Figure 2.
Single preimplantation embryos lacking the transgene contain L1 RNA (A–C) and L1 retrotransposition events (D). RT–PCR analysis and genotyping PCR on offspring of an L1LRE3 mouse (A), an L1RP rat (B), and an L1 GF 21 mouse (C). RNA isolated from single morulae or blastocysts was subjected to RT–PCR to detect L1 RNA from the L1 transgene. For genotyping, DNA from each embryo was subjected to genotyping PCR. To exclude the possibility of a false negative genotype for the transgene, each embryo was genotyped by two independent primer sets for the L1 transgene, and three independent genomic loci were amplified as controls. In A–C, an asterisk denotes a transgene-negative, L1 RNA-positive preimplantation embryo. (D) Retrotransposition events in individual late blastocysts. Single blastocysts of the L1RP mouse were subjected to genotyping PCR. For semiquantification, mouse DNA that carries one retrotransposition event per diploid genome (Ostertag et al. 2002) was used as a calibrator DNA. (Middle) The amount of DNA of each blastocyst used in the intron-flanking PCR was presumed to be 0.1–0.5 ng. Thus, retrotransposition events appear to be present in much less than one copy per cell. (RT) Reverse transcriptase; (Tg) transgene; (Rtn) retrotransposition event; (WT) wild-type animal; (M) 1-kb plus DNA Ladder (Invitrogen).