Polymerase chain reaction (PCR) analysis of the genomic DNA of <i>Recombination activating gene-1</i>-knockout fetal fibroblasts and tail tissues.

<p>(A) Polymerase chain reaction (PCR) analysis of <i>Recombination activating gene-1</i> (<i>Rag-1</i>)-knockout (KO) genomic DNA to detect the inserted vectors. The lane marked T6-12 displays results from the original porcine fetal fibroblasts used for preparation of the KO cells. The lanes marked WT displays results from the non-transgenic wild-type controls. Lanes marked #95-2 and <i>Rag</i>^+/− PFF display results from <i>Rag-1</i> heterozygous KO cells and <i>Rag-1</i> heterozygous KO porcine fetal fibroblasts. Lanes marked #3-55 and <i>Rag</i>^−/− PFF display results from <i>Rag-1</i> homozygous KO cells and <i>Rag-1</i> homozygous KO porcine fetal fibroblasts. Lanes marked <i>Rag</i>^−/− No. 1 and No. 2 display the results from the tails of the two <i>Rag-1</i> homozygous KO neonatal piglets. Lanes marked WT No. 1 and No. 2 display results from the tails of non-transgenic wild-type control piglets. Primers P 1, P 2, and P 3 were used for PCRs. Lane 1 detects the heterozygous KO of targeted allele 1, and lane 2 detects the homozygous KO of targeted allele 2. (B) PCR of genomic DNA to demonstrate the deletion of the <i>Rag-1</i> region of Exon 2 by homologous recombination. WT is shown as a nontransgenic wild-type control. <i>Rag-1</i> heterozygous and homozygous KO are shown (^+/−) and (^−/−). The lane marked T6-12 displays results from the original porcine fetal fibroblasts used to prepare the KO cells. Lanes marked #95-2 and <i>Rag</i>^+/− PFF display results from the <i>Rag-1</i> heterozygous KO cells and <i>Rag-1</i> heterozygous KO porcine fetal fibroblasts. Lanes marked #3-55 and <i>Rag</i>^−/− PFF display results from the <i>Rag-1</i> homozygous KO cells and <i>Rag-1</i> homozygous KO porcine fetal fibroblasts. Lanes marked <i>Rag</i>^−/− pig No. 1 and No. 2 display results from the tails of the <i>Rag-1</i> homozygous KO neonatal piglets. Lanes marked wild-type pig No. 1 and No. 2 display results from the tails of non-transgenic wild-type control piglets. The primer set consisting of Pc 1 and Pc 2 was used for the PCR analysis. The 27-base pair (bp) deletion detected in Exon 2 including open reading frame was caused by homologous recombination.