Unactivated oocytes as cytoplast recipients for nuclear transfer

Abstract

A method of reconstituting an animal embryo involves transferring a diploid nucleus into an oocyte which is arrested in the metaphase of the second meiotic division. The oocyte is not activated at the time of transfer, so that the donor nucleus is kept exposed to the recipient cytoplasm for a period of time. The diploid nucleus can be donated by a cell in either the GO or G1 phase of the cell cycle at the time of transfer. Subsequently, the reconstituted embryo is activated. Correct ploidy is maintained during activation, for example, by incubating the reconstituted embryo in the presence of a microtubule inhibitor such as nocodazole. The reconstituted embryo may then give rise to one or more live animal births. The invention is useful in the production of transgenic animals as well as non-transgenics of high genetic merit.

1 . A method of reconstituting an animal embryo, the process comprising transferring a diploid nucleus into an oocyte which is arrested in the metaphase of the second meiotic division without concomitantly activating the oocyte, keeping the nucleus exposed to the cytoplasm of the recipient for a period of time sufficient for the embryo to become capable of giving rise to a live birth and subsequently activating the reconstituted embryo while maintaining correct ploidy. 
   
   
       2 . A method as claimed in  claim 1 , in which the animal is an ungulate species. 
   
   
       3 . A method as claimed in  claim 2 , in which the animal is a cow or bull, pig, goat, sheep, camel or water buffalo. 
   
   
       4 . A method as claimed in any one of  claims 1  to  3 , in which the donor nucleus is genetically modified. 
   
   
       5 . A method as claimed in any one of  claims 1  to  4 , wherein the diploid nucleus is donated by a quiescent cell. 
   
   
       6 . A method as claimed in any one of  claims 1  to  5 , wherein the recipient oocyte is enucleate. 
   
   
       7 . A method as claimed in any one of  claims 1  to  6 , wherein nuclear transfer is achieved by cell fusion. 
   
   
       8 . A method as claimed in any one of  claims 1  to  7 , wherein the animal is a cow or bull and wherein the donor nucleus is kept exposed to the recipient cytoplasm for a period of from 6 to 20 hours prior to activation. 
   
   
       9 . A method as claimed in any one of  claims 1  to  8 , wherein correct ploidy is maintained during activation by microtubule inhibition. 
   
   
       10 . A method as claimed in  claim 9 , wherein microtubule inhibition is achieved by the application of nocodazole. 
   
   
       11 . A method as claimed in any one of  claims 1  to  8 , wherein correct ploidy is maintained during activation by microtubule stabilisation. 
   
   
       12 . A method as claimed in  claim 11 , wherein microtubule stabilisation is achieved by the application of taxol. 
   
   
       13 . A method of preparing an animal, the method comprising:
 (a) reconstituting an animal embryo as claimed in any preceding claim;   (b) causing an animal to develop to term from the embryo; and   (c) optionally, breeding from the animal so formed.   
   
   
       14 . A method as claimed in  claim 13 , wherein the animal embryo is further manipulated prior to full development. of the embryo. 
   
   
       15 . A method as claimed in  claim 14 , wherein more than one animal is derived from the embryo. 
   
   
       16 . A reconstituted animal embryo which is capable of giving rise to a live birth and is prepared by a method as claimed in any one of  claims 1  to  12 . 
   
   
       17 . An animal prepared by a method as claimed in any one of  claims 13  to  15 . 
   
   
       18 . An animal developed from an embryo as claimed in  claim 16 .