Fig. 3.
Generation of live born PSEN1M146I RMCE piglets. a Piglets generated by RMCE and SCNT. Four of 20 live born piglets are shown. b Southern blot analysis of genomic DNA isolated from 16 RMCE piglets and pig #2772 digested with SpeI. A 670-bp Neor fragment was used as probe. Lanes 1–16 represent 16 RMCE piglets, lane 17 pig #2772, lane 18 wt pig, lane 19 wt pig DNA mixed with PSEN1M146I plasmid DNA, and lane 20 molecular weight marker. The blue arrow marks the band in pig #2772 absent in RMCE piglets. c Southern blot analyses as in b except for the use of a PSEN1M146I probe. The blue arrow marks the PSEN1M146I transgene present in RMCE piglets. Three other bands present in the wt pig and pig #2772 are marked with black arrows (endogenous PSEN1). Positive control band (lane 19) is marked with a black triangle. d Top panel Schematic drawing of the RMCE targeted acceptor locus B. Black arrows indicate positions of primers used to reveal RMCE. f Arrow marks the forward genomic primer upstream of LIR and x marks the reverse primer specific of either PSEN1M146I or GFP. Lower panel PCR on genomic DNA from two RMCE piglets (lanes 1, 2, 5, and 6) and pig #2772 (lanes 3 and 7). Lanes 4 and 8 are water controls. f Primer was used with primer x, PSEN1M146I or GFP, in lanes 1–4 and 5–8, respectively. M is a 1 kb ladder. e Expression of bi-cistronic PSEN1M146I-IRES-Pac mRNA in fibroblasts of five RMCE piglets. Lanes 1–5 PCR on cDNA synthesized from fibroblast RNA, lanes 6–10 control PCR on –RT templates, lane 11 water control (W), and lane 12 positive control (P). M, 0.1 kb ladder