Transgenic modification of spermatogonial stem cells using lentiviral vectors

The continuous production of spermatazoa throughout the reproductive lifetime of a male depends on the maintenance of a pool of progenitor cells called spermatogonial stem cells (SSCs). SSCs represent a very small fraction of the cellular population in the testes and lack definitive molecular markers for their identification. The discovery of conditions that allow one to propagate mouse SSCs in vitro essentially indefinitely has truly facilitated studies of the molecular mechanisms regulating SSC function. While multiple conditions for culturing SSCs have now been described, here we detail a method for culturing SSCs that uses a simpler medium than the original formulation. As with numerous other primary and stem cell cultures, it is difficult to introduce DNA into cultured SSCs using standard transfection approaches. However, VSV-G pseudotyped lentivirus efficiently infects cultured SSCs with minimal toxicity. Here we present protocols for producing lentivirus and stably modifying the genome of cultured SSCs using lentiviral vectors.