (n = 19) from the same petrochemical installation were screened by polymerase chain reaction (PCR) followed by oligonucleotide hybridization (ONH) for the presence of mutations in the H, K, and NRAS cellular proto-oncogenes. A KRAS mutation was detected in one individual from the exposed group who was haematologically normal at the time of sampling. The presence of this mutation was confirmed by nude mouse tumorigenicity assay and positively identified as a K13 Gly-Asp substitution by cloning and sequencing.

  16. 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.

  17. Specific sequence deletions in two classes of murine leukemia virus-related proviruses in the mouse genome.

    PubMed

    Ch'ang, L Y; Yang, W K; Myer, F E; Koh, C K; Boone, L R

    1989-02-01

    Characteristic long terminal repeats (LTR) of approximately 700 and 750 bp were found, respectively, in the two classes (polytropic and modified polytropic) of murine leukemia virus (MuLV)-related nonecotropic nonxenotropic proviral sequences in eight individual molecular clones of RFM/Un mouse chromosomal DNA fragments. Three proviral clones, two polytropic and one modified polytropic, contained sequence deletions in the viral structural genes. Nucleotide sequence analysis revealed that 7-bp direct repeats occur at both ends of deleted sequences in intact structures and one of the repeats remains in genomes with the deletion. Specifically, the deleted sequences were a 1487-bp gag-pol sequence with ACTGCCC repeat, a 113-bp mid-pol sequence with CAGGCAA repeat, and a 1811-bp env sequence with GGTCCAG repeat. The same specific sequence deletions were found in both classes of MuLV-related proviral structures. Examination of chromosomal DNA from eight inbred laboratory mouse strains and six wild mouse species showed that a minor population of proviruses with these specific deletions were present in Mus musculus and Mus spretus, all of which contain prominent 700-bp LTR polytropic proviral structures. The 750-bp LTR modified polytropic proviral structures were phylogenetically more restricted, being equally predominant in Mus musculus domesticus mice, but minor to undetectable in Mus spretus subspecies, and absent in other wild mouse populations.

  18. Quantitative proteomics reveals protein kinases and phosphatases in the individual phases of contextual fear conditioning in the C57BL/6J mouse.

    PubMed

    Šmidák, Roman; Mayer, Rupert Laurenz; Bileck, Andrea; Gerner, Christopher; Mechtcheriakova, Diana; Stork, Oliver; Lubec, Gert; Li, Lin

    2016-04-15

    A series of protein kinases and phosphatases (PKPs) have been linked to contextual fear conditioning (cFC) but information is mainly derived from immunochemical studies. It was therefore decided to use an explorative label-free quantitative proteomics approach to concomitantly determine PKPs in hippocampi of mice in the individual phases of cFC. C57BL/6J mice were divided into four groups: three training groups representing the acquisition, consolidation and retrieval phases of cFC and a foot shock control group. Using this approach we identified 32 protein kinases or phosphatases/phosphatase subunits with significantly changed protein levels in one or more training groups as compared to foot shock control. These include members of PKP signalling modules of mitogen-activated protein kinase (MAP3K10, RAF1, KSR2), Ca2+/calmodulin-dependent protein kinase (CaMKIIα, DAPK1), protein kinase C (PRKCD) and protein phosphatases 1, 2A, 2B(3) previously implicated in various learning paradigms. In addition, our analysis showed protein kinases WNK1, LYN, VRK1, ABL1, CDK4, CDKL3, SgK223 and ADCK1, and protein phosphatases PTPRF, ACP1, DNAJC6, SSH2 and UBASH3B that have not been directly linked to fear memory processes so far. Determination of PKPs in the individual cFC phases represents a valuable resource for interpretation of previous and design of future studies on PKPs in memory mechanisms.

  19. Effects of cloning and root-tip size on observations of fungal ITS sequences from Picea glauca roots

    Treesearch

    Daniel L. Lindner; Mark T. Banik

    2009-01-01

    To better understand the effects of cloning on observations of fungal ITS sequences from Picea glauca (white spruce) roots two techniques were compared: (i) direct sequencing of fungal ITS regions from individual root tips without cloning and (ii) cloning and sequencing of fungal ITS regions from individual root tips. Effect of root tip size was...

  20. To clone alone: the United Nations' Human Cloning Declaration.

    PubMed

    Isasi, Rosario M; Annas, George J

    2006-01-01

    The United Nations labored for almost four years to create a treaty governing human cloning. In 2005 that effort was abandoned, and instead the United Nations' General Assembly adopted a "Declaration on Human Cloning".

  1. MXS-Chaining: A Highly Efficient Cloning Platform for Imaging and Flow Cytometry Approaches in Mammalian Systems.

    PubMed

    Sladitschek, Hanna L; Neveu, Pierre A

    2015-01-01

    The continuous improvement of imaging technologies has driven the development of sophisticated reporters to monitor biological processes. Such constructs should ideally be assembled in a flexible enough way to allow for their optimization. Here we describe a highly reliable cloning method to efficiently assemble constructs for imaging or flow cytometry applications in mammalian cell culture systems. We bioinformatically identified a list of restriction enzymes whose sites are rarely found in human and mouse cDNA libraries. From the best candidates, we chose an enzyme combination (MluI, XhoI and SalI: MXS) that enables iterative chaining of individual building blocks. The ligation scar resulting from the compatible XhoI- and SalI-sticky ends can be translated and hence enables easy in-frame cloning of coding sequences. The robustness of the MXS-chaining approach was validated by assembling constructs up to 20 kb long and comprising up to 34 individual building blocks. By assessing the success rate of 400 ligation reactions, we determined cloning efficiency to be 90% on average. Large polycistronic constructs for single-cell imaging or flow cytometry applications were generated to demonstrate the versatility of the MXS-chaining approach. We devised several constructs that fluorescently label subcellular structures, an adapted version of FUCCI (fluorescent, ubiquitination-based cell cycle indicator) optimized to visualize cell cycle progression in mouse embryonic stem cells and an array of artificial promoters enabling dosage of doxycyline-inducible transgene expression. We made publicly available through the Addgene repository a comprehensive set of MXS-building blocks comprising custom vectors, a set of fluorescent proteins, constitutive promoters, polyadenylation signals, selection cassettes and tools for inducible gene expression. Finally, detailed guidelines describe how to chain together prebuilt MXS-building blocks and how to generate new customized MXS

  2. 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)

  3. Secure the Clones

    NASA Astrophysics Data System (ADS)

    Jensen, Thomas; Kirchner, Florent; Pichardie, David

    Exchanging mutable data objects with untrusted code is a delicate matter because of the risk of creating a data space that is accessible by an attacker. Consequently, secure programming guidelines for Java stress the importance of using defensive copying before accepting or handing out references to an internal mutable object. However, implementation of a copy method (like clone()) is entirely left to the programmer. It may not provide a sufficiently deep copy of an object and is subject to overriding by a malicious sub-class. Currently no language-based mechanism supports secure object cloning. This paper proposes a type-based annotation system for defining modular copy policies for class-based object-oriented programs. A copy policy specifies the maximally allowed sharing between an object and its clone. We present a static enforcement mechanism that will guarantee that all classes fulfill their copy policy, even in the presence of overriding of copy methods, and establish the semantic correctness of the overall approach in Coq. The mechanism has been implemented and experimentally evaluated on clone methods from several Java libraries.

  4. 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.

  5. 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)

  6. 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.

  7. 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.

  8. 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)

  9. 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)

  10. [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.

  11. To Clone or Not To Clone: Method Analysis for Retrieving Consensus Sequences In Ancient DNA Samples

    PubMed Central

    Winters, Misa; Barta, Jodi Lynn; Monroe, Cara; Kemp, Brian M.

    2011-01-01

    The challenges associated with the retrieval and authentication of ancient DNA (aDNA) evidence are principally due to post-mortem damage which makes ancient samples particularly prone to contamination from “modern” DNA sources. The necessity for authentication of results has led many aDNA researchers to adopt methods considered to be “gold standards” in the field, including cloning aDNA amplicons as opposed to directly sequencing them. However, no standardized protocol has emerged regarding the necessary number of clones to sequence, how a consensus sequence is most appropriately derived, or how results should be reported in the literature. In addition, there has been no systematic demonstration of the degree to which direct sequences are affected by damage or whether direct sequencing would provide disparate results from a consensus of clones. To address this issue, a comparative study was designed to examine both cloned and direct sequences amplified from ∼3,500 year-old ancient northern fur seal DNA extracts. Majority rules and the Consensus Confidence Program were used to generate consensus sequences for each individual from the cloned sequences, which exhibited damage at 31 of 139 base pairs across all clones. In no instance did the consensus of clones differ from the direct sequence. This study demonstrates that, when appropriate, cloning need not be the default method, but instead, should be used as a measure of authentication on a case-by-case basis, especially when this practice adds time and cost to studies where it may be superfluous. PMID:21738625

  12. Cloning Components of Human Telomerase.

    DTIC Science & Technology

    1998-07-01

    nuclear factor NF90 homolog. (5 clones). RNA binding protein. Poorly understood. 3. FRG1 . Poorly understood. 4. DEK. Weak homology to Tetrahymena p95...least some of the clones for poorly understood genes (e.g. Hax-1, FRG1 , NF90, NF45, KIAA0098, KIAA0026, BAC397c4). Aim II. Functional Cloning of the

  13. Water relations of populus clones

    SciTech Connect

    Pallardy, S.G.; Kozlowski, T.T.

