CUSTOMIZED TRANSGENESIS VIA MODIFICATION OF SPERMATOGONIAL STEM CELLS

Spermatogonia mediated transgenesis is becoming an increasingly popular method of genetic modification in animals. It guarantees direct germline transmission because spermatogonial stem cells are the unipotent type of stem cell and can differentiate only into the mature spermatozoa. Additionally, modified spermatogonial stem cells can be clonally expanded. Clone or several clones can be specifically identified for the presence of the desired alteration, expanded in vitro and transplanted in vivo. Since expression of DAZL gene as well as DAZL promotor driven transgene is restricted to germ cell line, we expected that DAZL promoter-driven transgenic tamoxifen inducible ERT2CreERT2 can be utilized for the germline-specific controllable Cre mediated recombination of targeted DNA flanked by loxP sites. Here we describe a transposon-mediated transformation of spermatogonial stem cells and generation of transgenic rat line, DAZL-ERT2CreERT2 facilitated by transplantation of modified cells into testis of DAZL deficient recipients-producers. In obtained animal line tamoxifen inducible engineered ERT2CreERT2 recombinase is specifically expressed in germ cells at a level sufficient for liganddepended efficient recombination. Using male germ cells from a generated animal and its cross to GCS-EGFP transgenic rat, several reporter spermatogonial cell lines were prepared to test the performance of the transgene in vitro and in vivo. This transgenic rat line can be utilized not only for analysis of the genetic makeup of spermatogenesis but potentially for germline activation and transmission of desirable ERT2CreERT2 mediated rearrangements of tissue-specific genetic elements flanked by loxP sites located in the part of the spermatogonial genome, accessible for the recombinase.