User-Friendly Genetic Conditional Knockout Strategies by CRISPR/Cas9

Figure 2

Genotyping of Eed conditional knockout generated by a two-step targeting strategy. (a) Schematics of sequential targeting LNL and FNFL cassettes to the Eed gene in two steps. (b) The genotyping results of sequential targeting LNL and FNFL cassettes to the Eed gene. In clones with biallelic insertion of LNL cassettes, a 2.29 kb DNA fragment was amplified without detecting the 431 bp wild-type amplicon with primers F1 and R1 (b1). When the neo^r cassette was depleted upon 4-OHT treatment, we detected a 497 bp PCR amplicon with primers F1 and R1, which was slightly larger than the 431 bp wild-type allele due to the remaining LoxP site (b2). For FNFL homozygous insertion, the PCR amplicon changed from the 263 bp wild-type allele to the 2.16 kb targeting allele using primers F2 and R2 (b3). Next, Dox-inducible FNFL depletion results in the 311 bp DNA fragment, which was slightly larger than the 263 bp wild-type allele because of the remaining LoxP and FRT sites (b4). Finally, when the targeting clones were treated with 4-OHT, a 1.3 kb PCR amplicon was amplified with primers F3 and R3 instead of detecting a 2.38 kb DNA fragment (b5), indicating that the 2nd exon of Eed was deleted.

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