    1981-02-01

    Stomatal aperture and water balance in the field of eight Populus clones varying in growth rate were closely related to environmental factors and clonal differences were clearly expressed. Leaf water potential (psi) was influenced by solar radiation, leaf conductance, evaporative demand, and soil moisture content. The effects of soil moisture on psi were greatly modified by atmospheric conditions and stomatal conductance. Several slow-growing clones exhibited extended periods of psi below that of rapidly growing clones, despite high evaporative demand and the much greater transpiring surfaces of the fast-growing clones. Stomata of all clones responded to changes in light intensity and vapor pressure gradient (VPG). Pronounced stomatal sensitivity to VPG of two rapidly growing clones of common parentage, and the resultant capacity of these clones to moderate water deficits under high evaporative demand, were associated with drought resistance in one of the parents. Seasonal maximum leaf conductance was positively related to growth in several clones, suggesting that rapidly growing clones possess the capacity to carry on higher rates of gas exchange under favorable conditions. Analysis of changes in psi with changes in transpirational flux density (TFD) showed that for four clones, psi change per unit change in TFD decreased as TFD increased, indicating plant adaptation for prevention of damaging psi even at high TFD. More rapidly growing clones exhibited a larger initial rate of decline in psi with TFD, but reduced the rate of decline more than slow-growing clones as TFD increased. (Refs. 41).

  14. 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

  15. Three concepts of cloning in human beings.

    PubMed

    Cui, Ke-Hui

    2005-07-01

    Human cloning, organ cloning and tissue cloning are various types of cloning that occur at different levels with different methodologies. According to three standards of terminology for an embryo (fertilization through germ cells, development in the uterus and having the potential to produce a human life), tissue cloning and type I organ cloning will not produce an embryo. In contrast, human cloning and type II organ cloning will produce an embryo. Thus, only non-germinal tissue cloning and type I organ cloning are beyond the ethical question and will not change human beings as a species. Using cloned tissues to make new tissues or organs is promising for the future of medicine.

  16. Systematic Expression Profiling of the Mouse Transcriptome Using RIKEN cDNA Microarrays

    PubMed Central

    Bono, Hidemasa; Yagi, Ken; Kasukawa, Takeya; Nikaido, Itoshi; Tominaga, Naoko; Miki, Rika; Mizuno, Yosuke; Tomaru, Yasuhiro; Goto, Hitoshi; Nitanda, Hiroyuki; Shimizu, Daisuke; Makino, Hirochika; Morita, Tomoyuki; Fujiyama, Junshin; Sakai, Takehito; Shimoji, Takashi; Hume, David A.; Hayashizaki, Yoshihide; Okazaki, Yasushi

    2003-01-01

    The number of known mRNA transcripts in the mouse has been greatly expanded by the RIKEN Mouse Gene Encyclopedia project. Validation of their reproducible expression in a tissue is an important contribution to the study of functional genomics. In this report, we determine the expression profile of 57,931 clones on 20 mouse tissues using cDNA microarrays. Of these 57,931 clones, 22,928 clones correspond to the FANTOM2 clone set. The set represents 20,234 transcriptional units (TUs) out of 33,409 TUs in the FANTOM2 set. We identified 7206 separate clones that satisfied stringent criteria for tissue-specific expression. Gene Ontology terms were assigned for these 7206 clones, and the proportion of `molecular function' ontology for each tissue-specific clone was examined. These data will provide insights into the function of each tissue. Tissue-specific gene expression profiles obtained using our cDNA microarrays were also compared with the data extracted from the GNF Expression Atlas based on Affymetrix microarrays. One major outcome of the RIKEN transcriptome analysis is the identification of numerous nonprotein-coding mRNAs. The expression profile was also used to obtain evidence of expression for putative noncoding RNAs. In addition, 1926 clones (70%) of 2768 clones that were categorized as “unknown EST,” and 1969 (58%) clones of 3388 clones that were categorized as “unclassifiable” were also shown to be reproducibly expressed. PMID:12819129

  17. Mammalian cloning: advances and limitations.

    PubMed

    Solter, D

    2000-12-01

    For many years, researchers cloning mammals experienced little success, but recent advances have led to the successful cloning of several mammalian species. However, cloning by the transfer of nuclei from adult cells is still a hit-and-miss procedure, and it is not clear what technical and biological factors underlie this. Our understanding of the molecular basis of reprogramming remains extremely limited and affects experimental approaches towards increasing the success rate of cloning. Given the future practical benefits that cloning can offer, the time has come to address what should be done to resolve this problem.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Emergence of clones in sexual populations

    NASA Astrophysics Data System (ADS)

    Neher, Richard A.; Vucelja, Marija; Mézard, Marc; Shraiman, Boris I.

    2012-05-01

    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.

  4. Cell-free cloning using φ29 DNA polymerase

    PubMed Central

    Hutchison, Clyde A.; Smith, Hamilton O.; Pfannkoch, Cynthia; Venter, J. Craig

    2005-01-01

    We describe conditions for rolling-circle amplification (RCA) of individual DNA molecules 5–7 kb in size by >109-fold, using φ29 DNA polymerase. The principal difficulty with amplification of small amounts of template by RCA using φ29 DNA polymerase is “background” DNA synthesis that usually occurs when template is omitted, or at low template concentrations. Reducing the reaction volume while keeping the amount of template fixed increases the template concentration, resulting in a suppression of background synthesis. Cell-free cloning of single circular molecules by using φ29 DNA polymerase was achieved by carrying out the amplification reactions in very small volumes, typically 600 nl. This procedure allows cell-free cloning of individual synthetic DNA molecules that cannot be cloned in Escherichia coli, for example synthetic phage genomes carrying lethal mutations. It also allows cell-free cloning of genomic DNA isolated from bacteria. This DNA can be sequenced directly from the φ29 DNA polymerase reaction without further amplification. In contrast to PCR amplification, RCA using φ29 DNA polymerase does not produce mutant jackpots, and the high processivity of the enzyme eliminates stuttering at homopolymer tracts. Cell-free cloning has many potential applications to both natural and synthetic DNA. These include environmental DNA samples that have proven difficult to clone and synthetic genes encoding toxic products. The method may also speed genome sequencing by eliminating the need for biological cloning. PMID:16286637

  5. Cell-free cloning using phi29 DNA polymerase.

    PubMed

    Hutchison, Clyde A; Smith, Hamilton O; Pfannkoch, Cynthia; Venter, J Craig

    2005-11-29

    We describe conditions for rolling-circle amplification (RCA) of individual DNA molecules 5-7 kb in size by >10(9)-fold, using phi29 DNA polymerase. The principal difficulty with amplification of small amounts of template by RCA using phi29 DNA polymerase is "background" DNA synthesis that usually occurs when template is omitted, or at low template concentrations. Reducing the reaction volume while keeping the amount of template fixed increases the template concentration, resulting in a suppression of background synthesis. Cell-free cloning of single circular molecules by using phi29 DNA polymerase was achieved by carrying out the amplification reactions in very small volumes, typically 600 nl. This procedure allows cell-free cloning of individual synthetic DNA molecules that cannot be cloned in Escherichia coli, for example synthetic phage genomes carrying lethal mutations. It also allows cell-free cloning of genomic DNA isolated from bacteria. This DNA can be sequenced directly from the phi29 DNA polymerase reaction without further amplification. In contrast to PCR amplification, RCA using phi29 DNA polymerase does not produce mutant jackpots, and the high processivity of the enzyme eliminates stuttering at homopolymer tracts. Cell-free cloning has many potential applications to both natural and synthetic DNA. These include environmental DNA samples that have proven difficult to clone and synthetic genes encoding toxic products. The method may also speed genome sequencing by eliminating the need for biological cloning.

  6. Cloning and cryptography with quantum continuous variables

    NASA Astrophysics Data System (ADS)

    Cerf, N. J.; Iblisdir, S.; van Assche, G.

    2002-02-01

    The cloning of quantum variables with continuous spectra is investigated. We define a Gaussian 1-to-2 cloning machine that copies equally well two conjugate variables such as position and momentum or the two quadrature components of a light mode. The resulting cloning fidelity for coherent states, namely F=2/3, is shown to be optimal. An asymmetric version of this Gaussian cloner is then used to assess the security of a continuous-variable quantum key distribution scheme that allows two remote parties to share a Gaussian key. The information versus disturbance tradeoff underlying this continuous quantum cryptographic scheme is then analyzed for the optimal individual attack. Methods to convert the resulting Gaussian keys into secret key bits are also studied. Finally, the extension of the Gaussian cloner to optimal N-to-M continuous cloners is discussed, and it is shown how to implement these cloners for light modes using a phase-insensitive optical amplifier and beam splitters. In addition, a phase-conjugate input cloner is defined, yielding M clones and M' anticlones from N replicas of a coherent state and N' replicas of its phase-conjugate (with M'-M=N'-N). This novel kind of cloners is shown to outperform the standard N-to-M cloners in some cases.

  7. Experimental Eavesdropping Based on Optimal Quantum Cloning

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

    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)PLRAAN1050-2947]. 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.

  8. 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.

