Targeted Gene Correction in Albino Rats

For Tyr-repair of albino rats, a targeting ssAAV vector plasmid, pssAAV_rTyr-repair, was constructed. Tyr gene was amplified by PCR using the primers listed in Table S1 (Wistar Tyr amplification) using peripheral blood cells of Crlj:WI as a template. Tyr homology arms were amplified by PCR using the primers listed in Table S1 (5′-homology arm and 3′-homology arm) using the template as above. These homology arms include the Tyr_repair cassette, which has corrected sequences containing an in-frame silent mutation to provide an SnaBI site for RFLP analysis. Homology arms were inserted into pUC19 using an In-Fusion HD Cloning Kit. After confirmation of the sequence, homology arms and rTyr-repair cassette were excised using restriction endonucleases, NheI-HF and MluI-HF (New England Biolabs, Japan, Tokyo, Japan), and then ligated with pAAV_MCS2. AAV6_rTyr-repair was conducted as reported previously^{33 (link)}.