Genotyping and expression analysis of Mx1 transgene in trangenic pigs. (A) PCR to identify piglets with genomic integration of the Mx1 transgene. Upper panel: PCR analysis with primers Mx1-2 and Mx1-7; Lower panel: PCR analysis with primers Mx1-1 and Mx1-5. The lanes are: M, marker; N, non-transgenic pig; #4-1, #4-2, #4-3, #4-4, #4-5, transgenic pigs; P, plasmid DNA. (B) Identification of mRNA level of Mx1 in the transgenic piglets using real time RT-PCR. Lanes 1–3 are naturally bred piglets, lanes 4–13 are cloned pigs without transgene integration, and lanes 14–18 are 5 cloned pigs that contained the transgene. Porcine GAPDH was used as reference control. Values represent the mean ± s.d. from triplicate experiments. Statistically significant P values are noted with an asterisk (*P < 0.001. One-way natural breeding pig was used to generate the P values). (C) Detecting the presence of Mx1 protein in fibroblasts of transgenic pigs by western blot. Cell lysates from HEK293T cells transiently transfected with pVAX-Mx1 were used as a positive control. (D) Expression of the Mx1 protein in various organs of transgenic and non-transgenic pigs. The arrowhead indicates the uncleaved transgenic Mx1-2A-EGFP protein and the arrow indicates the cleaved Mx1 protein.