Nuclear transfer embryo formation method

Abstract

A nuclear transfer embryo is formed by destabilizing microtubules of an oocyte, whereby essentially all endogenous chromatin collects at a second polar body during meiosis of an oocyte. The oocyte is fused with the nucleus of a donor somatic cell of the same species of said oocyte prior to cessation of extrusion of the second polar body from the oocyte, thereby forming the nuclear transfer embryo. In one embodiment, the nuclear transfer embryo is employed to impregnate an animal, such as a mammal. In another embodiment, the donor nucleus is transgenic.

1 - 20 . (canceled)  
   
   
       21 . A method of cloning a mammal, comprising: 
 a) destabilizing microtubules of an oocyte, whereby essentially all endogenous genetic material collects at a second polar body during meiosis of said oocyte; and    b) combining the oocyte with at least the nucleus of a donor cell of the same species of said oocyte prior to cessation of extrusion of the second polar body from said oocyte, thereby forming a nuclear transfer embryo.    c) impregnating a mammal of the same species as the nuclear transfer embryo with the nuclear transfer embryo under conditions suitable for gestation of the cloned mammal; and    d) gestating the embryo, thereby causing the embryo to develop into the cloned mammal.    
   
   
       22 . (canceled)  
   
   
       23 . A method of producing a transgenic mammal, comprising the steps of: 
 a) destabilizing microtubules of an oocyte, whereby essentially all endogenous genetic material collects at a second polar body during meiosis of said oocyte; and    b) combining the oocyte with at least the nucleus of a donor cell of the same species of said oocyte prior to cessation of extrusion of the second polar body for said oocyte, thereby forming a nuclear transfer embryo.    c) impregnating a mammal of the same species as the nuclear transfer embryo with the nuclear transfer embryo under conditions suitable for gestation of the transgenic mammal; and    d) gestating the embryo, thereby causing the embryo to develop into the transgenic mammal.    
   
   
       24 . A method of cloning a mammalian fetus, comprising the steps of: 
 a) destabilizing microtubules of an oocyte, whereby essentially all endogenous genetic material collects at a second polar body during meiosis of said oocyte; and    b) combining the oocyte with at least the nucleus of a donor somatic cell of the same species of said oocyte prior to cessation of extrusion of the second polar body from said oocyte, thereby forming a nuclear transfer embryo.    c) impregnating a mammal of the same species as the nuclear transfer embryo with the nuclear transfer embryo under conditions suitable for gestation of the cloned mammalian fetus; and    d) gestating the embryo, thereby causing the embryo to develop into the cloned mammalian fetus.    
   
   
       25 . (canceled)  
   
   
       26 . A method of cloning a non-human mammal, comprising the steps of: 
 a) destabilizing microtubules of an oocyte, whereby essentially all endogenous genetic material collects at a second polar body during meiosis of said oocyte; and    b) combining the oocyte with at least the nucleus of a donor somatic cell of the same species of said oocyte prior to cessation of extrusion of the second polar body from said oocyte, thereby forming a nuclear transfer embryo;    c) impregnating a non-human mammal of the same species as the nuclear transfer embryo with the nuclear transfer embryo under conditions suitable for gestation of the cloned non-human mammal; and    d) gestating the embryo, thereby causing the embryo to develop into the cloned non-human mammal.    
   
   
       27 . A method of producing a transgenic non-human mammal, comprising the steps of: 
 a) destabilizing microtubules of an oocyte, whereby essentially all endogenous genetic material collects at a second polar body during meiosis of said oocyte; and    b) combining the oocyte with at least the nucleus of a donor somatic cell of the same species of said oocyte prior to cessation of extrusion of the second polar body from said oocyte, thereby forming a nuclear transfer embryo.    c) impregnating a non-human mammal of the same species as the nuclear transfer embryo with the nuclear transfer embryo under conditions suitable for gestation of the transgenic mammal; and    d) gestating the embryo, thereby causing the embryo to develop into the transgenic non-human mammal.    
   
   
       28 . A method of cloning a non-human mammalian fetus, comprising the steps of: 
 a) destabilizing microtubules of an oocyte, whereby essentially all endogenous genetic material collects at a second polar body during meiosis of said oocyte; and    b) combining the oocyte with at least the nucleus of a donor somatic cell of the same species of said oocyte prior to cessation of extrusion of the second polar body from said oocyte, thereby forming a nuclear transfer embryo.    c) impregnating a non-human mammal of the same species as the nuclear transfer embryo with the nuclear transfer embryo under conditions suitable for gestation of the cloned mammalian fetus; and    d) gestating the embryo, thereby causing the embryo to develop into the cloned non-human mammalian fetus.    
   
   
       29 . A method of producing a protein of interest in an animal, comprising the steps of: 
 a) destabilizing microtubules of an oocyte, whereby essentially all endogenous genetic material collects at a second polar body during meiosis of said oocyte; and    b) combining the oocyte with at least the nucleus of a donor cell of the same species of said oocyte prior to cessation of extrusion of the second polar body from said oocyte, thereby forming a nuclear transfer embryo.    c) impregnating a mammal of the same species as the nuclear transfer embryo with the nuclear transfer embryo under conditions suitable for gestation of the cloned mammal;    d) gestating the embryo, thereby causing the embryo to develop into the cloned mammal; and    e) purifying the protein of interest from the cloned animal.    
   
   
       30 . The method of  claim 29 , wherein purification of the protein of interest is expressed in tissue, cells or a bodily secretion of the cloned animal.  
   
   
       31 . The method of  claim 29 , wherein the tissue, cells or bodily secretion is selected from the group consisting of: milk, blood, urine, hair, mammary gland, muscle, viscera.  
   
   
       32 . The method of  claim 31 , wherein said viscera is selected from the group consisting of: brain, heart, lung, kidney, pancreas, gall bladder, liver, stomach, eye, colon, small intestine, bladder, uterus and testes.  
   
   
       33 . A method of producing a heterologous protein in a transgenic animal comprising the steps of: 
 a) destabilizing microtubules of an oocyte, whereby essentially all endogenous genetic material collects at a second polar body during meiosis of said oocyte; and    b) combining the oocyte with at least the nucleus of a donor cell of the same species of said oocyte prior to cessation of extrusion of the second polar body from said oocyte, thereby forming a nuclear transfer embryo.    c) impregnating a mammal of the same species as the nuclear transfer embryo with the nuclear transfer embryo under conditions suitable for gestation of a transgenic cloned mammal;    d) gestating the embryo, thereby causing the embryo to develop into the transgenic cloned mammal; and    e) purifying the protein of interest from the transgenic cloned animal.    
   
   
       34 . The method of  claim 33 , wherein the genetically engineered nucleus includes an operatively linked promoter.  
   
   
       35 . The method of  claim 34 , wherein said promoter is selected from the group consisting of: a host endogenous promoter, an exogenous promoter and a tissue-specific promoter.  
   
   
       36 . The method of  claim 35 , wherein said tissue-specific promoter is selected from the group consisting of: mammary-specific promoter, blood-specific promoter, muscle-specific promoter, neural-specific promoter, skin-specific promoter, hair-specific promoter and urinary-specific promoter.  
   
   
       37 - 40 . (canceled)