Cloning pigs using donor nuclei from differentiated cells

Abstract

An improved method of nuclear transfer involving the transplantation of donor differentiated pig cell nuclei into enucleated pig oocytes is provided. The resultant nuclear transfer units are useful for multiplication of genotypes and transgenic genotypes by the production of fetuses and offspring. Production of genetically engineered or transgenic pig embryos, fetuses and offspring is facilitated by the present method since the differentiated cell source of the donor nuclei can be genetically modified and clonally propagated.

1 . A method of cloning a pig fetus, comprising: 
 (i) inserting a desired differentiated pig cells or cell nuclei into enucleated, metaphase-II pig oocytes, under conditions suitable for the formation of nuclear transfer (NT) units;    (ii) activating the resultant nuclear transfer units; and    (iii) synchronously transferring 20 or more of said activated NT units into a host pig such that one or more of the NT units develops into a fetus.    
     
     
         2 . (canceled)  
     
     
         3 . The method according to  claim 1 , wherein a desired DNA is inserted, removed or modified in said differentiated pig cell or nucleus, thereby resulting in the production of a genetically altered NT unit.  
     
     
         4 . (canceled)  
     
     
         5 . The method according to  claim 1 , which comprises culturing said activated nuclear transfer unit until greater than the 2-cell developmental stage.  
     
     
         6 . The method according to  claim 1 , wherein the differentiated pig cell or nucleus is derived from mesoderm.  
     
     
         7 . The method according to  claim 1 , wherein the differentiated pig cell or nucleus is derived from ectoderm.  
     
     
         8 . The method according to  claim 1 , wherein the differentiated pig cell or nucleus is derived from endoderm.  
     
     
         9 . The method according to  claim 1 , wherein the differentiated pig cell or nucleus is a fibroblast cell or cell nucleus.  
     
     
         10 . The method according to  claim 1 , wherein the differentiated pig cell or nucleus is an adult cell or cell nucleus.  
     
     
         11 . The method according to  claim 1 , wherein the differentiated pig cell or nucleus is an embryonic or fetal cell or cell nucleus.  
     
     
         12 . The method according to  claim 1 , wherein the enucleated oocyte is matured prior to enucleation.  
     
     
         13 . The method according to  claim 1 , wherein the fused nuclear transfer unit is activated by exposure to two electrical pulses.  
     
     
         14 . The method according to  claim 1 , wherein the fused nuclear transfer unit is activated by exposure to a single electrical pulse.  
     
     
         15 . The method according to  claim 1 , wherein the fused nuclear transfer unit is activated by exposure to at least one activating factor derived from sperm cells.  
     
     
         16 . The method according to  claim 3 , wherein microinjection is used to insert a heterologous DNA.  
     
     
         17 . The method according to  claim 3 , wherein electroporation is used to insert a heterologous DNA.  
     
     
         18 - 78 . (canceled)  
     
     
         79 . A method of cloning a pig, comprising: 
 (i) inserting desired differentiated pig cells or cell nuclei into enucleated, metaphase-II pig oocytes, under conditions suitable for the formation of nuclear transfer (NT) units;    (ii) activating the resultant nuclear transfer units; and    (iii) synchronously transferring 20 or more of said activated NT units into a host pig such that one or more of the NT units develops into a pig.    
     
     
         80 . The method according to  claim 79 , wherein a desired DNA is inserted, removed or modified in said differentiated pig cell or nucleus, thereby resulting in the production of a genetically altered NT unit.  
     
     
         81 . The method according to  claim 79 , which comprises culturing said activated nuclear transfer unit until greater than the 2-cell developmental stage.  
     
     
         82 . The method according to  claim 79 , wherein the differentiated pig cell or nucleus is derived from mesoderm.  
     
     
         83 . The method according to  claim 79 , wherein the differentiated pig cell or nucleus is derived from ectoderm.  
     
     
         84 . The method according to  claim 79 , wherein the differentiated pig cell or nucleus is derived from endoderm.  
     
     
         85 . The method according to  claim 79 , wherein the differentiated pig cell or nucleus is a fibroblast cell or cell nucleus.  
     
     
         86 . The method according to  claim 79 , wherein the differentiated pig cell or nucleus is an adult cell or cell nucleus.  
     
     
         87 . The method according to  claim 79 , wherein the differentiated pig cell or nucleus is an embryonic or fetal cell or cell nucleus.  
     
     
         88 . The method according to  claim 79 , wherein the enucleated oocyte is matured prior to enucleation.  
     
     
         89 . The method according to  claim 79 , wherein the fused nuclear transfer unit is activated by exposure to two electrical pulses.  
     
     
         90 . The method according to  claim 79 , wherein the fused nuclear transfer unit is activated by exposure to a single electrical pulse.  
     
     
         91 . The method according to  claim 79 , wherein the fused nuclear transfer unit is activated by exposure to at least one activating factor derived from sperm cells.  
     
     
         92 . The method according to  claim 80 , wherein microinjection is used to insert a heterologous DNA.  
     
     
         93 . The method according to  claim 80 , wherein electroporation is used to insert a heterologous DNA.