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. 2019 Dec 1;33(23-24):1635–1640. doi: 10.1101/gad.328963.119

Figure 1.

Figure 1.

Generation of Dmpk knockin mice carrying CTG 3′UTR expansions. (A) CRISPR/Cas9-mediated recombination of HDR templates containing a CTG expansion flanked by homology arms at the Dmpk 3′UTR. External (blue) and internal (red) hybridization probes are indicated together with forward (F1) and reverse (R1) PCR primers. (B) Repeat dimerization by Golden Gate Assembly followed by RCA. Plasmids containing a repeat expansion flanked by BsaI and BbsI were linearized by SapI, linear constructs were separately digested by BsaI or BbsI, and repeat-containing fragments were gel purified, ligated, and amplified by RCA. This process generates a new construct carrying 2n-2 repeats, where “n” is the initial expansion size. (C) PCR of CTG202, CTG402, and CTG802 HDR templates. Linearized RCA products yielded a 2-kb backbone band (bb) and upper bands corresponding to the recombination template (dashed white lines indicate the isolated HDR templates). (D) PCR genotyping of Dmpk+/+ (wild-type), Dmpk+/170, and Dmpk+/480 mice.