  9. Clonal Diversity and Clone Formation in the Parthenogenetic Caucasian Rock Lizard Darevskia dahli

    PubMed Central

    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

  10. Age, microbiota, and T cells shape diverse individual IgA repertoires in the intestine

    PubMed Central

    Lindner, Cornelia; Wahl, Benjamin; Föhse, Lisa; Suerbaum, Sebastian; Macpherson, Andrew J.; Prinz, Immo

    2012-01-01

    Intestinal immunoglobulin A (IgA) ensures host defense and symbiosis with our commensal microbiota. Yet previous studies hint at a surprisingly low diversity of intestinal IgA, and it is unknown to what extent the diverse Ig arsenal generated by somatic recombination and diversification is actually used. In this study, we analyze more than one million mouse IgA sequences to describe the shaping of the intestinal IgA repertoire, its determinants, and stability over time. We show that expanded and infrequent clones combine to form highly diverse polyclonal IgA repertoires with very little overlap between individual mice. Selective homing allows expanded clones to evenly seed the small but not large intestine. Repertoire diversity increases during aging in a dual process. On the one hand, microbiota-, T cell–, and transcription factor RORγt–dependent but Peyer’s patch–independent somatic mutations drive the diversification of expanded clones, and on the other hand, new clones are introduced into the repertoire of aged mice. An individual’s IgA repertoire is stable and recalled after plasma cell depletion, which is indicative of functional memory. These data provide a conceptual framework to understand the dynamic changes in the IgA repertoires to match environmental and intrinsic stimuli. PMID:22249449

  11. Divergent ecology of sympatric clones of the asexual gecko, Lepidodactylus lugubris.

    PubMed

    Bolger, Douglas T; Case, Ted J

    1994-12-01

    We report differences in the thermal biology, elevational, temporal and geographic distributions of sympatric clones of the widespread asexual house gecko, Lepidodactylus lugubris. The two most common L. lugubris clones in Fiji, clones 2NA and 2NB, differ significantly in preferred temperature as measured in a laboratory heat gradient, but were similar in critical thermal maximum and minimum. Significant differences were found in the relative frequency of clones 2NA, 2NB, and a third Fijian clone, clone 3NB, at seven sites along an elevational gradient in Fiji. Clone 2NB was not collected at sites above 235 m, consistent with its higher preferred temperature, whereas clone 2NA was captured as high as 835 m. Clone 3NB was extremely rare at sealevel (1% of all individuals at three sites below 100 m), but predominated at the two highest-elevation sites (42% and 100%). Clones 2NA and 2NB did not differ significantly in their activity time or ambient activity temperature at low-elevation sites. Clone 3NB however, was active on significantly cooler nights at two of those sites. These significant inter-clonal differences in spatial and temporal distribution should allow a more complete utilization of resources by the assemblage of clones than by any single clonal genotype, and may promote coexistence of clones at a within-island and within-site scale. Clone 2NA, which is the most common clone in Fiji and has the broadest elevational distribution, also has the widest geographic distribution. It was the predominant clone at 27 of 34 sites surveyed in nine Pacific archipelagoes. This suggests that the ecological attributes that favor this clone in Fiji also favor it elsewhere in the Pacific despite differing environmental conditions and clonal composition in those areas.

  12. 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

  13. 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.

  14. MouseBook: an integrated portal of mouse resources.

    PubMed

    Blake, Andrew; Pickford, Karen; Greenaway, Simon; Thomas, Steve; Pickard, Amanda; Williamson, Christine M; Adams, Niels C; Walling, Alison; Beck, Tim; Fray, Martin; Peters, Jo; Weaver, Tom; Brown, Steve D M; Hancock, John M; Mallon, Ann-Marie

    2010-01-01

    The MouseBook (http://www.mousebook.org) databases and web portal provide access to information about mutant mouse lines held as live or cryopreserved stocks at MRC Harwell. The MouseBook portal integrates curated information from the MRC Harwell stock resource, and other Harwell databases, with information from external data resources to provide value-added information above and beyond what is available through other routes such as International Mouse Stain Resource (IMSR). MouseBook can be searched either using an intuitive Google style free text search or using the Mammalian Phenotype (MP) ontology tree structure. Text searches can be on gene, allele, strain identifier (e.g. MGI ID) or phenotype term and are assisted by automatic recognition of term types and autocompletion of gene and allele names covered by the database. Results are returned in a tabbed format providing categorized results identified from each of the catalogs in MouseBook. Individual result lines from each catalog include information on gene, allele, chromosomal location and phenotype, and provide a simple click-through link to further information as well as ordering the strain. The infrastructure underlying MouseBook has been designed to be extensible, allowing additional data sources to be added and enabling other sites to make their data directly available through MouseBook.

  15. The effect of high-fat diet on the composition of the gut microbiota in cloned and non-cloned pigs of lean and obese phenotype

    PubMed Central

    Pedersen, Rebecca; Andersen, Anders Daniel; Hermann-Bank, Marie Louise; Stagsted, Jan; Boye, Mette

    2013-01-01

    The aim of this study was to investigate the effect of high-far-high-energy diet on cloned and non-cloned domestic pigs of both lean and obese phenotype and to evaluate if the lean cloned pigs had a lower inter-individual variation as compared with non-cloned pigs. The microbiota of colon and terminal ileum was investigated in cloned and non-cloned pigs that received a high-far-high-energy diet with either restricted or ad libitum access to feed, resulting in lean and obese phenotypes, respectively. The fecal microbiota of lean pigs was investigated by terminal restriction fragment length polymorphism (T-RFLP). The intestinal microbiota of lean and obese cloned and non-cloned pigs was analyzed by quantitative real time PCR and a novel high-throughput qPCR platform (Fluidigm). Principal component analysis (PCA) of the T-RFLP profiles revealed that lean cloned and non-cloned pigs had a different overall composition of their gut microbiota. The colon of lean cloned pigs contained relatively more bacteria belonging to the phylum Firmicutes and less from the phylum Bacteroidetes than obese cloned pigs as estimated by qPCR. Fluidigm qPCR results revealed differences in specific bacterial groups in the gut microbiota of both lean and obese pigs. Our results suggest that high-far-high-energy diet is associated with changes in the gut microbiota even in the absence of obesity. Overall, the cloned pigs had a different gut microbiota from that of non-cloned pigs. To our knowledge this is the first study to investigate the gut microbiota of cloned domestic pigs of lean and obese phenotype. PMID:23974297

  16. 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.

  17. Mouse phenotyping.

    PubMed

    Fuchs, Helmut; Gailus-Durner, Valérie; Adler, Thure; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Calzada-Wack, Julia; Da Silva-Buttkus, Patricia; Neff, Frauke; Götz, Alexander; Hans, Wolfgang; Hölter, Sabine M; Horsch, Marion; Kastenmüller, Gabi; Kemter, Elisabeth; Lengger, Christoph; Maier, Holger; Matloka, Mikolaj; Möller, Gabriele; Naton, Beatrix; Prehn, Cornelia; Puk, Oliver; Rácz, Ildikó; Rathkolb, Birgit; Römisch-Margl, Werner; Rozman, Jan; Wang-Sattler, Rui; Schrewe, Anja; Stöger, Claudia; Tost, Monica; Adamski, Jerzy; Aigner, Bernhard; Beckers, Johannes; Behrendt, Heidrun; Busch, Dirk H; Esposito, Irene; Graw, Jochen; Illig, Thomas; Ivandic, Boris; Klingenspor, Martin; Klopstock, Thomas; Kremmer, Elisabeth; Mempel, Martin; Neschen, Susanne; Ollert, Markus; Schulz, Holger; Suhre, Karsten; Wolf, Eckhard; Wurst, Wolfgang; Zimmer, Andreas; Hrabě de Angelis, Martin

    2011-02-01

    Model organisms like the mouse are important tools to learn more about gene function in man. Within the last 20 years many mutant mouse lines have been generated by different methods such as ENU mutagenesis, constitutive and conditional knock-out approaches, knock-down, introduction of human genes, and knock-in techniques, thus creating models which mimic human conditions. Due to pleiotropic effects, one gene may have different functions in different organ systems or time points during development. Therefore mutant mouse lines have to be phenotyped comprehensively in a highly standardized manner to enable the detection of phenotypes which might otherwise remain hidden. The German Mouse Clinic (GMC) has been established at the Helmholtz Zentrum München as a phenotyping platform with open access to the scientific community (www.mousclinic.de; [1]). The GMC is a member of the EUMODIC consortium which created the European standard workflow EMPReSSslim for the systemic phenotyping of mouse models (http://www.eumodic.org/[2]). Copyright © 2010 Elsevier Inc. All rights reserved.

  18. Generation of five high-complexity painting probe libraries from flow-sorted mouse chromosomes

    SciTech Connect

    Weier, H.U.G. |; Polikoff, D.; Greulich, K.M.

    1994-06-01

    Mouse metaphase chromosomes were purified by flow sorting from the murine fibroblast cell line Mus spretus clone 5A. The authors sorted chromosomes that fell into five individual peaks based on the Hoechst 33258/chromomycin A3 DNA histogram: three peaks corresponding to the least amount of DNA and two peaks representing chromosomes with the most DNA content. This is the first example of the successful application of bivariate flow karyotyping to murine chromosome sorting. They then applied primer-directed in vitro DNA amplification using the polymerase chain reaction (PCR) to generate and label larger amounts of chromosome-specific DNA. In situ hybridization showed specific binding of the PCR products to mouse chromosomes Y, 19, 18, 3, and X as well as chromosomes 1 and 2. The combination of chromosome sorting from the M. spretus cell line and PCR proved to be highly valuable for generation of pools of DNA fragments that exhibit specific binding to mouse chromosomes and can be used to identify and delineate mouse metaphase chromosomes. 13 refs., 3 figs.

  19. 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.

  20. Guide to molecular cloning techniques

    SciTech Connect

    Berger, S.L.; Kimmel, A.R.

