Retrovirus - Spermatogonial Stem Cell-mediated Transgenic Approach

Recombinant retrovirus-spermatogonial stem cell approach is the common used method of transgenic animal production. In order to select an efficiently mediated method and ideal recipicent cell, a new method of transgenic mice had been established in this study. Recombinant retrovirus from PA-3 X I Ocfiulml, carrying NeoR, together with typan blus and polybrene, were injected into contorted seniniferous tubule of the mice (14-16 day) to infect spermatogonial stem cells in vivo. After 45 days, sperm were harvested from injected mice, it抯 qualities were examined. Three double primers were used in PCR to examine the genomic DNA of sperm. The results showed that injection liquid didn抰 have contrary effect on the qualities of sperm and the generation ability of male mice. Nine out often samples were transgenic, NeoR gene were confirmed to be integrated into the germ cell. Female mice were mated with the male respectively, and 48 offspring were produced. PCR and SothernRblotting were used to Screen Neo gene from the genomic DNAsamples extracted from the 48 offspring tails. PCR showed that nine were demonstrated to be transgenic, Sothern blotting examined thatfour of them including three females and one male were transgenic. Four founders were further mated with the nontransgenic mice respetively. Fifteen offsping were profticed and PCR were used toRscreen Neo gene integration. Four of five offsping from number one female founder were transgenie mice. The results showed,seminiferous tuble that were infected by recombinant retrovirus integrated Neo R gene into spermatogonial stem cell, and brougt it into eggs through sperm cell, so transgenic mice, carrying NeoR, were produced. The integration rate was 8.3%. As a result, the method appears to make a foundation to construct transgenic animal that not only for mice but also for large animal.