Transgenic animals

Abstract

The present invention provides improved methods and compositions for the generation of transgenic non-human animals. The present invention permits the introduction of exogenous nucleic acid sequences into the genome of unfertilized eggs (e.g., pre-maturation oocytes and pre-fertilization oocytes) by microinjection of infectious retrovirus into the perivitelline space of the egg. The methods of the present invention provide an increased efficiency of production of transgenic animals with a reduced rate of generating animals which are mosaic for the presence of the transgene.

1 . A composition comprising a stably maintained recombinant mammalian zygote, wherein said zygote comprises a polynucleotide containing the proviral form of a retroviral vector integrated into the genome of said zygote.  
     
     
         2 . The composition of  claim 1 , wherein said mammalian zygote is a bovine zygote.  
     
     
         3 . The composition of  claim 1 , wherein said proviral form of said retroviral vector encodes a protein of interest.  
     
     
         4 . The method of  claim 1 , wherein said recombinant retroviral vector comprises Moloney murine leukemia virus long terminal repeat.  
     
     
         5 . A method for introducing a polynucleotide contained within the genome of a recombinant retrovirus into the genome of a mammalian zygote, comprising: 
 a) providing: 
 i) a mammalian zygote having a plasma membrane and a zona pellucida, said plasma membrane and said zona pellucida defining a perivitelline space;  
 ii) an aqueous solution comprising a polynucleotide contained within the genome of a recombinant retrovirus; and  
   b) introducing said solution comprising said polynucleotide contained within the genome of a recombinant retrovirus into said perivitelline space under conditions which permit the introduction of said polynucleotide contained within the genome of a recombinant retrovirus into the genome of said zygote, such that said polynucleotide is stably maintained.    
     
     
         6 . The method of  claim 5 , wherein the efficiency of said introduction of said polynucleotide contained within the genome of a recombinant retrovirus into the genome of said zygote, such that said polynucleotide is stably maintained is at least twenty percent.  
     
     
         7 . The method of  claim 5 , wherein said polynucleotide contained within the genome of a recombinant retrovirus encodes a protein of interest.  
     
     
         8 . The method of  claim 7 , further comprising the step of transferring said zygote into a mammalian female recipient that is hormonally synchronized to simulate early pregnancy, thereby giving a transferred embryo.  
     
     
         9 . The method of  claim 8 , further comprising the step of allowing said transferred embryo to develop to term.  
     
     
         10 . The method of  claim 7 , further comprising the step of identifying at least one transgenic offspring.  
     
     
         11 . A transgenic animal produced according to the method of  claim 10 .  
     
     
         12 . The method of  claim 10 , wherein said recombinant retrovirus comprises Moloney murine leukemia virus long terminal repeat.  
     
     
         13 . The method of  claim 12 , wherein said protein of interest is expressed by said transgenic offspring.  
     
     
         14 . The method of  claim 13 , wherein said protein of interest is expressed in at least one body fluid of said transgenic offspring.  
     
     
         15 . The method of  claim 13 , wherein said expression of said protein of interest is preferentially mammary-specific expression.  
     
     
         16 . The method of  claim 5 , wherein said recombinant retrovirus comprises a heterologous membrane-associated protein.  
     
     
         17 . The method of  claim 16 , wherein said heterologous membrane-associated protein is a G glycoprotein selected from a virus within the family Rhabdoviridae.  
     
     
         18 . The method of  claim 17 , wherein said G glycoprotein is selected from the group comprising the G glycoprotein of vesicular stomatitis virus, Piry virus, Chandipura virus, Spring viremia of carp virus, Rabies virus, and Mokola virus.  
     
     
         19 . A method for producing a transgenic non-human animal, wherein the genome of said transgenic non-human animal comprises a polynucleotide encoding a recombinant retrovirus and at least one protein of interest, comprising the steps of: 
 a) providing: 
 i) a non-human mammalian zygote having a plasma membrane and a zona pellucida, said plasma membrane and said zona pellucida defining a perivitelline space;  
 ii) an aqueous solution comprising a polynucleotide contained within the genome of a recombinant retrovirus;  
   b) introducing said solution comprising said polynucleotide contained within the genome of a recombinant retrovirus into said perivitelline space under conditions which permit the introduction of said polynucleotide contained within the genome of a recombinant retrovirus into the genome of said zygote, such that said polynucleotide is stably maintained in a recombinant zygote;    c) transferring said recombinant zygote into a non-human female mammalian recipient that is hormonally synchronized to simulate early pregnancy, thereby giving a transferred embryo;    d) allowing said transferred embryo to develop to term to produce a transgenic animal.    
     
     
         20 . The method of  claim 19 , wherein the said at least one protein of interest is expressed by said transgenic animal.  
     
     
         21 . The method of  claim 19 , wherein said recombinant retrovirus comprises Moloney murine leukemia virus long terminal repeat.  
     
     
         22 . The method of  claim 21 , wherein the efficiency of said introduction of said polynucleotide contained within the genome of a recombinant retrovirus into the genome of said zygote, such that said polynucleotide is stably maintained is at least twenty percent.  
     
     
         23 . The method of  claim 21 , wherein the expression of said polynucleotide is preferentially mammary-specific expressed.  
     
     
         24 . The method of  claim 20 , comprising the further step of mating said transgenic animal to a non-transgenic animal under conditions such that transgenic offspring are produced.  
     
     
         25 . The method of  claim 24 , wherein said transgenic offspring express said polynucleotide.  
     
