Dramatic reduction of mice used for genetic manipulations employing novel technology for gene-targeting.

This proposal describes an alternative to traditional procedures for creating knockout mouse strains, developed by Prof. M. Ohtsuka (Tokai University, Japan) in 2018 and thereafter established at Harvard Medical School by Prof. T. Kobayashi, who recently visited our laboratory to help us set up the technology required.

Referred to as i-Gonad, this approach is based on genetic manipulations of DNA in a single cell mouse embryos directly in the womb.This methodology reduces the numbers of mice required to create novel mutant strains by more than 80%. Furthermore, this reduction can be even greater when targeting several genes.

For instance, when creating triple knockouts from heterozygous parents with traditional procedures, only 1 in 64 of the offspring is triple homozygous, meaning that 63 mice are sacrificed as by-products of the breeding scheme. Here we will check if these by-products can be dramatically reduced by i-Gonads.

It theoretically allows introducing novel genetic modifications directly into established mutant strains.

This should dramatically reduce the number of mice utilized for expansion breeding (starting from 25 folds).The ability to introduce mutations associated with specific human diseases, along with the capacity to manipulate several genes at once and do it on genetically altered background, will not only reduce the number of mice required for numerous experiments, but also make novel types of experiments possible with no loss in scientific rigor.