MAMMALIAN GENE MODIFICATION METHOD USING ELECTROPORATION

摘要

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.

主权项

1: A mammalian gene modification method, comprising:
immersing a zygote as defined in the following item (A) into a solution containing a nucleic acid molecule as defined in the following item (B); applying a square-wave electric pulse as defined in the following item (C) to the solution once or two or more times so that the square-wave electric pulse has a total electric energy of from 0.2 J/100 μL to 7.5 J/100 μL; then applying a square-wave electric pulse as defined in the following item (D) two or more times; and then applying a square-wave electric pulse as defined in the following item (E) two or more times: (A) a pronuclear stage zygote of a mammal, except for a human, with an intact zona pellucida; (B) RNA which functions so as to exhibit endonuclease activity against an arbitrary region of genomic DNA in a sequence-specific manner; (C) a square-wave electric pulse having a voltage per pulse of 375 V/cm or more; (D) a square-wave electric pulse having a voltage per pulse of 250 V/cm or less and an electric energy per pulse of from 0.01 J/100 μL to 3.6 J/100 μL; and (E) a square-wave electric pulse which is opposite in polarity to the electric pulse as defined in the item (D) and has a voltage per pulse of 250 V/cm or less and an electric energy per pulse of from 0.01 J/100 μL to 3.6 J/100 μL.