    1987-01-01

    This book includes the following selections: requirements for a molecular biology laboratory; general methods for isolating and characterizing nucleic acids; enzymatic techniques and recombinant DNA technology; restriction enzymes; growth and maintenance of bacteria; genetic cloning, preparation and characterization of RNA; preparation of cDNA and the generation of cDNA libraries; selections of clones from libraries; and identification and characterization of specific clones.

  1. 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.

  2. Evaluation of a Pooled Strategy for High-Throughput Sequencing of Cosmid Clones from Metagenomic Libraries

    PubMed Central

    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

  3. 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.

  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 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

  6. 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.

  7. 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.

  8. 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

  9. Adoptive immunity in mice challenged with L1210/DTIC clones.

    PubMed

    Canti, G; Ricci, L; Marelli, O; Franco, P; Nicolin, A

    1987-01-01

    New antigenic specificities, not detectable on parental cells, have been induced by many investigators in mouse lymphomas by treatment with the antitumor agent 5(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC). The antigens are transmissible, after withdrawal of the drug treatment, as an inheritable character. The mechanism of induction, the molecular nature, and the number of the new antigenic specificities have not been completely elucidated. Four clones from murine leukemia L1210 isolated and expanded in vitro were treated in vivo with DTIC and the new sublines were studied in detail. The four drug-treated sublines studied exhibited strong immunogenicity since they were rejected by syngeneic animals. Immunosuppressed animals challenged with 10(7) A/DTIC or P/DTIC cells were reciprocally protected by the adoptive transfer of spleen cells from donors that had rejected a lethal challenge of A/DTIC or P/DTIC clone. In a similar fashion, the adoptive transfer of spleen cells obtained from animals that had rejected the Q/DTIC or the R/DTIC clones protected immunosuppressed mice challenged with Q/DTIC or R/DTIC cells. No antitumor activity was observed in cross-protective schedules other than those indicated. It was been concluded that (a) the L1210 leukemia line does not have antigenic cells, (b) four DTIC-treated clone sublines were rejected by compatible hosts, and (c) two mutually exclusive sets of antigens were expressed in four antigenic clone sublines.

  10. Cloning an expressed gene shared by the human sex chromosomes

    SciTech Connect

    Darling, S.M.; Banting, G.S.; Pym, B.; Wolfe, J.; Goodfellow, P.N.

    1986-01-01

    The existence of genes shared by mammalian sex chromosomes has been predicted on both evolutionary and functional grounds. However, the only experimental evidence for such genes in humans is the cell-surface antigen encoded by loci on the X and Y chromosomes (MIC2X and MIC2Y, respectively), which is recognized by the monoclonal antibody 12E7. Using the bacteriophage lambdagt11 expression system in Escherichia coli and immunoscreening techniques, the authors have isolated a cDNA clone whose primary product is recognized by 12E7. Southern blot analysis using somatic cell hybrids containing only the human X or Y chromosomes shows that the sequences reacting with the cDNA clone are localized to the sex chromosomes. In addition, the clone hybridizes to DNAs isolated from mouse cells that have been transfected with human DNA and selected for 12E7 expression on the fluorescence-activated cell sorter. The authors conclude that the cDNA clone encodes the 12E7 antigen, which is the primary product of the MIC2 loci. The clone was used to explore sequence homology between MIC2X and MIC2Y; these loci are closely related, if not identical.

  11. 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.

  12. 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.

  13. Cloning and Characterization of the Mouse Hepatitis Virus Receptor

    DTIC Science & Technology

    1991-02-11

    interactions, the cell surface receptor is a protein with a binding domain that interacts with a soluble ligand. For virus-receptor interactions, the...cell surface receptor is either a protein, a carbohydrate , or a lipid molecule. The ligand is the viral attachment protein. Three major criteria...The examples of utilization of viral receptors to block infection are the use of the soluble , secreted forms of the CD4 molecule and fragments of

  14. Cloning and Characterization of the Mouse Hepatitis Virus Receptor

    DTIC Science & Technology

    1991-02-11

    Alexis Basile and Kathy Wilson for their technical assistance and for creating a nice enviroment to work in, and Dr. Michael Pensiero for his...Schreyer, S. Carrel and J. P. Mach. (1984). "Monodonal antibodies Identify a CEA crossreacting antigen of 95 kD (NCA-95) distinct in antigenicity and

  15. 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

  16. Frequent Occurrence of Highly Expanded but Unrelated B-Cell Clones in Patients with Multiple Myeloma

    PubMed Central

    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

  17. 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.

  18. Reproductive cloning and arguments from potential.

    PubMed

    Oakley, Justin

    2006-01-01

    The possibility of human reproductive cloning has led some bioethicists to suggest that potentiality-based arguments for fetal moral status become untenable, as such arguments would be committed to making the implausible claim that any adult somatic cell is itself a potential person. In this article I defend potentiality-based arguments for fetal moral status against such a reductio. Starting from the widely-held claim that the maintenance of numerical identity throughout successive changes places constraints on what a given entity can plausibly be said to have the potential to become, I argue that the cell reprogramming that takes place in reproductive cloning is such that it produces a new individual, and so adult somatic cells cannot be potential persons.

  19. Trypanosoma cruzi: infectivity modulation of a clone after passages through different hosts.

    PubMed

    Pérez Brandán, Cecilia; Padilla, Angel Marcelo; Diosque, Patricio; Basombrío, Miguel Angel

    2006-10-01

    Although Trypanosoma cruzi virulence can be modified through passages in vivo or long-term in vitro culture, the mechanisms involved are poorly understood. Here we report modifications in the infectivity of a T. cruzi clone after passages in different hosts without detectable changes in parasite genetic patterns. A clone was obtained from a T. cruzi IIe isolate and showed to be less virulent than the original isolate (p<0.05). This clone was enzymatically similar to the original isolate as shown by multilocus enzyme electrophoresis. Infection of this clone was compared by successive passages in mice and guinea pigs. The mouse-passaged subline became more virulent for both host species compared to the guinea pig-passaged subline (p<0.05). The clone line displayed similar random amplified polymorphic DNA patterns before and after passages in different hosts suggesting that alterations in virulence could be a result of a differential expression of virulence factors.

  20. Aberrant DNA methylation imprints in aborted bovine clones.

    PubMed

    Liu, Jing-He; Yin, Shen; Xiong, Bo; Hou, Yi; Chen, Da-Yuan; Sun, Qing-Yuan

    2008-04-01

    Genomic imprinting plays a very important role during development and its abnormality may heavily undermine the developmental potential of bovine embryos. Because of limited resources of the cow genome, bovine genomic imprinting, both in normal development and in somatic cell nuclear transfer (SCNT) cloning, is not well documented. DNA methylation is thought to be a major factor for the establishment of genomic imprinting. In our study, we determined the methylation status of differential methylated regions (DMRs) of four imprinted genes in four spontaneously aborted SCNT-cloned fetuses (AF). Firstly, abnormal methylation imprints were observed in each individual to different extents. In particular, Peg3 and MAOA were either seriously demethylated or showed aberrant methylation patterns in four aborted clones we tested, but Xist and Peg10 exhibited relatively better maintained methylation status in AF1 and AF4. Secondly, two aborted fetuses, AF2 and AF3 exhibited severe aberrant methylation imprints of four imprinted genes. Finally, MAOA showed strong heterogeneous methylation patterns of its DMR in normal somatic adult tissue, but largely variable methylation levels and relatively homogeneous methylation patterns in aborted cloned fetuses. Our data indicate that the aborted cloned fetuses exhibited abnormal methylation imprints, to different extent, in aborted clones, which partially account for the higher abortion and developmental abnormalities during bovine cloning.

  1. Clone history shapes Populus drought responses

    PubMed Central

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

    2011-01-01

    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

  2. The NEUROD gene maps to human chromosome 2q32 and mouse chromosome 2

    SciTech Connect

    Tamimi, R.; Dyer-Montgomery, K.; Hernandez, R.; Tapscott, S.J.

    1996-06-15

    The Neurod gene is a basic-helix-loop-helix gene that regulates neurogenesis and is identical to the hamster beta2 gene that was cloned as a regulator of insulin transcription. Here we report the cloning of human NEUROD and mapping of the gene to human chromosome 2q32 and to mouse chromosome 2. 12 refs., 1 fig.

  3. Learning, memory and exploratory similarities in genetically identical cloned dogs

    PubMed Central

    Shin, Chi Won; Kim, Geon A; Park, Won Jun; Park, Kwan Yong; Jeon, Jeong Min; Oh, Hyun Ju; Kim, Min Jung

    2016-01-01

    Somatic cell nuclear transfer allows generation of genetically identical animals using donor cells derived from animals with particular traits. To date, few studies have investigated whether or not these cloned dogs will show identical behavior patterns. To address this question, learning, memory and exploratory patterns were examined using six cloned dogs with identical nuclear genomes. The variance of total incorrect choice number in the Y-maze test among cloned dogs was significantly lower than that of the control dogs. There was also a significant decrease in variance in the level of exploratory activity in the open fields test compared to age-matched control dogs. These results indicate that cloned dogs show similar cognitive and exploratory patterns, suggesting that these behavioral phenotypes are related to the genotypes of the individuals. PMID:27030191

  4. Learning, memory and exploratory similarities in genetically identical cloned dogs.

    PubMed

    Shin, Chi Won; Kim, Geon A; Park, Won Jun; Park, Kwan Yong; Jeon, Jeong Min; Oh, Hyun Ju; Kim, Min Jung; Lee, Byeong Chun

    2016-12-30

    Somatic cell nuclear transfer allows generation of genetically identical animals using donor cells derived from animals with particular traits. To date, few studies have investigated whether or not these cloned dogs will show identical behavior patterns. To address this question, learning, memory and exploratory patterns were examined using six cloned dogs with identical nuclear genomes. The variance of total incorrect choice number in the Y-maze test among cloned dogs was significantly lower than that of the control dogs. There was also a significant decrease in variance in the level of exploratory activity in the open fields test compared to age-matched control dogs. These results indicate that cloned dogs show similar cognitive and exploratory patterns, suggesting that these behavioral phenotypes are related to the genotypes of the individuals.

