Method of constructing nucleus-implanted egg, parthenogenetic embryo and parthenogenetic mammal

Abstract

Disclosed is a method for constructing a nucleus-implanted egg, a parthenogenetic embryo and for producing a parthenogenetic mammal each having 2 haploid genome sets originating in mammalian ova, and provides methods of constructing a nucleus-implanted egg having a haploid genome set derived from primitive ovarian follicle egg (ng ovum) and a haploid genome set from MII phase (second meiosis metaphase) egg (fg ovum), a parthenogenetic embryo and a parthenogenetic mammal, including steps (1) introducing ng ovum into a nucleus-deleted deleted germinal vesicle stage (GV) egg, developing the obtained egg to MII phase by in vitro maturing and culturing to prepare a first nucleus-implanted egg, and (2) extracting MII phase chromosome from the first nucleus-implanted egg and introducing it into other fg ovum to prepare a second nucleus-implanted egg, wherein a ng or fg ovum from which an imprinted gene undergoing epigenetic modification during sperm generation is used.

1 . A method of constructing a nucleus-implanted egg of a non-human mammal, the nucleus-implanted egg having a haploid genome set derived from ng ovum and a haploid genome set from fg ovum, which comprises the steps of
 (1) introducing a primitive ovarian follicle egg (ng ovum) into a nucleus-deleted egg in a germinal vesicle stage (GV stage egg) and then developing them to MII phase (second meiosis metaphase) by in vitro maturing and culturing to prepare a first nucleus-implanted egg, and   (2) extracting MII phase chromosome from said first nucleus-implanted egg and introducing it into other MII phase egg (fg ovum) to prepare a second nucleus-implanted egg,   wherein ovum from which an imprinted gene that undergoes gene modification posteriori during the generation of sperm is deleted is used as the ng ovum or fg ovum.   
   
   
       2 . The method of  claim 1 , wherein the primitive ovarian follicle egg (ng ovum) is an oocyte of a neonate. 
   
   
       3 . The method of  claim 1 , wherein the other MII phase egg is an ovulation ovum. 
   
   
       4 . The method of  claim 1 , wherein the imprinted gene is at least one gene selected from the group consisting of H19, Gtl2 and Ras-grf1 genes. 
   
   
       5 . The method of  claim 1  or  4 , wherein the ovum from which the imprinted gene is deleted is derived from a gene-deleted non-human mammal. 
   
   
       6 . A method of constructing a parthenogenetic embryo, which comprises activating the second nucleus-implanted egg obtained by the method recited in  claim 1  and then culturing the second nucleus-implanted egg in vitro to develop the same. 
   
   
       7 . A method of constructing a parthenogenetic non-human mammal, which comprises implanting the parthenogenetic embryo obtained by the method recited in  claim 6  into the uterus of a non-human mammal and growing the same.