Figure 5. Generation of Mitf knockout pigs by zygote injection of R1 sgRNA and Cas9 mRNA.

(A) Newborn wild type pig (upper panel) and piglets carrying Mitf gene mutation (bottom panel). The photographs were taken by the author, Xianlong Wang. (B) RFLP agarose gel electrophoresis showing PCR product of target region derived from two piglets digested with BsaJI restriction enzymes. (C) Sanger sequencing of the targeting site in mutant pigs. The wild-type (WT) sequence is shown at the top, where the sgRNA sequence is labeled in red and the PAM sequence in purple. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. Deleted bases are marked with colons, and inserted bases are gray. (D) Western blot for MITF protein expression in the skin tissues of tail from wild type and mutant piglets. GAPDH was used as a loading control.