Production of recombinant polypeptides by bovine species and transgenic methods

Abstract

Transgenes for producing recombinant polypeptides transgenic bovine species. A transgene for producing recombinant polypeptides in the milk of transgenic bovine species comprises at least one expression regulation sequence, a secretory DNA sequence encoding a secretory signal sequence which is functional in mammary secretory cells of the bovine species and a recombinant DNA sequence encoding the recombinant polypeptide. Also included are methods for producing transgenic bovine species. The method includes introducing the above transgene into an embryonal target cell of a bovine species, transplanting the transgenic embryonic target cell formed thereby into a recipient bovine parent and identifying at least one female offspring which is capable of producing the recombinant polypeptide in its milk. The invention also includes transgenic bovine species capable of producing recombinant polypeptides in transgenic milk as well as the milk from such transgenic bovine species and food formulations containing one or more recombinant polypeptide. Methods are also provided for producing transgenic non-human mammals having a desirable phenotype. The method comprises first methylating a transgene followed by introduction into fertilized oocytes. The oocytes are then cultured to form pre-implantation embryos. Thereafter, at least one cell is removed from each of the pre-implantation embryos and the DNA digested with a restriction endonuclease capable of cleaving the methylated transgene but incapable of cleaving the unmethylated form of the transgene. Those pre-implantation embryos which have integrated the transgene contain DNA which is resistant to cleavage by the restriction endonuclease in the region containing the transgene.

1 .- 28 . (canceled)  
     
     
         29 . A transgenic bovine species capable of producing a recombinant polypeptide in at least one cell type of said animal.  
     
     
         30 . A transgenic bovine species capable of producing recombinant polypeptide in the milk of said transgenic species.  
     
     
         31 . The transgenic bovine species of  claim 30  wherein said recombinant polypeptide is a homologous polypeptide from bovine species.  
     
     
         32 . The transgenic bovine species of  claim 30  wherein said recombinant polypeptide is a heterologous polypeptide.  
     
     
         33 . The transgenic bovine species of  claim 32  wherein said heterologous polypeptide is selected from the group consisting of human milk proteins, human serum proteins, and industrial enzymes.  
     
     
         34 . The transgenic bovine species of  claim 33  wherein said heterologous polypeptide is a human milk protein.  
     
     
         35 . The transgenic bovine species of  claim 34  wherein said human milk protein is selected from the group consisting of secretory immunoglobulins, lysozyme, lactoferrin, lactoglobulin, a-lactalbumin and bile salt-stimulated lipase.  
     
     
         36 . The transgenic bovine species of  claim 35  wherein said milk protein is lactoferrin or lysozyme.  
     
     
         37 . The transgenic bovine species of  claim 33  wherein said heterologous polypeptide is a human serum protein.  
     
     
         38 . The transgenic bovine species of  claim 37  wherein said human serum protein is selected from the group consisting of albumin, immunoglobulin, Factor VIII, Factor IX and Protein C.  
     
     
         39 . The transgenic bovine species of  claim 38  wherein said serum protein is albumin.  
     
     
         40 . The transgenic bovine species of  claim 33  wherein said heterologous polypeptide is an industrial enzyme selected from the group consisting of proteases, lipases, chitinases and ligninases.  
     
     
         41 . Milk from transgenic bovine species containing a recombinant polypeptide.  
     
     
         42 . The milk of  claim 41  wherein said recombinant polypeptide is a homologous polypeptide from bovine species.  
     
     
         43 . The milk of  claim 41  wherein said recombinant polypeptide is a heterologous polypeptide.  
     
     
         44 . The milk of  claim 43  wherein said heterologous polypeptide is selected from the group consisting of human milk proteins, human serum proteins, and industrial enzymes.  
     
     
         45 . The milk of  claim 44  wherein said heterologous polypeptide is a human milk protein.  
     
     
         46 . The milk of  claim 45  wherein said human milk protein is selected from the group consisting of secretory immunoglobulins, lysozyme, lactoferrin, lactoglobulin, α-lactalbumin and bile salt-stimulated lipase.  
     
     
         47 .- 97 . (canceled)