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. 2017 Feb 8;7:42244. doi: 10.1038/srep42244

Figure 3. Generation of Nesprin-1α2 R257H knock-in mice.

Figure 3

(a) Sequence of CRISPR RNA (crRNA, red) to target the Cas9 nuclease to the arginine (R) 257 region in exon 4 in the Nesprin-1α2 (SYNE1) mouse gene. The protospacer adjacent motif (PAM) is shown in green. (b) Design of the mutation-specific primers to detect the R257 to histidine (H) amino acid substitution. (c) PCR screening using mutant-specific primers identified six of 17 newborn mice contained the correct gene variant allele. (d) Sub-cloning sequencing shows an individual clone has either wildtype (WT) or R257H knock-in (KI) alleles. A representative sequencing analysis of one founder (#13) of the six PCR-positive mice is shown. (e) Germline transmission analysis of the variant alleles to the F1 generation was achieved by backcrossing F0 knock-in mice with wildtype C57/B6 mice. All F0 mice showed successful germline transmission with an average efficiency of 52.8% (ranging from 36% to 71% among different founders, n = 3). A representative genotyping screen of offspring from one founder (#13) is shown.