Fig. 3

Generation of mice expressing zymogen-locked endogenous matriptase by ZFN-mediated gene editing. a Structure of donor DNA (top), wildtype St14 allele with the ZFN binding site (middle), and edited St14 allele (bottom) containing the base pair substitutions of interest. Exons are indicated as gray boxes and introns as black lines. The highlighted part of the donor DNA shows the dinucleotide substitution (indicated in red letters and underlined), the ZFN binding site (blue letters), and the synonymous nucleotide changes of the donor DNA (red letters) to avoid ZFN cleavage of the donor DNA. The ZFN binding site is shown below in capital letters with the ZFN cleavage site in red. Introduction of the donor DNA at the ZFN target site results in a c.1841–1842 GC → AA dinucleotide substitution. b Sequence analysis of exon 16 from St14 ^+/+ (top panel) and St14 ^zym/zym (bottom panel) mice confirms the GC → AA dinucleotide substitution causing the arginine 614 to glutamine substitution in endogenous matriptase (indicated by red letters in nucleotide and amino acid sequence). c and d Genotyping of St14 ^zym/zym mice. c The GC → AA dinucleotide substitution in endogenous St14 gene causes the loss of a Cac8I restriction endonuclease cleavage site and the generation of a SmlI restriction endonuclease cleavage site. d PCR amplification of genomic DNA from the targeted St14 exon 14 region from St14 ^+/+ (lanes 2 and 6), St14 ^zym/zym (lanes 3 and 7), and St14 ^zym/+ (lanes 4 and 8) mice. The amplified DNA was digested with SmlI in lanes 2–4 and with Cac8I in lanes 6–8. Position of molecular weight markers (lanes 1 and 5) are indicated on the left (bp)