     
         26 . The method of  claim 25 , wherein said expression of said polynucleotide is mammary-specific expression.  
     
     
         27 . A method for expressing a protein of wherein said protein of interest is encoded by a polynucleotide contained within the genome of a recombinant retrovirus, comprising the steps of: 
 a) providing: 
 i) a non-human mammalian zygote having a plasma membrane and a zona pellucida, said plasma membrane and said zona pellucida defining a perivitelline space;  
 ii) an aqueous solution comprising a polynucleotide encoding a protein of interest contained within the genome of a recombinant retrovirus; and  
   b) introducing said solution comprising said polynucleotide encoding a protein of interest contained within the genome of a recombinant retrovirus into said perivitelline space under conditions which permit the introduction of said polynucleotide contained within the genome of a recombinant retrovirus into the genome of said zygote, such that said polynucleotide is stably maintained;    c) allowing said zygote to develop into viable non-human animal, under conditions such that said protein of interest is expressed by said non-human animal.    
     
     
         28 . The method of  claim 27 , wherein said recombinant retrovirus comprises Moloney murine leukemia virus long terminal repeat.  
     
     
         29 . The method of  claim 27 , wherein said introduction of said polynucleotide contained within the genome of a recombinant retrovirus into the genome of said zygote, such that said polynucleotide is stably maintained is at least twenty percent.  
     
     
         30 . The method of  claim 27 , wherein said polynucleotide contained within the genome of a recombinant retrovirus encodes a viral protein.  
     
     
         31 . The method of  claim 30 , wherein said viral protein is hepatitis B surface antigen.  
     
     
         32 . The protein of interest expressed according to the method of  claim 27 .  
     
     
         33 . The method of  claim 27 , further comprising the step of d) harvesting said expressed protein of interest.  
     
     
         34 . The method of  claim 27 , wherein said expressed protein is expressed in the body fluids of said non-human animal.  
     
     
         35 . The method of  claim 34 , wherein said body fluids are selected from the group consisting of blood, milk, semen, and urine.  
     
     
         36 . A method for expressing a protein of interest wherein said protein of interest is encoded by a polynucleotide contained within the genome of a recombinant retrovirus, and said polynucleotide is integrated into the genome of a mammalian unfertilized oocyte, comprising the steps of: 
 a) providing: 
 i) an unfertilized mammalian egg comprising an oocyte having a plasma membrane and a zona pellucida, said plasma membrane and said zona pellucida defining a perivitelline space;  
 ii) an aqueous solution containing recombinant retrovirus, wherein said recombinant retrovirus comprises a polynucleotide encoding a protein of interest;  
   b) introducing said solution containing recombinant retrovirus into said perivitelline space under conditions which permit the infection of said oocyte to provide an infected oocyte;    c) contacting said infected oocyte with sperm under conditions which permit the fertilization of said infected oocyte to produce an embryo;    d) transferring said embryo into a hormonally synchronized mammalian recipient animal;    e) allowing said embryo to develop into at least one viable transgenic mammalian animal, under conditions such that said protein of interest is expressed by said transgenic mammalian animal.    
     
     
         37 . The method of  claim 36 , wherein said unfertilized oocyte is a pre-maturation oocyte.  
     
     
         38 . The method of  claim 37 , further comprising following the introduction of said solution containing infectious retrovirus into said pre-maturation oocyte, the further step of culturing said infected pre-maturation oocyte under conditions which permit the maturation of said pre-maturation oocyte.  
     
     
         39 . The method of  claim 36 , wherein said unfertilized oocyte is a pre-fertilization oocyte.  
     
     
         40 . The method of  claim 36 , further comprising identifying at least one transgenic offspring.  
     
     
         41 . The method of  claim 36 , wherein said mammal is a bovine.  
     
     
         42 . The method of  claim 36 , wherein said recombinant retrovirus comprises Moloney murine leukemia virus long terminal repeat.  
     
     
         43 . The method of  claim 42 , wherein said expression of said protein of interest is preferentially mammary specific expression.  
     
     
         44 . The method of  claim 43 , wherein said introduction of said polynucleotide contained within the genome of a recombinant retrovirus into the genome of said infected oocyte, such that said polynucleotide is stably maintained is greater than twenty percent.  
     
     
         45 . The method of  claim 36 , wherein said polynucleotide contained within the genome of a recombinant retrovirus encodes a viral protein.  
     
     
         46 . The method of  claim 45 , wherein said viral protein is hepatitis B surface antigen.  
     
     
         47 . The protein of interest expressed according to the method of  claim 36 .  
     
     
         48 . The method of  claim 36 , further comprising the step of f) harvesting said expressed protein of interest.  
     
     
         49 . The method of  claim 36 , wherein said expressed protein is expressed in the body fluids of said mammalian animal.  
     
     
         50 . The method of  claim 49 , wherein said body fluids are selected from the group consisting of blood, milk, semen, and urine.  
     
     
         51 . The method of  claim 36 , wherein said recombinant retrovirus comprises a heterologous membrane-associated protein.  
     
     
         52 . The method of claim  51 , wherein said heterologous membrane-associated protein is a G glycoprotein selected from a virus within the family Rhabdoviridae.  
     
     
         53 . The method of claim  52 , wherein said G glycoprotein is selected from the group comprising the G glycoprotein of vesicular stomatitis virus, Piry virus, Chandipura virus, Spring viremia of carp virus and Mokola virus.