  5. 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

  6. 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.

  7. [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.

  8. 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

  9. [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.

  10. 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

  11. Chromosomal localization of mouse bullous pemphigoid antigens, BPAG1 and BPAG2: Identification of a new region of homology between mouse and human chromosomes

    SciTech Connect

    Copeland, N.G.; Gilbert, D.J.; Jenkins, N.A. ); Li, K.; Sawamura, D.; Chu, Monli; Uitto, J. ); Giudice, G.J. )

    1993-01-01

    Two bullous pemphigoid antigens, BPAG1 and BPAG2, have been recently cloned and mapped to human chromosomes 6p12-p11 and 10q24.3, respectively. In this study, we localized the corresponding mouse genes by interspecific backcross analysis. Bpag-1 mapped to the proximal region of mouse chromosome 1, identifying a new region of homology between human chromosome 6 and mouse chromosome 1. Bpag-2 mapped to the distal end of mouse chromosome 19 in a region of homology to human chromosome 10q. These assignments confirm and extend the relationships between the human and the mouse chromosomes. 13 refs., 1 fig.

  12. Selection of High-Producing Clones Using FACS for CHO Cell Line Development.

    PubMed

    Gallagher, Clair; Kelly, Paul S

    2017-01-01

    Cell line development aims to generate and select clones with desirable characteristics. One of the most important parameters for biopharmaceutical cell selection is cell-specific productivity (Qp) or the quantity of product produced per cell per day. Fluorescence-activated cell sorting (FACS) is a powerful, high-throughput technique that facilitates multiparametric characterization and isolation of individual cell clones from heterogeneous populations. Here, we describe a FACS-based method for section of high-producing CHO cell clones.

  13. 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.

  14. Optimal cloning of pure states, testing single clones

    NASA Astrophysics Data System (ADS)

    Keyl, M.; Werner, R. F.

    1999-07-01

    We consider quantum devices for turning a finite number N of d-level quantum systems in the same unknown pure state σ into M>N systems of the same kind, in an approximation of the M-fold tensor product of the state σ. In a previous paper it was shown that this problem has a unique optimal solution, when the quality of the output is judged by arbitrary measurements, involving also the correlations between the clones. We show in this paper, that if the quality judgment is based solely on measurements of single output clones, there is again a unique optimal cloning device, which coincides with the one found previously.

  15. 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?

  16. Phospholipid epitopes for mouse antibodies against bromelain-treated mouse erythrocytes.

    PubMed Central

    Kawaguchi, S

    1987-01-01

    The reactivity of mouse antibodies against bromelain-treated mouse erythrocytes (BrMRBC) with phospholipid epitopes was assessed by ELISA, using four clones of monoclonal anti-BrMRBC antibodies that had idiotypes distinct from one another. The four antibodies could bind to low-density lipoproteins (LDL) from human and chicken, but not to LDL from mouse and rat. As to liposomes of natural phospholipids, all the clones reacted with liposomes of phosphatidylcholine, and some of them could react with liposomes of sphingomyelin, phosphatidylglycerol, phosphatidylic acid or cardiolipin. For liposomes of synthetic phosphatidylcholine with different fatty acids, the length of carbon chains and the number of unsaturated carbon chains of the fatty acids markedly affected the binding of each monoclonal antibody to the liposomes. The addition of dicetyl phosphate or stearylamine to phosphatidylcholine liposomes changed the reactivity of the liposomes. These results support the view that mouse anti-BrMRBC antibodies can recognize appropriately spaced phosphorylcholine residues on the surface of phospholipid liposomes, LDL and cells. The four clones had similar capacities for binding to LDL as well as to BrMRBC, but they had obviously different capacities for binding to phospholipid liposomes; the epitopes on phospholipid liposomes used in the present study were not so perfect as to react well with every anti-BrMRBC antibody. PMID:2443446

  17. Limitations on cloning in classical mechanics

    NASA Astrophysics Data System (ADS)

    Fenyes, Aaron

    2012-01-01

    In this paper, we show that a result precisely analogous to the traditional quantum no-cloning theorem holds in classical mechanics. This classical no-cloning theorem does not prohibit classical cloning, we argue, because it is based on a too-restrictive definition of cloning. Using a less popular, more inclusive definition of cloning, we give examples of classical cloning processes. We also prove that a cloning machine must be at least as complicated as the object it is supposed to clone.

  18. Cloning operator and its applications

    NASA Astrophysics Data System (ADS)

    Voicu, Liviu I.; Myler, Harley R.; Toma, Cristian E.

    1998-03-01

    A novel genetic operator called cloning is introduced and tested in different applications of genetic algorithms. Essentially, the cloning monotonically increases the lengths of the chromosomes during the evolution. It is argued that, under these circumstances, the cloning operator can accommodate a multiresolution search strategy, where the search starts at coarser scales and is subsequently mapped to finer scales upon achieving some in-scale performance criteria. Although the practical implementation of cloning is application dependent, a few general requirements are stated. In the remainder of the paper, different implementations of the cloning operator are introduced and employed in distinct applications, namely, function optimization, object support reconstruction from the support of its autocorrelation and the shortest path problem in planar graphs. The first two cases present typical multiresolution approaches to search problems and their results show consistent improvements in convergence speed with respect to classical genetic algorithms. In the last problem, a cloning operator is incorporated in an evolutionary algorithm that builds a set of valid paths in a planar graph. It is demonstrated that cloning can enhance the ability of a genetic algorithm to explore the search space efficiently in some applications.

  19. A novel nucleo-cytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp

    PubMed Central

    2011-01-01

    Background Unisexual vertebrates have been demonstrated to reproduce by gynogenesis, hybridogenesis, parthenogenesis, or kleptogenesis, however, it is uncertain how the reproduction mode contributes to the clonal diversity. Recently, polyploid gibel carp has been revealed to possess coexisting dual modes of unisexual gynogenesis and sexual reproduction and to have numerous various clones. Using sexual reproduction mating between clone D female and clone A male and subsequent 7 generation multiplying of unisexual gynogenesis, we have created a novel clone strain with more than several hundred millions of individuals. Here, we attempt to identify genetic background of the novel clone and to explore the significant implication for clonal diversity contribution. Methods Several nuclear genome markers and one cytoplasmic marker, the mitochondrial genome sequence, were used to identify the genetic organization of the randomly sampled individuals from different generations of the novel clone. Results Chromosome number, Cot-1 repetitive DNA banded karyotype, microsatellite patterns, AFLP profiles and transferrin alleles uniformly indicated that nuclear genome of the novel clone is identical to that of clone A, and significantly different from that of clone D. However, the cytoplasmic marker, its complete mtDNA genome sequence, is same to that of clone D, and different from that of clone A. Conclusions The present data indicate that the novel clone is a nucleo-cytoplasmic hybrid between the known clones A and D, because it originates from the offspring of gonochoristic sexual reproduction mating between clone D female and clone A male, and contains an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm) from the maternal clone D. It is suggested to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division. Significantly, the selected nucleo-cytoplasmic hybrid female

  20. Seamless Ligation Cloning Extract (SLiCE) cloning method.

    PubMed

    Zhang, Yongwei; Werling, Uwe; Edelmann, Winfried

    2014-01-01

    SLiCE (Seamless Ligation Cloning Extract) is a novel cloning method that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (15-52 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from bacterial artificial chromosomes or other sources. SLiCE is highly cost-effective and demonstrates the versatility as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. We established a DH10B-derived E. coli strain expressing an optimized λ prophage Red recombination system, termed PPY, which facilitates SLiCE with very high efficiencies.

  1. Cloning Sequencing and Structural Manipulation of the Enterotoxin D and E Genes from Staphylococcus aureus

    DTIC Science & Technology

    1990-05-30

    sign in mice injected with toxin samples from these clones. Only the mouse injected with whole cells from KS1866 showed flaking and peeling of the... epidermis . Even the E. coli strains containing pJJ825 (KSI825 and 15 KSI858) did not show exfoliation. It is possible that the E. coli strains do not

  2. Molecular Cloning of Adenosinediphosphoribosyl Transferase.

    DTIC Science & Technology

    1987-09-08

    AD-RIB5 458 NOLECULNA CLONING OF AOENOSINEDXPNOSPHORIBOSyL 1/1 TRNSFERASEMU CAILIFORNIA UNIV SRN FRANCISCO E KUN US SEP 8? WFOSR-TR-87-0982 SWFOSR-B5...ACCESSION NO.D,. 03261102F 2312 A~5 11. TITLE (include Securqt Classification) 0 Molecular Cloning of Adenosinediphosphoribosyl Transferase 12. PERSONAL...I’:- AFOSR.Tlt. 8 7 - 0 9 8,2 0IL * pi AFOSR- 85 -0377 PROGRESS REPORT Molecular Cloning of Adenosinediphosphoribosyl Transferase 5." Period of

  3. 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.

  4. 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.

