Fig. 5

Generation of cows with site-specific gene replacement using SEGCPN. a Procedure for generating cows with site-specific gene replacement using SEGCPN. b Schematic representation of the PCR analysis of the cows with site-specific gene replacement. Primers P14 and P15 amplified a 2.7-kb product, primers P17 and P18 amplified a 2.6-kb product, and primers P14 and P17 amplified a 6.8-kb product to confirm positive gene replacement in cows. M, 1-kb DNA ladder; C1–C3, gene replacement cows; WT, wild-type cow. c Southern blotting analysis of the gene replacement cows. The red rectangular box represents the positive band size obtained using different enzymes. Using SpeI, the expected fragment size was 8.2 kb; using the BamHI, the expected fragment size was 4.4 kb. C1–C3, gene replacement cows; WT, wild-type cow. d DNA sequencing between endogenous and exogenous DNA corresponding to homologous recombination in the gene replacement cows. e Identification of mosaicism in a gene replacement cow. P17/P18, identification of precise gene replacement in various cow tissues. Neo-F/R, the marker gene detected in various cow tissues. Cell, positive gene-targeted clone; lanes 3–12, various tissues of the gene replacement cow; WT, wild-type tissue; P, donor vector; H₂O, negative control. f Image of the gene replacement cows. g Analysis of the expression of recombinant human α-LA. Whey protein from the gene replacement cows and wild-type cows was separated by native PAGE and then subjected to blotting and hybridization with an anti-HLA antibody. Lane 1, bovine α-LA; lane 2, bovine whey; lanes 3–4, transgenic whey from the gene replacement cows; lane 5, human whey; lane 6, human α-LA