ES cells can proliferate without limit, and even after prolonged culture, retain the ability to form cells ranging from cardiac muscle to nerve to blood; potentially any cell type that makes up the body. The recent derivation of nonhuman primate (NHP), i.e. rhesus monkey and common marmoset, and human ES cells has widespread implications for human developmental biology, drug discovery, drug testing, and transplantation medicine. Because of the close embryological similarities between humans and old world monkeys, rhesus monkey ES cells and rhesus monkeys provide an extremely accurate, necessary model system for developing human ES cell-based therapies. ? ? The efficient genetic manipulation of primate ES cells is essential to: 1) elucidate gene function both during differentiation and in specific differentiated cells; 2) direct the differentiation of ES cells to specific lineages by the manipulation of transcription factors; 3) purify desired differentiated cell types from a mixed population of ES cell derivatives by introducing selectable markers; 4) use the differentiated derivatives of primate ES cells as vehicles for gene therapy; and 5) modulate the immune response to transplanted ES cell derivatives. Unfortunately, primate ES cells are extremely difficult to transfect. Transfection methods routinely used for mouse ES cells fail for primate ES cells. Recently, a series of pseudotyped, self-inactivating lentiviral vectors with internal promoters were tested. These are the first vectors tested that allow the derivation of rhesus and marmoset ES cell lines stably expressing a foreign gene. To improve these vectors for primate ES cells, and to use them as tools to dissect mechanisms of primate ES cell self-renewal, the following Specific Aims will be accomplished to: 1) generate lentiviral vectors with improved long term, site independent expression in primate ES cells and their differentiated derivatives by testing the effects of insulator sequences and scaffold attachment regions; 2) use a functional ES cell screen to isolate novel genomic DNA fragments that promote long term, integration site independent expression of lentiviral vectors; 3) use lentiviral vectors to test the role of specific genes involved in the self-renewal of other stem cells (Beta-catenin, ID-1, N-myc, Notch, STAT-3) in promoting the self-renewal of primate ES cells; and 4) use a lentiviral cDNA expression cloning strategy to identify novel genes produced by fibroblasts, or by the ES cells themselves that promote ES cell self-renewal.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Resource-Related Research Projects (R24)
Project #
5R24RR016209-02
Application #
6637900
Study Section
National Center for Research Resources Initial Review Group (RIRG)
Program Officer
Harding, John D
Project Start
2002-07-01
Project End
2007-06-30
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
2
Fiscal Year
2003
Total Cost
$430,987
Indirect Cost
Name
University of Wisconsin Madison
Department
Veterinary Sciences
Type
Other Domestic Higher Education
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Ramezani, Ali; Hawley, Teresa S; Hawley, Robert G (2008) Combinatorial incorporation of enhancer-blocking components of the chicken beta-globin 5'HS4 and human T-cell receptor alpha/delta BEAD-1 insulators in self-inactivating retroviral vectors reduces their genotoxic potential. Stem Cells 26:3257-66
Hawley, Robert G (2008) Does retroviral insertional mutagenesis play a role in the generation of induced pluripotent stem cells? Mol Ther 16:1354-5
Ramezani, Ali; Hawley, Teresa S; Hawley, Robert G (2008) Reducing the genotoxic potential of retroviral vectors. Methods Mol Biol 434:183-203
Hong, Sunghoi; Hwang, Dong-Youn; Yoon, Soonsang et al. (2007) Functional analysis of various promoters in lentiviral vectors at different stages of in vitro differentiation of mouse embryonic stem cells. Mol Ther 15:1630-9
Yang, Zhaoqing; Gagarin, Dmitry; Ramezani, Ali et al. (2007) Resistance to fas-induced apoptosis in cells from human atherosclerotic lesions: elevated Bcl-XL inhibits apoptosis and caspase activation. J Vasc Res 44:483-94
Owens, Bronwyn M; Hawley, Teresa S; Spain, Lisa M et al. (2006) TLX1/HOX11-mediated disruption of primary thymocyte differentiation prior to the CD4+CD8+ double-positive stage. Br J Haematol 132:216-29
Hawley, Robert G; Ramezani, Ali; Hawley, Teresa S (2006) Hematopoietic stem cells. Methods Enzymol 419:149-79
Ramezani, Ali; Hawley, Teresa S; Hawley, Robert G (2006) Stable gammaretroviral vector expression during embryonic stem cell-derived in vitro hematopoietic development. Mol Ther 14:245-54
Riz, Irene; Hawley, Robert G (2005) G1/S transcriptional networks modulated by the HOX11/TLX1 oncogene of T-cell acute lymphoblastic leukemia. Oncogene 24:5561-75
Akimov, Sergey S; Ramezani, Ali; Hawley, Teresa S et al. (2005) Bypass of senescence, immortalization, and transformation of human hematopoietic progenitor cells. Stem Cells 23:1423-33

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