Maximal Donor Germline Transmission from DAZL-Deficient Rats.

Abstract

<p>(A) Southern blot analysis of progeny from Wildtype (WT) and DAZL-Deficient (Dazl) recipient rats transplanted with 50,000 <i>GCS-EGFP</i> spermatogonia/right testis at passage 13 (i.e. 158 days in culture); left testes of each animal were not transplanted. At 75 days post-transplantation recipients (R) were paired with WT females (F) and allowed to produce pups by natural breeding (See R942-R949 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone-0006308-t001" target="_blank">Table 1</a>). Shown are blots from representative litters probed for <i>EGFP</i> to distinguish progeny produced by donor cells. OMP = loading control. Genomic DNA Controls were from untreated <i>GCS-EGFP</i> and <i>DAZL</i>-Deficient transgenic rats. LTR = PCR primers specific for lentiviral transgene in <i>DAZL</i>-deficient rats. GAPDH = PCR loading control. (B) Bright field and green fluorescence images of testes from Wildtype (<i>Left</i>) and DAZL-deficient (<i>Right</i>) recipient rats at 212 days post-transplantation. Scale bar = 1 cm. (C) Graph of germline transmission rates for the donor, <i>GCS-EGFP</i> transgene from Wildtype and DAZL-deficient recipient rats transplanted with 50,000 <i>GCS-EGFP</i> spermatogonia/right testis at passage 13; left testes were not transplanted. <i>DAZL</i>-deficient recipients transmitted the <i>GCS-EGFP</i> transgene to 100%+/−0% of progeny (+/−SEM, n = 3 recipients; 9 litters), with 73 of 73 total F1 pups born from donor cells. Wildtype recipients transmitted the <i>GCS-EGFP</i> transgene to 14%+/−5.9% of progeny (+/−SEM, n = 3 recipients; 9 litters), with 16 of 116 total F1 pups born from donor cells. (D) Genealogy tree showing stable transmission of donor haplotypes from <i>DAZL</i>-Deficient recipients (F0) R989 and R990 to F1 and F2 progeny. Recipients were each transplanted with 150,000 rat spermatogonia/testis from line RSGL-GCS9 at passage 17 (See R988-R990 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone-0006308-t001" target="_blank">Table 1</a>). Spermatogonial line RSGL-GCS9 was derived from a rat homozygous for the <i>GCS-EGFP</i> transgene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone.0006308-Wu1" target="_blank">[36]</a>. Thus, F1 progeny represent half-siblings; some of which were crossed to re-derive transgenic F2 progeny homozygous for the <i>tgGCS-EGFP</i> allele.</p