Transgenic rat models will be developed by genetic modification and transplantation of male germ line stem cells. Rats are widely used models of basic biology, and human physiology and disease, but the study of genes and their functions in the rat lags far behind progress in the mouse. While production and analysis of gain-of-function (transgenic, knock-in) and loss-of-function (gene targeted knockout) mutant mice have become routine and provide the most direct way to correlate specific genes with phenotypes, transgenic production in rats and other species is inefficient, with knockout technology unavailable. Development of rat genomic resources (the Rat Expressed Sequence Tag [EST] program and the Rat Genome program), combined with decades of phenotypic and physiological data, provide unprecedented opportunities for establishing rat models of mammalian biology. Spermatogonial stem cells constitute a unique vehicle for modifying the germ line, and it has been established that genetic manipulation and transplantation of this male germ line stem cell provides an alternative means for the production of transgenic mice. Since the spermatogenic process is well conserved across mammalian species, it is hypothesized that this new technology will be readily translated to rats. Furthermore, development of conditions for maintaining or expanding spermatogonial stem cells in culture will improve efficiency and expand the repertoire of genetic approaches that can be implemented. This technology will be developed in the rat because the rat is an established model for human physiology and disease, but the results should have broad implications for the development of transgenesis in other species. The recently described spermatogonial transplantation technique will be used to address and enable the following specific aims: 1) produce donor rat testis cell populations enriched for spermatogonial stem cells and develop appropriate rat recipient models; 2) introduce genetic modifications into donor rat spermatogonial stem cells and recover the new genotype in progeny of recipient rats; 3) develop conditions for maintaining and expanding rat spermatogonial stem cells in culture to increase their accessibility for genetic modification; and 4) immortalize rat spermatogonial stem cells by introducing the gene encoding telomerase reverse transcriptase (TERT) to establish the foundation for generating targeted genetic changes.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Research Project (R01)
Project #
5R01RR018500-03
Application #
6917058
Study Section
Special Emphasis Panel (ZRR1-CM-3 (01))
Program Officer
Rall, William F
Project Start
2003-09-15
Project End
2008-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
3
Fiscal Year
2005
Total Cost
$380,000
Indirect Cost
Name
Magee-Women's Research Institute and Foundation
Department
Type
DUNS #
119132785
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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Sheng, Yi; Lin, Chih-Cheng; Yue, Junming et al. (2010) Generation and characterization of a Tet-On (rtTA-M2) transgenic rat. BMC Dev Biol 10:17
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Hermann, Brian P; Sukhwani, Meena; Lin, Chih-Cheng et al. (2007) Characterization, cryopreservation, and ablation of spermatogonial stem cells in adult rhesus macaques. Stem Cells 25:2330-8
Ryu, Buom-Yong; Orwig, Kyle E; Oatley, Jon M et al. (2007) Efficient generation of transgenic rats through the male germline using lentiviral transduction and transplantation of spermatogonial stem cells. J Androl 28:353-60
Buageaw, Anyanee; Sukhwani, Meena; Ben-Yehudah, Ahmi et al. (2005) GDNF family receptor alpha1 phenotype of spermatogonial stem cells in immature mouse testes. Biol Reprod 73:1011-6

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