Figure 4.

Generation of mutant mice with a 5-Mb inversion. (A) Schematic representation of the 5-Mb region upstream of Sox9. The black line represents a genome sequence. Homology arms are indicated with black (arm1) and gray (arm3) boxes. PCR primers are indicated with arrows. The inverted region is denoted by a box containing arrowheads. (B) Agarose gel electrophoretogram images of PCR products amplified using 3F2/1F1 (upper panel), 3R1/1R2 (middle panel), 3F3/1F2 (lower left panel), and 3R3/1R4 (lower right panel) primer sets and DNA prepared from the embryos. Sample ID is indicated at top of each panel. Arrows and arrowheads indicate positive and nonspecific signals, respectively. M: a 100-bp DNA ladder (markers); N: no-template control. (C) Sequences of inversion junctions. Results of direct sequencing of PCR products shown in (B). Corresponding sequences of arms and inverted sequences are shown at the top of each panel. Parts of gRNA recognition sequences are underlined. Asterisks indicate an unknown sequence. The obelisk indicates a genomic sequence nonhomologous to the arms or to an internal sequence flanked by sgRNA3 and sgRNA1. Black and gray arrows correspond to those in (A). The inverted region is indicated with a box containing arrowheads as shown in (A).