Kyoto University

Nepa Gene Co., Ltd.

Tomoji Mashimo

Takehito Kaneko

Yasuhiko Hayakawa

Kiyoshi Hayakawa

Kazuto Yoshimi

An object is to develop a technology enabling utilization, by only an extremely simple technique, of a technology which is widely applicable to mammals without requiring the utilization of an ES cell, and which involves modifying a certain gene by targeting a certain sequence on a genome (genome editing technology based on ZFN or the like). Provided is a technology for efficiently modifying an arbitrary target gene of a mammal, by immersing a pronuclear stage mammalian zygote with an intact zona pellucida into a solution containing a pair of molecules of mRNA having a certain sequence, and performing electroporation treatment through application of multiple square-wave pulses in three steps with the total electric energy of a first electric pulse adjusted within a predetermined range.

Mammalian gene modification method using electroporation

This invention provides a method for knock-in of a donor DNA into the genome of a cell, comprising introducing at least one artificial nuclease system capable of cleaving target sequence(s) of the cell genome, the donor DNA, and two single-stranded oligonucleotides (ssODNs) into the cell, the artificial nuclease system cleaving the target sequence(s) on the cell genome, the two ssODNs each complementary to one of the ends generated by the target sequence cleavage in the cell genome and to one of the introduction ends of the donor DNA, the donor DNA being knocked-in at the cleavage site via the two ssODNs.

Growing community of inventors

Kyoto, Japan

Tomoji Mashimo