Fig. 4

Generation of bulls with EGFP labeling at the SRY locus using SEGCPN. a Procedure for generating bulls with EGFP-labeling at the SRY locus using SEGCPN. b Schematic representation of the PCR analysis of the EGFP-labeled bovine. Primers P6 and P7 amplified a 2.2-kb product, primers P10 and P11 amplified a 1.9-kb product, and primers P6 and P11 amplified a 3.8-kb product to confirm positive EGFP-labeled bulls. M, 1-kb DNA ladder; C1–C3, EGFP-labeled bulls; WT, wild-type bull. c DNA sequencing between endogenous and exogenous DNA corresponding to homologous recombination in the EGFP-labeled bulls. d Southern blotting analysis of the EGFP-labeled bulls. The red rectangular box represents the positive band size obtained using different enzymes. Upon digestion with BglII, a band of 8.4 kb resulting from the targeted introduction of pSRY-EGFP was detected; using BsrGI, the expected fragment size was 3.3 kb; using PstI, the expected fragment size was 4.0 kb. C1–C3, EGFP-labeled bulls; WT, wild-type bull. e Identification of mosaicism in an EGFP-labeled bull. P10/P11, identification of precise EGFP-labeling in various bull tissues. Neo-F/R, the marker gene detected in various bull tissues. Cell, positive gene-targeted clone; lanes 3–11, various tissues of an EGFP-labeled bull; WT, wild-type tissue; P, donor vector; H₂O, negative control. f Photos of semen from the EGFP-labeled bulls and WT bulls. g RT-PCR analysis of EGFP expression in tissues from EGFP-labeled bulls. GAPDH was used as a reference