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.

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.

Abstract: 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.

Abstract: 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.

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.

Abstract: The present invention provides a composition for freezing or freeze-drying spermatozoa or sperm heads thereof, wherein the composition enables the spermatozoa or sperm heads thereof to maintain chromosome integrity at a temperature of about +4° C. to about ?200° C. Also provided is a container containing the composition. The present invention further provides a method for maintaining chromosome integrity of spermatozoa or sperm heads during freezing or freeze-drying. The present invention also provides methods for freezing or freeze-drying spermatozoa to obtain at least one spermatozoan capable of fertilizing an oocyte to produce a live offspring. Also provided are frozen or freeze-dried spermatozoa produced by these methods, and containers containing the frozen or freeze-dried spermatozoa. Finally, the present invention provides methods for producing a live mammalian offspring from an oocyte fertilized with a rehydrated freeze-dried (or thawed or freeze-thawed) spermatozoan.

Abstract: The invention provides methods for producing transgenic embryos and animals with a higher efficiency than presently available procedures based on Intracytoplasmic Sperm Injection (ICSI) of nucleic acid complexed with recombinase.

Abstract: The present invention provides a composition for freezing or freeze-drying spermatozoa or sperm heads thereof, wherein the composition enables the spermatozoa or sperm heads thereof to maintain chromosome integrity at a temperature of about +4° C. to about −200° C. Also provided is a container containing the composition. The present invention further provides a method for maintaining chromosome integrity of spermatozoa or sperm heads during freezing or freeze-drying. The present invention also provides methods for freezing or freeze-drying spermatozoa to obtain at least one spermatozoan capable of fertilizing an oocyte to produce a live offspring. Also provided are frozen or freeze-dried spermatozoa produced by these methods, and containers containing the frozen or freeze-dried spermatozoa. Finally, the present invention provides methods for producing a live mammalian offspring from an oocyte fertilized with a rehydrated freeze-dried (or thawed or freeze-thawed) spermatozoan.