1991 — 2000 |
Bishop, Colin Edward |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Biology of the Mouse Y Chromosome @ University of Tennessee Health Sci Ctr
The Sxr (Sex reversed) region is a small fragment of the mouse Y chromosome short arm, transposed distal to the pairing/recombination region of the Y in XYSxr mutant mice. During male meiosis, Sxr is regularly transferred to the paternal X chromosome, giving rise to sterile XXSxr males. It has been shown that this region contains the genes controlling primary sex determination, Tdy; H-Y antigen expression, Hya (as defined by T cell killing and proliferation assays); a spermatogenesis gene, Spy; the Zinc finger genes Zfy-1 and Zfy-2 and Sry a prime candidate for Tdy. In addition we have compelling evidence for a new conserved Sxr gene with both X and Y linked copies, detected by an Sxr probe pY8. A deletion mutant of Sxr termed Sxrb has been described which has lost Zfy-2 sequences and Hya and Spy functions. Hence, both Hya and Spy are contained within the deletion and are now amenable to molecular analysis and cloning. The thrust of this (revised) grant proposal is (1) to construct a long-range map the Sxr region; (2) to produce a high resolution contig map of the entire Sxrb deletion using overlapping YAC clones; (3) to clone Hya and investigate its relationship to Spy using transgenic technology, and (4) to characterize the new Y-X common gene. YACs spanning the Sxrb deletion will be transfected into an XXSxrb (H-Y negative) cell line and colonies screened with cloned proliferative helper and killer lines for the expression of H-Y. Positive clones containing the Hya gene will be analyzed for their DNA content with Sequence Tagged Site (STS) probes taken from the relevant YAC clone and exonic sequences of Hya sought using species conservation, etc. The cloning of Hya will allow us to investigate its specific function and more broadly the role of the some 30 minor transplantation antigens that have been identified in both mouse and man. A new fundamental field of study could thus be opened up. The cloning of the new Sxr gene(s) from the Y and X, initially detected with Sxr probe pY8 and a detailed analysis of this finding, is new and relevant to the field as a whole. The program itself is designed to generate as much valuable scientific information as possible. The isolation and characterization of overlapping YAC clones covering the entire Sxrb deletion and the isolation of evenly spaced STS's will give a high resolution map of this important region and enable us to examine the strong possibility that there may exist as yet unknown genes in this area of the Y. The long-range map of the Sxr region is essentially a map of the short arm of the mouse Y, containing a minimum of six important genes. In addition to providing essential data on the size of the Sxrb deletion, all chromosomal maps such as these will be of central importance in the wider context of the "genome project".
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0.943 |
2009 — 2010 |
Bishop, Colin Edward |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Development of Normal and Dystrophic Canine Ips Lines @ Wake Forest University Health Sciences
A large number of genetic diseases occur in dogs mainly due to founder effects and population bottlenecks resulting from centuries-old inbreeding practices. These include von Willebrand disease, severe combined immunodeficiency disorder, X-linked Duchenne type muscular dystrophy, pyruvate kinase deficiency, muscle type phosphofructokinase deficiency, hemophilia and narcolepsy. More than fifty-eight percent of dog genetic diseases, such as Duchenne-type muscular dystrophy, are true homologues of human diseases caused by mutations in the same gene. In addition, many of the more than 400 modern dog breeds show a high prevalence of more complex multigenic diseases, including allergies, cancers, blindness, heart disease, cataracts, epilepsy, hip dysplasia and deafness. Because of obvious parameters such as lifespan and size, dogs represent an important model for human disease. In dogs, the physiology, disease presentation and clinical response often parallels that of the human more closely than the corresponding mouse model. They therefore provide a unique preclinical model to test the usefulness of regenerative medicine and tissue engineering approaches to the treatment and cure of Human disease. Pivotal to these new approaches will be the use of the differentiated derivatives of embryonic stem cells (ES). The recent demonstration that is possible to generate autologous, pluripotent stem cells (iPS) from skin fibroblasts, by relatively simple reprogramming steps is a major scientific advance which will have profound ramifications in many areas of basic research and applied clinical medicine. It is our central hypothesis that the generation and characterization f canine iPS cell lines will hasten the development of replacement tissues and organs in human. In order to address this hypothesis the following three specific aims are proposed (1) To generate induced pluripotent stem cell lines (iPS) from normal and dystrophic (GRMD) dogs. (2) Gene expression profiles of iPS lines will be characterized and compared to a blastocyst derived canine (3) To assess the pluripotency of canine ES and iPS lines in vivo and in vitro.
