Fig. 2

Generation of site-specific lactoferrin knock-in pigs using CRISPR/Cas9 technology. a Detection of site-specific insertion of selected clones using PCR genotyping. Lanes 1–12 represent 12 different clones, lanes 2, 3, 7, 9, 10 represent clone 25#, 27#, 68#, 75# and 81#, respectively; lane N represents the negative control wild-type cell line. b A genetically modified piglet at day 3 postpartum. c Detection of site-specific insertion of donor DNA in the genetically modified pigs using a PCR genotyping assay. Lanes 1–7 represent 7 individuals, and lane WT represents one negative control wild-type pig. WT, wild-type. d Nucleotide sequence analysis of junctions between endogenous and exogenous DNA corresponding to homologous recombination (HR) events. P5/P6 primers were used to amplify the specific region for the left- and right-hand junctions. Primary nucleotide sequence data corresponding to transition regions between the homologous arms of the targeting vector and the internal transgene DNA. LF, lactoferrin