Figure 2.

Generation of mutant mice with a 5-Mb deletion with or without a donor plasmid. (A) Schematic representation of the 5-Mb region upstream of Sox9. The black line represents a genome sequence. Homology arms in a donor plasmid 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 donor plasmid (left panel) and without the donor plasmid (right panel). Embryo IDs are shown above images with the # sign. M: a 1-kb DNA ladder (markers); N: no template control. (C) Sequences of the donor plasmid and of embryos injected with the donor plasmid are aligned. Positions corresponding to arms and ClaI recognition sequence are indicated at the top. (D) Sequences of the donor plasmid and of embryos injected without the donor plasmid are aligned. Positions corresponding to arms and ClaI recognition sequence are indicated at the top. Parts of gRNA recognition sequences are underlined. Black and gray boxes correspond to those in (A).