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1. (WO1991008216) PRODUCTION OF RECOMBINANT POLYPEPTIDES BY BOVINE SPECIES AND TRANSGENIC METHODS
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WHAT IS CLAIMED IS:

1. A transgene for producing a recombinant polypeptide in transgenic bovine species comprising at least one expression regulation DNA sequence functional in at least one cell-type of said bovine species operably linked to a recombinant DNA encoding a recombinant polypeptide, wherein said transgene is capable of directing the expression of said recombinant DNA sequence in at least said one cell-type of a bovine species containing said transgene to produce said recombinant polypeptide.

2. The transgene of Claim 1 wherein said expression regulation sequences comprise 5' and 3' expression regulation sequences from a serum albumin, said cell- type is liver cell, said recombinant polypeptide is human serum albumin and said transgene further comprises a secretory DNA sequence functional in said liver cells and operably linked to the recombinant DNA encoding said human serum albumin.

3. A transgene for producing a recombinant polypeptide in the milk of transgenic bovine species comprising at least one expression regulation DNA sequence functional in the mammary secretory cells of said bovine species, a secretory DNA sequence encoding a secretory signal sequence also functional in the mammary secretory cells of said bovine species and a recombinant DNA sequence encoding a recombinant polypeptide, said secretory DNA sequence being operably linked to said recombinant DNA sequence and to form a secretory-recombinant DNA sequence and said at least one expression regulation sequence being operably linked to said secretory-recombinant DNA sequence, wherein said transgene is capable of directing the expression of said secretory-recombinant DNA sequence in mammary secretory cells of bovine species containing said transgene to produce a form of recombinant polypeptide which when secreted from said mammary secretory cells produces recombinant polypeptide in he milk of said bovine species.

4. The transgene of Claim 1 or 3 further comprising a recombinant intervening sequence.

5. The transgene of Claim 4 wherein said recombinant intervening sequence is a hybrid intervening sequence.

6. The transgene of Claim 5 wherein said hybrid intervening sequence contains a permissive RNA splice signal.

7. The transgene of Claim 3 wherein said recombinant polypeptide is a homologous polypeptide from bovine species.

8. The transgene of Claim 7 wherein said homologous polypeptide is selected from the group consisting of caseins, lactoferrin, lysozyme, cholesterol hydrolase and serum albumin.

9. The transgene of Claim 3 wherein said recombinant polypeptide is a heterologous polypeptide.

10. The transgene of Claim 9 wherein said heterologous polypeptide is selected from the group consisting of human milk proteins, human serum proteins, and industrial enzymes.

11. The transgene of Claim 10 wherein said heterologous polypeptide is a human milk protein.

12. The transgene of Claim 11 wherein said human milk protein is selected from the group consisting of secretory immunoglobulins, lysozyme, lactoferrin, lactoglobulin, α-lactalbumin and bile salt-stimulated lipase.

13. The transgene of Claim 12 wherein said milk protein is lactoferrin.

14. The transgene of Claim 10 wherein said heterologous polypeptide is a human serum protein.

15. The transgene of Claim 14 wherein said human serum protein is selected from the group consisting of albumin, immunoglobulin. Factor VIII, Factor IX and Protein C.

16. The transgene of Claim 15 wherein said serum protein is albumin.

17. The transgene of Claim 10 wherein said heterologous polypeptide is an industrial enzyme selected from the group consisting of proteases, Upases, chitenases and ligninases.

18. The transgene of Claim 3 wherein said secretory DNA sequence is selected from the group consisting of DNA sequences encoding secretory signal sequences from human lactoferrin, human serum albumin, and secretory signal sequences from bovine αS1-casein, αS2-casein, β- casein, k-casein, α-lactalbumin, ß-lactoglobulin, and serum albumin.

19. The transgene of Claim 18 wherein said secretory DNA sequence is the DNA sequence encoding the signal secretion sequence of bovine αS1 casein.

20. The transgene of Claim 3 wherein said at least one expression regulation sequence comprises 5' expression regulation DNA sequences, wherein said 5' expression regulation DNA sequence is operably linked to the 5' end of said secretory-recombinant DNA sequence.

21. The transgene of Claim 20 wherein said 5' expression regulation DNA sequence is selected from the group consisting of 5' expression regulation sequence from bovine genes encoding αS1-casein, αS2-casein, ß-casein, k-casein, α-lactalbumin, and β-lactoglobulin.

22. The transgene of Claim 21 wherein said 5' expression regulation DNA sequence is a proximal 5' expression regulation sequence comprising the promoter of bovine αS1-casein.

23. The transgene of Claim 22 wherein said 5' expression regulation DNA sequence further comprises a distal 5' expression regulation sequence comprising 5'-flanking DNA sequence from bovine αS1-casein.

