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. 2020 Jun 29;10:10591. doi: 10.1038/s41598-020-67478-5

Figure 1.

Figure 1

Generation of Cln3Q352X mice. (A) Schematic of targeting vector. Guide RNAs were cloned into a guide RNA/cas9 expression vector by inserting double-stranded oligo cassettes into the Bbs I sites. gRNA and cassette sequences are demonstrated in Table 1. Each oligo cassette contained 20 bp gRNA sequences with a guanosine at the 5′ end for optimal expression, and adherent ends for cloning at Bbs I sites. (B) PCR amplification of transfected mouse N2A cells with each of the two gRNA vectors, and results of SURVEYOR assay showing non-homologous end joining frequency. (C) Schematic of single stranded oligo donor nucleotide (ssODN) DNA donor generation using mCln3.g14. Blue: mCln3.g14, Red: Q352X mutation, Yellow: silent leucine mutation. (D) Sequence chromatogram of cloned homozygous Cln3Q352X mouse. (E) Representative sequence chromatogram of heterozygous F1 Cln3Q352X mouse.