Figure 3.

Generation of mutant mice with a 5-Mb deletion using an ssODN template. (A) Schematic representation of the 5-Mb region upstream of Sox9. The black line represents genome sequence. Homology arms in the ssODN are indicated with black and gray boxes. PCR primers are indicated with arrows. Positions of sgRNA recognition sequences are denoted with scissors. (B) An agarose gel electrophoretogram image of PCR products amplified using 3F1 and 1R1 primers and DNA prepared from embryos injected with the ssODN template. Embryo IDs are shown above images with the # sign. M: a 1-kb DNA ladder (markers); N: no template control. (C) Sequences of the ssODN template and of embryos injected with the ssODN template are aligned. Positions corresponding to arms and ClaI recognition sequence are indicated at the top. Parts of gRNA recognition sequences are underlined. The asterisk indicates an unknown sequence. The obelisk indicates genomic sequence nonhomologous to the ssODN or to the internal sequence flanked by sgRNA3 and sgRNA1. Black and gray arrows correspond to those in (A).