Skip to main content
. 2013 Oct 22;8(10):e77696. doi: 10.1371/journal.pone.0077696

Figure 2. Targeted insertion of a restriction site using ZFNc-kit and gene-targeting ssODNs.

Figure 2

a. A schematic of the mouse c-kit locus and the ssODN sequences of different sizes designed to introduce an exogenous BglII restriction site (in blue) into the genome in vivo using ZFNc-kit and ssODNs.

b. Detection of introduced BglII site in founder animals. The representative gel electropherogram of genotyping results by BglII digestion of PCR product. F0 pups were genotyped by BglII digestion of the 592 bp PCR product amplified from tail genomic DNA. The PCR product from the wild type c-kit allele cannot be digested by BglII (WT), while the PCR product from the targeted mutant allele (10 out of 109 founders) can be digested by BglII into two distinct fragments (238 bp and 354 bp). The PCR primers are listed in Table S1.

c. Sequence analysis of the c-kit mutant allele of the 10 founders with correct BglII restriction bands. The target region of the mouse c-kit gene was PCR-amplified from tail genomic DNA of the 5-day-old founders and subjected to T-A cloning. Colonies were randomly picked for DNA sequencing. Precise BglII site insertion was detected in 8 out of 10 mutant founders. ZFN binding site is underlined. BglII sites are highlighted in blue. Indels are highlighted in red.