  5. Genetic identification of a novel hantavirus of the harvest mouse Reithrodontomys megalotis.

    PubMed Central

    Hjelle, B; Chavez-Giles, F; Torrez-Martinez, N; Yates, T; Sarisky, J; Webb, J; Ascher, M

    1994-01-01

    We have cloned the S genomic segment of a novel hantavirus of the harvest mouse Reithrodontomys megalotis. The virus is phylogenetically distinct from other hantaviruses. The new hantavirus was identified in harvest mice separated by approximately 1,000 km. A wood rat (Neotoma mexicana) was found to be infected with the harvest mouse hantavirus. PMID:8084007

  6. Brain-specific expression of MAP2 detected using a cloned cDNA probe

    PubMed Central

    1986-01-01

    We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA "walking" experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule. Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum. Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein. The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues. Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus. We conclude that the expression of MAP2 is brain-specific. Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse. The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both. Under conditions of low

  7. 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.

  8. Current Protocols in Mouse Biology Tissue-specific regulation of oncogene expression using Cre-inducible ROSA26 knock-in transgenic mice

    PubMed Central

    Carofino, Brandi L.; Justice, Monica J.

    2015-01-01

    Cre-inducible mouse models are often utilized for the spatial and temporal expression of oncogenes. With the wide number of Cre recombinase lines available, inducible transgenesis represents a tractable approach to achieve discrete oncogene expression. Here, we describe a protocol for targeting Cre-inducible genes using a loxP-STOP-loxP approach to the ubiquitously expressed ROSA26 locus. Gene targeting provides several advantages over standard transgenic techniques, including a known site of integration and previously characterized pattern of expression. Historically, an inherent instability of ROSA26 targeting vectors has hampered the efficiency of developing ROSA26 knock-in lines. In this protocol, we provide individual steps for utilizing Gateway recombination for cloning, and detailed instructions for screening targeted ES cell clones. By following this protocol, one can achieve germline transmission of a ROSA26 knock-in line within several months. PMID:26069083

  9. 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

  10. 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

  11. 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.

  12. 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)

  13. 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)

  14. 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.

  15. 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.

  16. 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)

  17. 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.

  18. Animal Cloning and Food Safety

    MedlinePlus

    ... Products For Consumers Home For Consumers Consumer Updates Animal Cloning and Food Safety Share Tweet Linkedin Pin ... safe to eat as food from conventionally bred animals. This conclusion stems from an extensive study of ...

  19. 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)

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Genetic switch to hypervirulence reduces colonization phenotypes of the globally disseminated group A streptococcus M1T1 clone.

    PubMed

    Hollands, Andrew; Pence, Morgan A; Timmer, Anjuli M; Osvath, Sarah R; Turnbull, Lynne; Whitchurch, Cynthia B; Walker, Mark J; Nizet, Victor

    2010-07-01

    The recent resurgence of invasive group A streptococcal disease has been paralleled by the emergence of the M1T1 clone. Recently, invasive disease initiation has been linked to mutations in the covR/S 2-component regulator. We investigated whether a fitness cost is associated with the covS mutation that counterbalances hypervirulence. Wild-type M1T1 group A Streptococcus and an isogenic covS-mutant strain derived from animal passage were compared for adherence to human laryngeal epithelial cells, human keratinocytes, or fibronectin; biofilm formation; and binding to intact mouse skin. Targeted mutagenesis of capsule expression of both strains was performed for analysis of its unique contribution to the observed phenotypes. The covS-mutant bacteria showed reduced capacity to bind to epithelial cell layers as a consequence of increased capsule expression. The covS-mutant strain also had reduced capacity to bind fibronectin and to form biofilms on plastic and epithelial cell layers. A defect in skin adherence of the covS-mutant strain was demonstrated in a murine model. Reduced colonization capacity provides a potential explanation for why the covS mutation, which confers hypervirulence, has not become fixed in the globally disseminated M1T1 group A Streptococcus clone, but rather may arise anew under innate immune selection in individual patients.

  7. A Genetic Switch to Hypervirulence Reduces Colonization Phenotypes of the Globally Disseminated Group A Streptococcus M1T1 Clone

    PubMed Central

    Hollands, Andrew; Pence, Morgan A.; Timmer, Anjuli M.; Osvath, Sarah R.; Turnbull, Lynne; Whitchurch, Cynthia B.; Walker, Mark J.; Nizet, Victor

    2010-01-01

    Background The recent resurgence of invasive group A streptococcal disease has been paralleled by the emergence of the M1T1 clone. Recently, invasive disease initiation to has been linked to mutations in the covR/S two-compnent regulator. Here we investigate if a fitness cost is associated with covS mutation that counterbalances hypervirulence. Methods Wild-type M1T1 GAS and an isogenic covS mutant derived from animal passage were compared for adherence to human laryngeal epithelial cells, keratinocytes or fibronectin, biofilm formation, and binding to intact mouse skin. Targeted mutagenesis of capsule expression from both strains was performed for analysis of its unique contribution to the observed phenotypes. Results The covS mutant bacteria showed reduced capacity to bind to epithelial cell layers as a consequence of increased capsule expression. The covS mutant strain also had reduced capacity to bind fibronectin and to form biofilms on plastic and epithelial cell layers. A defect in skin adherence of the covS mutant strain was demonstrated in a murine model. Conclusions Reduced colonization capacity provides a potential explanation as to why the covS mutation conferring hypervirulence has not become fixed in the globally-disseminated M1T1 GAS clone, but rather may arise anew under innate immune selection in individual patients. PMID:20507231

  8. [Cloning of four members of giant panda Dmrt genes].

    PubMed

    Shui, Yi; Yu, Hong-Shi; Xia, Lai-Xin; Guo, Yi-Qing; Cheng, Han-Hua; Zhou, Rong-Jia

    2004-05-01

    Sex determining genes Mab-3 of C. elegans and Doublesex of Drosophila contain a common DNA binding motif called DM (Doublesex and Mab-3) domain, both of which regulate similar aspects of sexual development. Human Doublesex-related gene DMRT1 has been identified, which also contains the conserved DM-related DNA-binding domain and plays an essential role in gonadal differentiation. We amplified genomic DNA of the giant panda using the DM degenerate primers and detected two bands, approximately 140 bp and 250 bp. After cloned into T-easy vector and sequenced, four sequences showed high homology with the DM domain. Amino acid sequence of the first clone is 100% identical with the Dmrt1 of human, mouse and pig, hence we named it as pDmrt1. The second clone is 96% identical with human DMRTB1, and the third one 100% with the Dmrt3 of mouse and medaka, which were named as pDmrtb1 and pDmrt3 respectively. The last sequence contains an intron of 116 bp within the DM domain, which encodes an amino acid sequence 100% identical with human DMRTC2, accordingly we named it as pDmrtc2. Based on similarities of amino acid sequences of the DM domain, Dmrt protein sequences from human, mouse and giant panda were included in a phylogenetic tree. They revealed seven distinct subgroups: Dmrt1, Dmrt2, Dmrt3, Dmrt4 (DMRTA1), Dmrt5 (DMRTA2), Dmrt6 (DMRTB1) and Dmrt7 (DMRTC2). Our results further reveal the unexpected complexity and the evolutionary conservation of the DM domain gene family in both invertebrates and vertebrates.

  9. [Cloning of vertebrates: successes and problems].

    PubMed

    Koniukhov, B V

    1997-12-01

    Cloning of vertebrates, in particular, amphibians and mammals, is discussed. In the last decade, significant progress was made cloning mammals, while cloning of adult amphibians remained problematical. Low-traumatic methods of enucleation of recipient oocytes and transplantation of donor nuclei were worked out. In 1997, an adult sheep was cloned in Great Britain, thus demonstrating the possibility of cloning adult mammals. However, methods of cloning mammals need improvement because of the high lethality of reconstructed embryos (nuclear transplants). The use of in vitro cultured low-differentiated stem cells to obtain donor nuclei seems promising. Works on human cloning are not expedient in the near future because of technical and ethical aspects.

  10. Mouse centromere mapping using oligonucleotide probes that detect variants of the minor satellite.

    PubMed

    Kipling, D; Wilson, H E; Mitchell, A R; Taylor, B A; Cooke, H J

    1994-03-01

    Cytologically, the centromere is found at the very end of most Mus musculus chromosomes, co-localizing with an array of minor satellite sequences. It is separated from the euchromatin of the long arm by a large domain of heterochromatin, composed in part of arrays of major satellite sequences. We used oligonucleotide probes that specifically detect regions of sequence variation found in certain cloned minor satellite sequences. They detect a limited subset of the minor satellite arrays in the mouse genome, based on both pulsed-field gel electrophoresis and in situ hybridization data, and provide direct molecular genetic markers for individual centromeres in some inbred mouse strains. Array size polymorphisms detected by these probes map to positions consistent with the centromeres of chromosomes 1 and 14 in the BXD recombinant inbred (RI) strains. The genetic distances between these minor satellite arrays and loci on the long arms of chromosomes 1 and 14 are consistent with repression of meiotic recombination in the heterochromatic domains separating them. The existence of chromosome-specific minor satellite sequences implies that the rate of sequence exchange between non-homologous chromosomes relative to the rate between homologous chromosomes is much lower than has previously been postulated. We suggest that the high degree of sequence homogeneity of mouse satellite sequences may instead reflect recent common ancestry.

  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. Growth and hematologic characteristics of cloned dogs derived from adult somatic cell nuclear transfer.

