A Novel Transgenic Rat Model for the Study of Germ Cell Biology




Cronkhite, Jennifer T.

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With over one million publications in scientific journals, the rat is a very important biological model in science. Unfortunately, since the introduction of genetic manipulation technology in the mouse, extension of this technology to the rat has proven to be very difficult. In an attempt to generate a transgenic line of rats expressing GFP in all cells of the body, a serendipitous integration of a ROSA-EGFP transgene resulted in exclusive expression of EGFP in the germ cells of both sexes. EGFP expression was uniform and robust in cleavage stage embryos beginning at the late 2-cell stage and continuing through blastocyst development where expression became restricted to cells of the inner cell mass. Subsequent analysis showed high EGFP expression exclusively in primordial, embryonic, and adult germ cells. This unique expression pattern makes this EGFP marked locus the first molecular marker of the germline lineage in both sexes in mammals. FISH was used to localize the transgene insertion to chromosome 11q11-q12, proximal to Grik1 and in close proximity to Ncam2. Analysis of the region did not identify known germ cell-specific genes but did identify 19 ESTs or transcribed loci present in testes, ovary, or pre-implantation libraries from mice or rats. The unique germ cell specific expression of EGFP in these transgenic rats makes them an excellent novel tool to study germ cell origin, development, and differentiation. To evaluate the utility of the transgenic line for germ cell transplantation studies, non-selected, freshly isolated seminiferous tubule cells were transferred to the testis of recipient males. The donor cell population colonized the testis at a surprisingly high efficiency within 30 days following transfer. Since EGFP is a vital marker, the colonization process can be followed in vivo and the extent of colonization quantified. This assay was then used to define when developing germ cells first acquire apparent stem cell activity, and to assess the plasticity of adult SP bone marrow cells to enter the germ lineage.

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