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1 |
2009 — 2010 |
Bishop, Colin Edward |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pluripotency of Amniotic Fluid-Derived Stem Cells @ Wake Forest University Health Sciences
Human pluripotent stem cells (hPSCs) win be crucial for the development of regenerative therapies, especially when autologous, specialized cells cannot be obtained in sufficient quantity. Despite great .promise, pluripotent embryonic stem (ES) cells have some recognized drawbacks. These include tumorigenicity and difficulty of histocompatibility matching. Other classes of hPSCs may overcome these limitations. In the agency-wide Program Announcement, amniotic fluid is noted specifically as a potential non-embryonic source for hPSCs. We recently described clonal human AFS cell lines and demonstrated that they are able to give rise to cell lineages that include representatives of each of the three embryonic germ layers. Others have described the use of defined genetic factors to reprogram somatic cells to an ES-like state, termed induced pluripotent stem (iPS) cells. We hypothesize that AFS cells represent a developmentally more advanced stage than ES and iPS cells, while nevertheless retaining a high degree of pluripotency. In particular, with support from preliminary data, we anticipate that AFS cells will not give rise to teratomas under conditions conducive to tumor formation by its and iPS cells, Therefore, critical comparison of genes expressed by AFS and ES or iPS cells should lead to significant insights into functions essential for pluripotency and also into those associated with teratoma formation. A confounding factor in such comparisons is that cell lines derived from different human beings are genetically diverse. In order to focus precisely on differences [unreadable] corresponding to developmental stage, we propose to compare matched pairs of AFS and iPS cells from the same individuals. This will be achieved by using defined factors to reprogram AFS cells to the more primitive ES-like state. (We refer to such reprogrammed cells iPS-AFS cells). In this revised version of our original R01 application we intend to hire two new employees from outside the University. A new junior post-doctoral fellow and a new junior technician.
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1 |
2009 — 2010 |
Bishop, Colin Edward |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Primate Ips Lines Using Retrovirus and Tat Protein Transduction @ Wake Forest University Health Sciences
DESCRIPTION (provided by applicant): The overriding hypothesis of this application is that the generation and characterization of non Human primate (NHP) induced pluripotent stem cell lines (iPS) will hasten the development of replacement tissues and organs in Humans. Macaque monkeys share an extremely close phylogenetic relationship with Man, and develop natural or experimentally induced disease states which accurately mimic the Human condition. NHPs can provide a unique preclinical model to test the usefulness of iPS cells and regenerative medicine approaches to the treatment and cure of disease, where other models may fail. The ability to generate autologous, pluripotent stem cells from skin fibroblasts, by relatively simple reprogramming steps, is a quantum scientific advance which will have major ramifications in many areas of basic research and applied clinical medicine. It is highly unlikely, however, that the use of viral vectors in the reprogramming protocol (as it exists now) will be acceptable for use in patients. It is vital, therefore, that alternative ways of reprogramming, without virus, be investigated. The short 11 amino acid protein transduction domain (PTD) derived from the HIV transactivator, TAT has been used to deliver large (~110kD), biologically active proteins directly into cells in vitro and in vivo. TAT fusion proteins and peptides have also been used to treat mouse models of cancer, inflammation and other diseases. It is our hypothesis that recombinant TAT and be able to reprogram cells into a pluripotent state. Once well characterized, lines derived by viral or protein transduction methods can be used in NHP preclinical models for a number of different diseases. We therefore propose to: (1) generate induced pluripotent stem cell lines (iPS) from the non-human primate, Macaca fascicularis (cynomolgus macaque) (2) To determine if M. fascicularis iPS cells can be produced non-virally using recombinant proteins modified with an HIV derived, TAT cell penetrating motif to aid in protein translocation across the plasma membrane and (3) characterize and compare iPS lines generated by viral and protein transduction.
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1 |