An efficient evaluation system for factors affecting the genome editing efficiency in mouse.

Genome editing technology is widely used in the field of laboratory animal science for the production of genetic disease models and the analysis of gene function. One of the major technical problems in genome editing is the low efficiency of precise knock-in by homologous recombination compared to simple knockout via non-homologous end joining. Many studies have focused on this issue, and various solutions have been proposed; however, they have yet to be fully resolved. In this study, we established a system that can easily determine the genotype at the mouse (Mus musculus) Tyr gene locus for genome editing both in vitro and in vivo. In this genome editing system, by designing the Cas9 cleavage site and donor template, wild-type, knockout, and knock-in genotypes can be distinguished by restriction fragment length polymorphisms of PCR products. Moreover, the introduction of the H420R mutation in tyrosinase allows the determination of knock-in mice with specific coat color patterns. Using this system, we evaluated the effects of small-molecule compounds on the efficiency of genome editing in mouse embryos. Consequently, we successfully identified a small-molecule compound that improves knock-in efficiency in genome editing in mouse embryos. Thus, this genome editing system is suitable for screening compounds that can improve knock-in efficiency.

Comparative study of doses of exogenous progesterone administration needed to delay parturition in Jcl:MCH(ICR) mice.

The effects of progesterone (P4) used in physiological studies and in delayed parturition in reproductive engineering were examined. A dose of 0.25, 0.5, 1, or 2 mg of P4 was repeatedly administered to Jcl:MCH(ICR) mice on days 17 and 18 of gestation, and plasma concentrations of P4 were investigated. The P4 concentrations in mothers and fetuses after administration of exogenous P4 were no differences between doses of 1 and 2 mg. Jcl:MCH(ICR) mothers administered a P4 dose of 1 mg did not give birth. Therefore, we consider 2 mg of P4 is an overdose and that it is evident that a dose of 1 mg P4 is sufficient to induce delayed parturition.