    PubMed

    Park, Jung Eun; Kim, Min Kyu; Kang, Jung Taek; Oh, Hyun Ju; Hong, So Gun; Kim, Dae Young; Jang, Goo; Lee, Byeong Chun

    2010-04-01

    Three viable female dogs, which have the same genotype, have been successfully produced by somatic cell nuclear transfer (SCNT); however, data on the growth pattern of cloned dogs are lacking. Thus, the aim of this study was (1) to assess growth parameters among those cloned dogs with measurement of body weight, height, and radiographic analysis of skull size and bone plate, and (2) to compare hematologic characteristics among the donor dog, cloned dogs, and age-matched control dogs. The cloned dogs were kept in the same environmental conditions. The body weight increased from 0.52, 0.46, and 0.52 kg at birth to 21.9, 22.9, and 20.4 kg at 68 weeks of age for individual cloned dogs, respectively. The withers height increased from 34.5, 32.6, and 35.2 cm at 8 weeks of age to 67.1 cm at 68 weeks of age in the three clones. The radiographic data demonstrated that patterns of bone growth were similar among cloned dogs, and all measured parameters of matured cloned dogs were similar with that of the fully grown donor dog. An age-specific pattern was identified on hematologic and serum biochemical measurements in both cloned dogs and age-matched controls. The parameters examined were within the normal reference ranges for healthy dogs. In conclusion, three genetically identical cloned dogs showed similar growth characteristics and had normal hematological and serum biochemical parameters.

  13. DNA methylation errors in cloned mice disappear with advancement of aging.

    PubMed

    Senda, Sho; Wakayama, Teruhiko; Arai, Yoshikazu; Yamazaki, Yukiko; Ohgane, Jun; Tanaka, Satoshi; Hattori, Naka; Yanagimachi, Ryuzo; Shiota, Kunio

    2007-01-01

    Cloned animals have various health problems. Aberrant DNA methylation is a possible cause of the problems. Restriction landmark genomic scanning (RLGS) that enabled us to analyze more than 1,000 CpG islands simultaneously demonstrated that all cloned newborns had aberrant DNA methylation. To study whether this aberration persists throughout the life of cloned individuals, we examined genome-wide DNA methylation status of newborn (19.5 dpc, n=2), adult (8-11 months old, n=3), and aged (23-27 months old, n=4) cloned mice using kidney cells as representatives. In the adult and aged groups, cloning was repeated using cumulus cells of the adult founder clone of each group as nucleus donor. Two newborn clones had three with aberrantly methylated loci, which is consistent with previous reports that all cloned newborns had DNA methylation aberrations. Interestingly, we could detect only one aberrantly methylated locus in two of the three adult clones in mid-age and none of four senescent clones, indicating that errors in DNA methylation disappear with advancement of animals' aging.

  14. 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.

  15. Molecular cloning of human terminal deoxynucleotidyltransferase.

    PubMed Central

    Peterson, R C; Cheung, L C; Mattaliano, R J; Chang, L M; Bollum, F J

    1984-01-01

    A cDNA of the human terminal deoxynucleotidyltransferase (TdT; "terminal transferase," EC 2.7.7.31) was isolated from a human lymphoblastoid cell cDNA library in lambda gt 11 by using immunological procedures. Four inserts containing 723 to 939 base pairs were recloned in pBR322 for hybridization and preliminary sequence studies. mRNA selected by hybridization to recombinant DNA was translated to a 58-kDa peptide that specifically immunoprecipitated with rabbit antibodies to calf terminal transferase and mouse monoclonal antibody to human terminal transferase. Blot hybridization of total poly(A)+ RNA from KM3 (TdT+) cells with nick-translated pBR322 recombinant DNA detected a message of about 2000 nucleotides, sufficient to code for the 580 amino acids in the protein. mRNA from terminal transferase- cells gave no signal in hybrid selection or RNA blot hybridization. The complete sequence of the 939-base-pair insert sequence was obtained from deletions cloned in pUC8. The DNA sequence contains an open reading frame coding for 238 amino acids, about 40% of the protein. Three peptides isolated by HPLC from tryptic digests of succinylated 58-kDa calf thymus terminal transferase were sequenced, providing 20, 18, and 22 residues of peptide sequence. A search of the translated sequence of the 939-base-pair insert shows three regions beginning after arginine that have greater than 90% homology with the sequence determined from the calf thymus terminal transferase peptides. These results provide unambiguous evidence that the human terminal transferase sequence has been cloned. Images PMID:6087320

  16. 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.

  17. Local cloning of entangled states

    NASA Astrophysics Data System (ADS)

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

    2010-08-01

    We investigate the conditions under which a set S of pure bipartite quantum states on a D×D 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.

  18. 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.

  19. 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.

  20. Aberrant Expression of TIMP-2 and PBEF Genes in the Placentae of Cloned Mice Due to Epigenetic Reprogramming Error

    PubMed Central

    Kim, Hong Rye; Lee, Jae Eun; Oqani, Reza Kheirkhahi; Kim, So Yeon; Wakayama, Teruhiko; Li, Chong; Sa, Su Jin; Woo, Je Seok; Jin, Dong Il

    2016-01-01

    Cloned mice derived from somatic or ES cells show placental overgrowth (placentomegaly) at term. We had previously analyzed cloned and normal mouse placentae by using two-dimensional gel electrophoresis and mass spectrometry to identify differential protein expression patterns. Cloned placentae showed upregulation of tissue inhibitor of metalloproteinase-2 (TIMP-2), which is involved in extracellular matrix degradation and tissue remodeling, and downregulation of pre-B cell colony enhancing factor 1 (PBEF), which inhibits apoptosis and induces spontaneous labor. Here, we used Western blotting to further analyze the protein expression levels of TIMP-2 and PBEF in cloned placentae derived from cumulus cells, TSA-treated cumulus cells, intracytoplasmic sperm injection (ICSI), and natural mating (NM control). Cloned and TSA-treated cloned placentae had higher expression levels of TIMP-2 compared with NM control and ICSI-derived placentae, and there was a positive association between TIMP-2 expression and the placental weight of cloned mouse concepti. Conversely, PBEF protein expression was significantly lower in cloned and ICSI placentae compared to NM controls. To examine whether the observed differences were due to abnormal gene expression caused by faulty epigenetic reprogramming in clones, we investigated DNA methylation and histone modification in the promoter regions of the genes encoding TIMP-2 and PBEF. Sodium bisulfite sequencing did not reveal any difference in DNA methylation between cloned and NM control placentae. However, ChIP assays revealed that the level of H3-K9/K14 acetylation at the TIMP-2 locus was higher in cloned placentae than in NM controls, whereas acetylation of the PBEF promoter was lower in cloned and ICSI placenta versus NM controls. These results suggest that cloned placentae appear to suffer from failure of histone modification-based reprogramming in these (and potentially other) developmentally important genes, leading to aberrant

  1. [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.

  2. 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

    2016-04-01

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

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. Differential Evolution Optimization of Diffraction Pattern Models with Clones

    NASA Astrophysics Data System (ADS)

    Lynch, Vickie; Buergi, Hans-Beat; Hauser, Juerg; Hoffmann, Christina; Michels-Clark, Tara; Miller, Steve

    2010-03-01

    With the TOPAZ single crystal diffractometer at the Spallation Neutron Source in operation, new computational methods are needed for analyzing the three-dimensional diffraction patterns recorded from disordered crystals. One such method uses a combination of differential evolution and Monte Carlo techniques to model the disorder and analyze the diffuse scattering. Software implementing this method originally developed at the University of Bern has been modified to use TeraGrid high performance computers. Since different model crystals produced from a given set of disorder parameters differ, the error of the fit to the data of such clones differs too. The performance of the differential evolution optimization in improving the fit of the model is being tested by using a variable number of clones for each individual gene set in a generation of differential evolution. A reference dataset with minimal noise has been generated for this purpose. Results of tests varying the number of clones will be presented.

  8. Human cloning and human dignity.

    PubMed

    Birnbacher, Dieter

    2005-03-01

    Judging from the official documents dealing with the moral and legal aspects of human reproductive cloning there seems to be a nearly worldwide consensus that reproductive cloning is incompatible with human dignity. The certainty of this judgement is, however, not matched by corresponding arguments. Is the incompatibility of reproductive with human dignity an ultimate moral intuition closed to further argument? The paper considers several ways by which the intuition might be connected with more familiar applications of the concept of human dignity, and argues that there is no such connection. It concludes that the central objections to human reproductive cloning are not objections relating to dignity but objections relating to risk, especially the risks imposed on children born in the course of testing the method's safety.

  9. Cloning of Plasmodium falciparum by single-cell sorting

    PubMed Central

    Miao, Jun; Li, Xiaolian; Cui, Liwang

    2010-01-01

    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. PMID:20435038

  10. 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. Copyright 2010 Elsevier Inc. All rights reserved.

  11. Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction.

