Mammalian gene modification method using electroporation

Abstract

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.

1 : A method of modifying a gene in a pronuclear stage zygote of a non-human mammal, 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 at least once 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-non-human mammal with an intact zona pellucida;   (B) RNA exhibiting, when associated with an endonuclease, 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;   (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; and   after performing the electroporation, culturing the resultant zygote into a 2- to 16-cell stage embryo in a medium, and then transplanting the embryo into an oviduct or a uterus of a female of the same species or an allied species of the non-human mammal to provide offspring.   
     
     
         2 . The method according to  claim 1 , wherein the non-human mammal is a primate. 
     
     
         3 . The method according to  claim 1 , wherein the non-human mammal is a swine. 
     
     
         4 . The method according to  claim 1 , wherein the non-human mammal comprises a species belonging to an order Rodentia. 
     
     
         5 . A method of creating a genetically modified individual of a non-human mammal, comprising modifying a gene in a pronuclear stage zygote of a non-human mammal, 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-non-human mammal with an intact zona pellucida;   (B) RNA exhibiting, when associated with an endonuclease, 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;   culturing the resultant zygote into a 2- to 16-cell stage embryo in a medium, and then transplanting the embryo into an oviduct or a uterus of a female of the same species to provide offspring.   
     
     
         6 . The method according to  claim 5 , wherein the non-human mammal is a primate. 
     
     
         7 . The method according to  claim 5 , wherein the non-human mammal is a swine. 
     
     
         8 . The method according to  claim 5 , wherein the non-human mammal comprises a species belonging to an order Rodentia. 
     
     
         9 . The method according to  claim 1 , wherein the nucleic acid molecule as defined in the following item (B) comprises a nucleic acid molecule as defined in the following item (b1) and a nucleic acid molecule as defined in the following item (b2):
 (B) RNA exhibiting, when associated with an endonuclease, endonuclease activity against an arbitrary region of genomic DNA in a sequence-specific manner;   (b1) mRNA encoding a protein having a sequence-specific DNA-binding domain, and a domain which exhibits restriction enzyme activity when forming a dimer with a restriction enzyme activity domain as defined in the following item (b2); and   (b2) mRNA encoding a protein having a sequence-specific DNA-binding domain which is a region in a vicinity of a genomic DNA region end to which the protein as defined in the item (b1) binds and which binds to a complementary strand thereof, and a domain which exhibits restriction enzyme activity when forming a dimer with the restriction enzyme activity domain as defined in the item (b1).   
     
     
         10 : The method according to  claim 1 , wherein the nucleic acid molecule as defined in the following item (B) comprises a nucleic acid molecule as defined in the following item (b3) and a nucleic acid molecule as defined in the following item (b4):
 (B) RNA exhibiting, when associated with an endonuclease, endonuclease activity against an arbitrary region of genomic DNA in a sequence-specific manner;   (b3) guide RNA having a complementary sequence of an arbitrary base sequence of the genomic DNA, and a sequence which specifically binds to a protein as defined in the following item (b4); and   (b4) mRNA encoding a protein which exhibits endonuclease activity when specifically binding to the guide RNA as defined in the item (b3).   
     
     
         11 . The method according to  claim 5 , wherein the nucleic acid molecule as defined in the following item (B) comprises a nucleic acid molecule as defined in the following item (b1) and a nucleic acid molecule as defined in the following item (b2):
 (B) RNA exhibiting, when associated with an endonuclease, endonuclease activity against an arbitrary region of genomic DNA in a sequence-specific manner;   (b1) mRNA encoding a protein having a sequence-specific DNA-binding domain, and a domain which exhibits restriction enzyme activity when forming a dimer with a restriction enzyme activity domain as defined in the following item (b2); and   (b2) mRNA encoding a protein having a sequence-specific DNA-binding domain which is a region in a vicinity of a genomic DNA region end to which the protein as defined in the item (b1) binds and which binds to a complementary strand thereof, and a domain which exhibits restriction enzyme activity when forming a dimer with the restriction enzyme activity domain as defined in the item (b1).   
     
     
         12 : The method according to  claim 5 , wherein the nucleic acid molecule as defined in the following item (B) comprises a nucleic acid molecule as defined in the following item (b3) and a nucleic acid molecule as defined in the following item (b4):
 (B) RNA exhibiting, when associated with an endonuclease, endonuclease activity against an arbitrary region of genomic DNA in a sequence-specific manner;   (b3) guide RNA having a complementary sequence of an arbitrary base sequence of the genomic DNA, and a sequence which specifically binds to a protein as defined in the following item (b4); and   (b4) mRNA encoding a protein which exhibits endonuclease activity when specifically binding to the guide RNA as defined in the item (b3).