Direct modification of spermatogonial stem cells using lentivirus vectors in vivo leads to efficient generation of transgenic rats

Spermatogonial stem cells(SSCs)transmit genetic information to the next progeny in males.Thus,SSCs are a potential target for germ I i ne modifications to gen erate tran sgenic an imals.In this study,we report a technique for the gen erati on of tran sgenic rats by in vivo manipulation of SSCs with a high success rate.SSCs in juvenile rats were transduced in vivo with high titers of lentivirus harbori ng enhan ced green fluoresce nt protei n and mated with wild-type females to create foun der rats.These founder rats expressed the transgene and passed on the transgene with an overall success rate of 50.0%.Subsequent generations of progeny from the founder rats both expressed and passed on the transgene.Thus,direct modification of SSCs in juvenile rats is an effective means of generating transgenic rats through the male germline.This technology could be adapted to larger animals,in which existing methods for gene modificatio n are in adequate or in applicable,resulti ng in the gen eration of tran sge nic an imals in a variety of species.

Characterization of transgenic mice expressing EGFP under the control of the monkey 20α-hydroxysteroid dehydrogenase promoter

To directly assess the molecular function of the monkey 20α-hydroxysteroid dehydrogenase(20α-HSD) promoter, we generated transgenic mice(tg) expressing enhanced green fluorescent protein(EGFP) under control of this promoter. We demonstrated that prostaglandin F2α induced 20α-HSD promoter activity in CHO cells in a dose-dependent manner. Furthermore, forskolin treatment markedly reduced 20α-HSD promoter activity, and prolactin exhibited weak inhibitory activity. The transgenic mouse obtained one positive founder male. The transgene was propagated in 10 successive generations without any notable defects to the progeny. EGFP and 20α-HSD in the tg mice were colocalized in the luteal cells of the ovary during late pregnancy. Strong EGFP and 20α-HSD protein signals were also detected in the adult testis. Immunohistochemical analysis revealed high EGFP levels in the seminiferous epithelium, whereas 20α-HSD was expressed in the seminiferous tubules. Our data suggest that the ovaries in monkey and mouse exhibit similar expression patterns of 20α-HSD during pregnancy. However, the expression pattern of EGFP in tg mice testis slightly differed from that of the endogenous 20α-HSD. Further investigation is required to elucidate the functional mechanisms underlying regulation of the monkey 20α-HSD promoter in the tg mice.