Fig. 3: Generation of knockin (ki) mouse models.
From: The Piezo channel is a mechano-sensitive complex component in the mammalian inner ear hair cell
a Introduction of the mutation into the Pz1 and Pz2 loci by gene targeting. a, Cartoon of a short segment of the C-terminal ___domain (CTD) of Pz protein showing the sequence residues 2493–2496 in Pz1 MFEE mutated to AAAA. The corresponding conserved sequence in Pz2 was at 2767-2770, mutated to AAAA. The plasma membrane appears as a blue rectangle. Amino acids are labeled as indicated: polar, hydrophobic, acidic, and basic residues. b CRISPR/Cas9-mediated genome engineering was used. The Pz conditional overexpression ki mouse line features a “CAG promoter-loxP-3*SV40 pA-loxP-Kozak-Mutant Pz1 CDS-P2A-EGFP-rBG pA″ cassette inserted into intron 1 of the ROSA26 locus. The Pz2 conditional overexpression knockin mouse line features a “CAG promoter-loxP-3*SV40 pA-loxP-Kozak-Mutant Pz2 CDS-P2A-tdTomato-rBG pA″ cassette also inserted into intron 1 of ROSA26 locus. c, d Tail genomic DNA was digested for Pz1 (Pz1-ki) genotyping. We developed a PCR assay (35 cycles) with a pair of primers (607 bp: F:5″-AAGCACGTTTCCGACTTGAGTTG-3″ and R:5″-GGGTGAGCATGTCTTTAATCTACC-3″). The primers for the mutant were (356 bp: F:5″-TTCAGGGTCAGCTTGCCGTAG-3″ and R:5″-CTGCCGTGTGTGGACCGCATCCT-3″). The mutant Pz2 (Pz2-ki) primers were: (199 bp: F:5″-ACCTCCTCGCCCTTGCTCACCAT-3″ and R:5″-TCGTGAGGGAGACAGGGGAGTTG-3″).
