Construction of 3p21.3-loxP Targeting Vector

The 3p21.3-loxP targeting vector was constructed as an X3.2 vector, which contains additional restriction enzyme sites (SpeI, BamHI, PacI, and SacI) of the AgeI site in the X3.1 vector (Kononenko et al. 2013 (link)), which contains a loxP site and a 3′ HPRT site. X3.2 vector constructed the annealing oligo 5′-CCGGTACTAGTCGGGATCCCCTTAATTAAGGGGAGCTCA-3′ and 5′-CCGGTGAGCTCCCCTTAATTAAGGGGATCCCGACTAGTA-3′ into subcloned into AgeI sites of the X3.1 vector. For the addition of the sgRNA target site in X3.2, the annealing oligo 5′-TAACCAAACACGTACGCGTACGATGCTAGCT-3′ and 5′-AGCATCGTACGCGTACGTGTTTGGTTAAT-3′ subcloned into PacI/SacI sites of the X3.2 vector (X3.3). For the addition of the marker gene, the blasticidin S-resistant gene was amplified using PCR with the pEBmulti-Bsd vector (Wako) as a template and primers (5′-GGAAGATCTCCAGCAGGCAGAAGTATGCAAAGCA-3′ and 5′-GGACTAGTCAAGTTTCGAGGTCGAGTGTCAGTC-3′), digested with BglII/SpeI (NEB, Hertfordshire, UK), and cloned into the X3.3 vector (3p21.3-loxP targeting vector).