Method for the production of nuclear transfer embryos

Abstract

Methods for the production of nuclear transfer embryos, nuclear transfer embryos and animals derived therefrom are described. The method generally comprises at least the steps of: providing at least one enucleated recipient cell; providing at least one donor cell or nucleus; providing a fusion media which is substantially free of calcium; placing said at least one enculeated recipient cell and at least one donor cell or nucleus in contact with one another to form couplets; and, fusing via electrofusion in said fusion media said at least one recipient cell with at least one donor cell or nucleus to form a nuclear transfer embryo.

1 . A method for the production of nuclear transfer embryos comprising at least the steps of: 
 providing at least one enucleated recipient cell;    providing at least one donor cell or nucleus;    placing said at least one enucleated recipient cell and at least one donor cell or nucleus in contact with one another to form couplets;    providing a fusion media which is substantially free of calcium; and    fusing via electrofusion, in said fusion media, said at least one recipient cell with at least one donor cell or nucleus to form a nuclear transfer embryo.    
     
     
         2 . A method as claimed in  claim 1  wherein said recipient and donor cells are porcine.  
     
     
         3 . A method as claimed in claims  1  or  2  wherein the parameters of said electrofusion are a single electrical pulse at between 100V to 200V for between 30 μs and 100 μs over an electrode gap of 1 mm.  
     
     
         4 . A method as claimed in claims  1  or  2  wherein the parameters of said electrofusion are a single electrical pulse at 1.5 kV/cm for 60 μs.  
     
     
         5 . A method as claimed in any one of  claims 1  to  4  wherein said recipient cell is a freshly ovulated or a follicular oocyte arrested at MII.  
     
     
         6 . A method as claimed in any one of  claims 1  to  4  wherein said recipient cell is an in vitro-matured oocyte.  
     
     
         7 . A method as claimed in any one of  claims 1  to  6  wherein the couplets are held in media which is substantially free of calcium for a period prior to electrofusion to form a nuclear transfer embryo.  
     
     
         8 . A method as claimed in  claim 7  wherein the period is at least approximately 15 minutes.  
     
     
         9 . A method as claimed in any one of  claims 1  to  8  further comprising the step of incubation in a media which is substantially free of calcium for a period following electrofusion.  
     
     
         10 . A method as claimed in  claim 9  wherein the period is at least approximately 15 minutes.  
     
     
         11 . A method as claimed in any one of  claims 1  to  10  wherein said donor cell or nucleus is a somatic cell.  
     
     
         12 . A method as claimed in  claim 11  wherein said donor cell is a fibroblast.  
     
     
         13 . A method of cloning animals comprising at least the steps of: 
 producing a nuclear transfer embryo according to a method as claimed in any one of  claims 1  to  12 ;    activating said nuclear transfer embryo to provide an activated embryo;    optionally allowing said activated embryo to undergo at least one round of cell division;    transferring activated and divided embryo to a synchronised female recipient animal;    allowing said synchronised female recipient animal to carry said embryo to full gestation to produce a cloned animal.    
     
     
         14 . A method as claimed in  claim 13  wherein said NT embryo is porcine and said recipient female animal and said cloned animal are pigs.  
     
     
         15 . A method as claimed in claims  13  or  14  wherein where the nuclear transfer embryo is constructed using in vivo-derived oocytes, said nuclear transfer embryo is held in a calcium-containing media with serum prior to activation thereof.  
     
     
         16 . A method as claimed in claims  13  or  14  wherein where the nuclear transfer embryo is constructed using in vitro-matured oocytes, said nuclear transfer embryo is held in a media substantially free of calcium for a period prior to activation thereof.  
     
     
         17 . A method as claimed in  claim 16  wherein the period is at least approximately 15 minutes.  
     
     
         18 . A method as claimed in any one of  claims 13  to  17  wherein said nuclear transfer embryos are activated no later than 5 hours post fusion.  
     
     
         19 . A method for the production of porcine nuclear transfer embryos comprising at least the steps of: 
 providing at least one enucleated recipient cell of porcine origin;    providing at least one donor cell or nucleus of porcine origin;    placing said at least one enucleated recipient cell and at least one donor cell or nucleus in contact with one another to form couplets;    optionally providing a first media which is substantially free of calcium;    optionally incubating said couplets in said first media for a period of preferably at least approximately 15 minutes;    providing a second media which is substantially free of calcium;    fusing via electrofusion, in said second media, said at least one recipient cell with at least one donor cell or nucleus to form a nuclear transfer embryo;    providing a third media which is substantially free of calcium; and    incubating said nuclear transfer embryo in said third media for a period preferably of at least approximately 15 minutes following electrofusion.    
     
     
         20 . A method of cloning pigs comprising at least the steps of: 
 producing a porcine nuclear transfer embryo according to  claim 19;     activating said nuclear transfer embryo to provide an activated embryo;    optionally allowing said activated embryo to undergo at least one round of cell division;    transferring activated and divided embryo to a synchronised female recipient animal;    allowing said synchronised female recipient animal to carry said embryo to full gestation to produce a cloned pig.