    PubMed

    Codner, Gemma F; Lindner, Loic; Caulder, Adam; Wattenhofer-Donzé, Marie; Radage, Adam; Mertz, Annelyse; Eisenmann, Benjamin; Mianné, Joffrey; Evans, Edward P; Beechey, Colin V; Fray, Martin D; Birling, Marie-Christine; Hérault, Yann; Pavlovic, Guillaume; Teboul, Lydia

    2016-08-05

    Karyotypic integrity is essential for the successful germline transmission of alleles mutated in embryonic stem (ES) cells. Classical methods for the identification of aneuploidy involve cytological analyses that are both time consuming and require rare expertise to identify mouse chromosomes. As part of the International Mouse Phenotyping Consortium, we gathered data from over 1,500 ES cell clones and found that the germline transmission (GLT) efficiency of clones is compromised when over 50 % of cells harbour chromosome number abnormalities. In JM8 cells, chromosomes 1, 8, 11 or Y displayed copy number variation most frequently, whilst the remainder generally remain unchanged. We developed protocols employing droplet digital polymerase chain reaction (ddPCR) to accurately quantify the copy number of these four chromosomes, allowing efficient triage of ES clones prior to microinjection. We verified that assessments of aneuploidy, and thus decisions regarding the suitability of clones for microinjection, were concordant between classical cytological and ddPCR-based methods. Finally, we improved the method to include assay multiplexing so that two unstable chromosomes are counted simultaneously (and independently) in one reaction, to enhance throughput and further reduce the cost. We validated a PCR-based method as an alternative to classical karyotype analysis. This technique enables laboratories that are non-specialist, or work with large numbers of clones, to precisely screen ES cells for the most common aneuploidies prior to microinjection to ensure the highest level of germline transmission potential. The application of this method allows early exclusion of aneuploid ES cell clones in the ES cell to mouse conversion process, thus improving the chances of obtaining germline transmission and reducing the number of animals used in failed microinjection attempts. This method can be applied to any other experiments that require accurate analysis of the genome for

  12. [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.

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

    PubMed

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

    2016-07-08

    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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. 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

  15. Cloning and expression of a novel neuropeptide Y receptor.

    PubMed

    Weinberg, D H; Sirinathsinghji, D J; Tan, C P; Shiao, L L; Morin, N; Rigby, M R; Heavens, R H; Rapoport, D R; Bayne, M L; Cascieri, M A; Strader, C D; Linemeyer, D L; MacNeil, D J

    1996-07-12

    The neuropeptide Y family of peptides, which includes neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP), are found in the central and peripheral nervous system and display a wide array of biological activities. These actions are believed to be mediated through pharmacologically distinct G protein-coupled receptors, and, to date, three members of the NPY receptor family have been cloned. In this study we describe the cloning and expression of a novel NPY receptor from mouse genomic DNA. This receptor, designated NPY Y5, shares 60% amino acid identity to the murine NPY Y1 receptor. The pharmacology of this novel receptor resembles that of the NPY Y1 receptor and is distinct from that described for the NPY Y2, Y3, and Y4 receptors. In situ hybridization of mouse brain sections reveals expression of this receptor within discrete regions of the hypothalamus including the suprachiasmatic nucleus, anterior hypothalamus, bed nucleus stria terminalis, and the ventromedial nucleus with no localization apparent elsewhere in the brain.

  16. 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

  17. 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)

  18. 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)

  19. Optimizing mouse models for precision cancer prevention

    PubMed Central

    Le Magnen, Clémentine; Dutta, Aditya; Abate-Shen, Cory

    2017-01-01

    As cancer has become increasingly more prevalent in our society, cancer prevention research has evolved toward placing a greater emphasis on reducing cancer deaths and minimizing the adverse consequences of having cancer. “Precision cancer prevention” takes into account the collaboration of intrinsic and extrinsic factors for influencing cancer incidence and aggressiveness in the context of the individual, as well as the recognition that such knowledge can improve early detection and more accurate discrimination of cancerous lesions. The premise of this review is that analyses of mouse models can greatly augment precision cancer prevention. However, as of now, mouse models, and particularly genetically-engineered mouse (GEM) models, have yet to be fully integrated into prevention research. Herein we discuss opportunities and challenges for “precision mouse modeling”, including their essential criteria of mouse models for prevention research, representative success stories, and opportunities for the more refined analyses in future studies. PMID:26893066

  20. Cloning of canine Toll-like receptor 7 gene and its expression in dog tissues.

    PubMed

    Okui, Yasuhumi; Kano, Rui; Maruyama, Haruhiko; Hasegawa, Atsuhiko

    2008-01-15

    Toll-like receptor 7 (TLR7) is activated by single strand RNA and imidazoquinoline compounds, and induces interferon production. In this study, canine TLR7 cDNA was cloned and sequenced. The full-length cDNA of canine TLR7 gene was 3419bp, encoding 1032 amino acids. The similarities of canine TLR7 with human and mouse TLR7 were 84 and 80% at the nucleotide sequence level, and 86 and 79% at amino acid sequence level, respectively. Further, the expression of TLR7 mRNA was investigated in canine normal tissues by semiquantitative RT-PCR analysis. The common expression level of TLR7 mRNA in tissues from three dogs examined was in large intestine, lung, pancreas, small intestine and skin, though the expression level in each tissue was varied among these healthy dogs. In other tissues (kidney, liver, lymph node, spleen, adrenal gland, and PBMCs), the level of TLR7 mRNA expression was different in individuals.

  1. Energy Values of Nine Populus Clones

    Treesearch

    Terry F. Strong

    1980-01-01

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

  2. Life-history consequences of adaptation to pollution. "Daphnia longispina clones historically exposed to copper".

    PubMed

    Agra, Ana Raquel; Soares, Amadeu M V M; Barata, Carlos

    2011-05-01

    The present study was conducted to assess life history effects of adaptation to pollution in Daphnia longispina clones historically exposed to an acid mine drainage from an abandoned pyrite mine. Four sensitive and resistant clones from reference and impacted populations were exposed to a range of copper exposure levels and their life history and physiological responses in terms of survival, reproduction, respiration and feeding rates compared. The most resistant clone was from 16 to 48 fold more tolerant to copper in terms of LC(50) and population growth rates than sensitive ones, respectively. The genetic differences between the resistant and sensitive clones were that in the polluted environment individuals of the resistant clone survived 50% better, reproduced 3 days earlier, grew 20% better, produced three more offspring per day and had population growth rates 45% greater. In the unpolluted environment, however, individuals from the resistant clone had the lowest reproduction and somatic growth rates but equivalent population growth rates, than sensitive daphnids. Thus, these life history changes did not translate into lower fitness in unpolluted environments in terms of population growth rates. Observed higher respiration rates of the most resistant clone support in part the energy cost hypothesis of tolerance, whereas increase feeding, reproduction and growth rates across copper exposure levels may also, indicate that resistant individuals need copper to fulfil they physiological demands thus supporting the metal deficiency hypothesis as well.

  3. 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.

  4. 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.

  5. How variable clones build an invariant retina.

    PubMed

    He, Jie; Zhang, Gen; Almeida, Alexandra D; Cayouette, Michel; Simons, Benjamin D; Harris, William A

    2012-09-06

    A fundamental question in developmental neuroscience is how a collection of progenitor cells proliferates and differentiates to create a brain of the appropriate size and cellular composition. To address this issue, we devised lineage-tracing assays in developing zebrafish embryos to reconstruct entire retinal lineage progressions in vivo and thereby provide a complete quantitative map of the generation of a vertebrate CNS tissue from individual progenitors. These lineage data are consistent with a simple model in which the retina is derived from a set of equipotent retinal progenitor cells (RPCs) that are subject to stochastic factors controlling lineage progression. Clone formation in mutant embryos reveals that the transcription factor Ath5 acts as a molecular link between fate choice and mode of cell division, giving insight into the elusive molecular mechanisms of histogenesis, the conserved temporal order by which neurons of different types exit the cell cycle. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. 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.

  7. 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…

  8. Reproductive cloning: past, present and future.

    PubMed

    Gurdon, J B

    2005-03-01

    This brief outline in reproductive cloning describes the background to these studies and then discusses successive aspects of the subject. These include abnormalities in cloned animals, therapeutic cloning and the ethics of this subject. A reference to further reading is provided.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Distinct herpesvirus resistances and immune responses of three gynogenetic clones of gibel carp revealed by comprehensive transcriptomes.

    PubMed

    Gao, Fan-Xiang; Wang, Yang; Zhang, Qi-Ya; Mou, Cheng-Yan; Li, Zhi; Deng, Yuan-Sheng; Zhou, Li; Gui, Jian-Fang

    2017-07-24

    Gibel carp is an important aquaculture species in China, and a herpesvirus, called as Carassius auratus herpesvirus (CaHV), has hampered the aquaculture development. Diverse gynogenetic clones of gibel carp have been identified or created, and some of them have been used as aquaculture varieties, but their resistances to herpesvirus and the underlying mechanism remain unknown. To reveal their susceptibility differences, we firstly performed herpesvirus challenge experiments in three gynogenetic clones of gibel carp, including the leading variety clone A(+), candidate variety clone F and wild clone H. Three clones showed distinct resistances to CaHV. Moreover, 8772, 8679 and 10,982 differentially expressed unigenes (DEUs) were identified from comparative transcriptomes between diseased individuals and control individuals of clone A(+), F and H, respectively. Comprehensive analysis of the shared DEUs in all three clones displayed common defense pathways to the herpesvirus infection, activating IFN system and suppressing complements. KEGG pathway analysis of specifically changed DEUs in respective clones revealed distinct immune responses to the herpesvirus infection. The DEU numbers identified from clone H in KEGG immune-related pathways, such as "chemokine signaling pathway", "Toll-like receptor signaling pathway" and others, were remarkably much more than those from clone A(+) and F. Several IFN-related genes, including Mx1, viperin, PKR and others, showed higher increases in the resistant clone H than that in the others. IFNphi3, IFI44-like and Gig2 displayed the highest expression in clone F and IRF1 uniquely increased in susceptible clone A(+). In contrast to strong immune defense in resistant clone H, susceptible clone A(+) showed remarkable up-regulation of genes related to apoptosis or d