Production of Transgenic Rats by Lentiviral Transduction of Male Germ-Line Stem Cells

• Published in: Proceedings of the National Academy of Sciences - PNAS 2002-11, Vol.99 (23), p.14931-14936
• Main Authors: Hamra, F. Kent, Gatlin, Joel, Chapman, Karen M., Grellhesl, Dana M., Garcia, J. Victor, Hammer, Robert E., Garbers, David L.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Animals, Genetically Modified - genetics; beta-Galactosidase - genetics; Biological Sciences; Cell culture techniques; Cell Transplantation; Cells; Cultured cells; Fertilization; Genetic Vectors; Germ cells; Germ Cells - physiology; Green Fluorescent Proteins; Homozygote; Immunohistochemistry; Lentivirus - genetics; Luminescent Proteins - genetics; Male; Mice; Molecular Sequence Data; Peptide Fragments - chemistry; Proteins - chemistry; Proteins - genetics; Rats; RNA-Binding Proteins; Somatic cells; Spermatogenesis; Stem cells; Testes; Testis - transplantation; Transgenes; Transgenic animals
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.222561399

Primary cultures of rat spermatogenic cells that did not bind to collagen matrices were able to colonize and form mature spermatozoa when transferred to testes of recipient males. Up to 73% of the progeny from matings with recipient males were derived from the transferred spermatogenic cells. Subsequently, two populations of germ cells were obtained by selection on laminin matrices. Both populations expressed the spermatogenic cell marker, DAZL, but not the somatic cell marker, vimentin. The cells that bound to laminin represented ≈5% of the total population and were greatly enriched in ability to colonize a recipient testis, suggesting an enrichment in germ-line stem cells. The colonization potential was maintained for at least 7 days in culture. These cells were subsequently transduced with a lentiviral enhanced GFP reporter vector and then transferred to WT recipient males. After mating, 26 of 44 pups were derived from the cultured donor germ cells, and 13 pups carried the lentiviral transgene. Based on Southern analysis, the transgene was integrated at a different genetic locus in each animal and was transmitted to ≈50% of pups in the F2generation. Thus, by using these procedures, ≈30% of pups in the F1generation inherited and stably transmitted a lentiviral transgene that integrated at various genomic sites.