Fig. 1.

Schematics of the strategy for inserting the neomycin and EGFP selection cassette into the MYL2 locus. a TALENs targeting site for the MYL2 gene and the homologous recombination events. Red arrow indicates the stop codon of MYL2 gene. After introduction of a double-strand break near the MYL2 exon 7 after the stop codon by TALENs, an IRES-Neo (donor 1) or P2A-EGFP (donor 2) along with an excisable PGK-Puromycin drug selection cassette sequence was inserted into the MYL2 locus downstream of the TAG stop codon (middle panel). The bottom panel shows the targeted genomic locus after Cre-mediated excision of the Puro selection cassette. Blue box, exon of the MYL2 gene. b Nested-PCR strategy to identify successfully targeted clones. Both gels indicated 10 out of 14 clones had at least one copy of the targeted allele (targeting efficiency was 71.5%). MYL2 myosin light chain 2, TALEN transcription activator-like (TAL) effector nuclease, IRES internal ribosome entry site, P2A self-cleaving peptide sequence, Neo neomycin resistant cassette, EGFP enhanced green fluorescent protein, PGK phosphoglycerol kinase promoter, Puro puromycin resistance gene, polyA polyadenylation sequence, 5’arm 5’-homology arms, 3’arm 3’-homology arms