TY - JOUR
T1 - Production of transgenic rats by lentiviral transduction of male germ-line stem cells
AU - Hamra, Franklin K
AU - Gatlin, Joel
AU - Chapman, Karen M.
AU - Grellhesl, Dana M.
AU - Garcia, J. Victor
AU - Hammer, Robert E
AU - Garbers, David L.
PY - 2002/11/12
Y1 - 2002/11/12
N2 - 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 F2 generation. Thus, by using these procedures, ≈30% of pups in the F1 generation inherited and stably transmitted a lentiviral transgene that integrated at various genomic sites.
AB - 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 F2 generation. Thus, by using these procedures, ≈30% of pups in the F1 generation inherited and stably transmitted a lentiviral transgene that integrated at various genomic sites.
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U2 - 10.1073/pnas.222561399
DO - 10.1073/pnas.222561399
M3 - Article
C2 - 12391306
AN - SCOPUS:0037069345
SN - 0027-8424
VL - 99
SP - 14931
EP - 14936
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 23
ER -