24. The transgene of Claim 20 further comprising 3' expression regulation sequences operably linked to the 3' end of said secretory-recombinant DNA sequence.

25. The transgene of Claim 24 wherein said 3' expression regulation sequence comprise 3' expression regulation sequence from bovine genes encoding αS1-casein, αS2-casein, ß-casein, k-casein, α-lactalbumin, and ß-lactoglobulin.

26. The transgene of Claim 25 wherein said 3' expression regulation DNA sequence comprises a 3' proximal expression regulation sequence from bovine αS1-casein.

27. The transgene of Claim 26 wherein said 3' expression regulation DNA sequence further comprises a 3' distal expression regulation sequence from bovine αS1-casein.

28. The transgene of Claim 27 wherein said distal 5' expression regulation DNA sequence comprises about a 30kb 5'-flanking region of bovine αS1-casein and said distal 3' expression regulation DNA sequence comprises about a 15kb 3'-flanking region of bovine αS1-casein.

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 trangenic species.

31. The transgenic bovine species of Claim 30 wherein said recombinant polypeptide is a homologous polypeptide from bovine species.

32. The transgene bovine species of Claim 30 wherein said recombinant polypeptide is a heterologous polypeptide.

33. The transgenic bovine species of Claim 30 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, α-lactalbumin and bile salt-stimulated lipase.

36. The transgenic bovine species of Claim 35 wherein said milk protein is lactoferrin.

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. The milk of Claim 46 wherein said milk protein is lactoferrin.

48. The milk of Claim 43 wherein said heterologous polypeptide is a human serum protein.

49. The milk of Claim 48 wherein said human serum protein is selected from the group consisting of albumin, immunoglobulin. Factor VIII, Factor IX and Protein C.

50. The milk of Claim 49 wherein said serum protein is albumin.

51. A food formulation comprising transgenic milk containing a recombinant polypeptide.

52. The food formulation of Claim 51 wherein said recombinant polypeptide is at least partially purified from said transgenic milk.

53. The food formulation of Claim 31 formulated with nutrients appropriate for infant formula.

54. A method for producing a transgenic bovine species capable of producing a recombinant polypeptide in the milk of said bovine species, said method comprising: introducing the transgene of Claim 1 into an embryonal target cell of a bovine species;
transplanting the transgenic embryonal target cell formed thereby or the embryo obtained herefrom into a recipient female bovine parent; and
identifying at least one female offspring which is capable of producing said recombinant polypeptide in the milk of said offspring.

55. A method for producing a transgenic non-human mammal having a desirable phenotype comprising:
(a) methylating a transgene capable of conferring said phenotype when incorporated into the cells of said transgenic non-human animal;
(b) introducing said methylated transgene into fertilized oocytes of said non-human mammal to permit integration of said transgene into the genomic DNA of said fertilized oocytes;
(c) culturing the individual oocytes formed hereby to pre-implantation embryos thereby replicating the genome of each of said fertilized oocytes;
(d) removing at least one cell from each of said preimplantation embryos and lysing said at least one cell to release the DNA contained therein;
(e) contacting said released DNA with a restriction endonuclease capable of cleaving said methylated transgene but incapable of cleaving the unmethylated form of said transgene formed after integration into and replication of said genomic DNA; and
(f) detecting which of said cells from said preimplantation embryos contain a transgene which is resistant to cleavage by said restriction endonuclease as an indication of which pre-implantation embryos have integrated said transgene.

56. The method of Claim 55 wherein said removal of at least one cell forms a first and second hemi-embryo for each of said pre-implementation embryos and each of said first hemi-embryos is lysed and analyzed according to steps (d) through (f), said method further comprising;
(g) cloning at least one of said second hemiembryos; and
(h) to form a multiplicity of transgenic embryos.

57. The method of Claim 56 further comprising transplanting more than one of said transgenic embryos into recipient female parents to produce a population containing at least two transgenic non-human animals having the same genotype.

58. The method of Claim 55 further comprising transplanting the remainder of said pre-implantation embryo containing a genomically integrated transgene into a recipient female parent and identifying at least one offspring having said phenotype.

59. The method of Claim 55 wherein said restriction endonuclease is DPNI and said transgene is methylated at N6 of the adenine of the sequence GATC contained within said transgene.

60. The method of Claim 59 wherein said detection utilizes a polymerase chain reaction using extension primers complementary to sequences upstream and downstream to said GATC sequence.

61. The method of Claim 59 wherein said non-human transgenic mammal is bovine species, said transgene encodes a recombinant polypeptide and said desired phenotype is the ability to produce said recombinant polypeptide in the milk of said bovine species.

62. The method of Claim 61 wherein said transgene is the transgene of Claim 3.