Production of transgenic rats by lentiviral transduction of male germ-line stem cells

Abstract

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 F 2 generation. Thus, by using these procedures, ≈30% of pups in the F 1 generation inherited and stably transmitted a lentiviral transgene that integrated at various genomic sites.

Efficient Enhancement of Lentiviral Transduction Efficiency in Murine Spermatogonial Stem Cells

“…Therefore lentiviral vector could be useful vector for transduction of cells that have a slow cell cycle, such as SSCs (Kim et al, 2010;Kubota et al, 2004;Nagano et al, 2002). In previous studies, transgenic animals were also produced by lentiviral transduction and transplantation of SSCs (Hamra et al, 2002;Ryu et al, 2007). …”

“…Several groups have produced transgenic animal using lentivirus-mediated SSC transduction (Hamra et al, 2002;Ryu et al, 2007). For most of these studies, the transgenic animals were produced by lentiviral infection of highly purified SSCs and transplanting them right into testes of the recipient animals.…”

Expression patterns of mitotic and meiotic cell cycle regulators in testicular cancer and development

“…In addition, we analyzed microarray hybridizations of RNA from murine laminin-binding germ cells (enriched in spermatogonia), laminin-nonbinding germ cells (enriched in spermatocytes), Sertoli cells and testicular interstitial cells. 22,23 The purity of the respective cell cultures was about 90%. 23 The primary hybridization data were extracted using Affymetrix Microarray Suite 5.0, and expression levels per chip were normalized to an average of 250.…”

Les cellules souches de la lignée germinale mâle : de l’approche fondamentale aux nouvelles technologies de transgenèse

“…Ces observations sont en accord avec la diminution des capacités de prolifération, migration et adhérence au collagène des cellules musculaires lisses vasculaires DDR1 -/-observées in vitro [6]. DDR2 est surexprimé dans les cellules étoilées du foie de rat après intoxication par le chlorure de carbone ou ligature du cholédoque [9], résultats à rapprocher du rôle mitogène de ce récepteur sur ces mêmes cellules in vitro [8]. Les DDR interviennent ainsi dans la répa-ration des lésions artérielles et probablement dans les remaniements secondaires à la formation des plaques d'athérosclérose.…”

“…Les critères cytologiques ou moléculaires pour l'identification et la purification de ces cellules sont encore fragiles. Cependant, les techniques performantes aujourd'hui dispo- Un travail récent a maintenant étendu au rat la transgenèse par transplantation germinale [9].…”