Transgenic bovine

• US 5,741,957 A
• Filed: 06/05/1995
• Issued: 04/21/1998
• Est. Priority Date: 12/01/1989
• Status: Expired due to Term

• Current Assignee: Pharming Intellectual Property B.V. (Pharming Group N.V.)
• Original Assignee: Pharming BV (Pharming Group N.V.)
• Inventors: Pieper, Frank, Heyneker, Herbert L., Platenburg, Gerard, Lee, Sang He, Strijker, Rein, Deboer, Herman A., Krimpenfort, Paul J. A.
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US08/461,333
• Time in Patent Office: 1,051 Days
• Field of Search: 800/2, 800/DIG. 1, 536/23.1, 536/23.4, 536/24.1, 435/69.1, 435/325, 435/320.1, 935/36
• US Class Current: 800/7
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2227/101 Bovine
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: A23C 9/20 Dietetic milk products not covered by groups A23C9/12 - A23C9/18
• CPC Class Codes: C07K 14/4732 Casein in foodstuffs A23J
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA
• CPC Class Codes: C07K 14/79 Transferrins, e.g. lactoferrins, ovotransferrins
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/15 chimeric enhancer/promoter combination
• CPC Class Codes: C12N 2830/42 being an intron or intervening sequence for splicing and/or stability of RNA
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 2840/44 : being a specific part of the splice mechanism, e.g. donor, acceptor
• CPC Class Codes: C12N 9/2462 : Lysozyme (3.2.1.17)
• CPC Class Codes: C12N 9/6464 : Protein C (3.4.21.69)
• CPC Class Codes: C12Y 304/21069 : Protein C activated (3.4.21.69)

Abstract

A transgenic bovine is disclosed whose somatic and germ cells contain a transgene, wherein the transgene comprising a mammary gland specific promoter, a mammary gland specific enhancer, a DNA sequence encoding a signal sequence functional in bovine mammary gland secretory cells and a DNA sequence encoding a heterologous polypeptide of interest wherein the transgenic bovine expresses the transgene such that the polypeptide of interest is detectable in milk produced by the transgenic bovine.

Claims

1. A transgenic bovine whose somatic and germ cells contain a transgene, wherein the transgene comprises in operable association:

a mammary gland specific promoter;

a mammary gland specific enhancer;

a DNA sequence encoding a signal sequence functional in bovine mammary gland secretory cells; and

a DNA sequence encoding a heterologous polypeptide of interest;

wherein the transgenic bovine or a female descendant of the transgenic bovine expresses the transgene in mammary secretory cells such that the polypeptide of interest is detectable in milk produced by the transgenic bovine or a female descendant of the transgenic bovine.

2. A method of producing a polypeptide, comprising recovering milk from the transgenic bovine or female descendent of claim 1, wherein the milk contains the polypeptide.

3. The method of claim 2, comprising transplanting a preimplantation stage embryo comprised of the transgene into a recipient female bovine, wherein the female bovine gestates the embryo to give birth to the transgenic bovine.

4. The method of claim 3, comprising prior to transplanting:

obtaining an ovum from bovine ovaries;

maturing the ovum in vitro;

fertilizing the mature ovum in vitro to form a zygote;

introducing the transgene into the zygote in vitro;

maturing the zygote to a preimplantation stage embryo in vitro.

5. The method of claim 2, further comprising purifying the polypeptide from the milk.

6. The method of claim 5, further comprising formulating the polypeptide as a pharmaceutical.

7. The method of claim 2, wherein the polypeptide is human lactoferrin.

8. The transgenic bovine of claim 1, wherein the promoter is within a 5'"'"' expression regulation sequence from a bovine gene, and wherein the transgene further comprises a 3'"'"' untranslated sequence from a gene of interest and a 3'"'"' flanking sequence from a bovine gene.

9. The transgenic bovine of claim 8, wherein the transgene further comprises an intervening sequence.

10. The transgenic bovine of claim 9, wherein the intervening sequence is a hybrid intervening sequence.

11. The transgenic bovine of claim 10, wherein the hybrid intervening sequence comprises a 5'"'"' portion of a bovine α

-S₁ -casein intervening sequence and a 3'"'"' portion of an IgG heavy chain intervening sequence.

12. The transgenic bovine of claim 11, wherein the 3'"'"' portion is a 3'"'"' splice signal sequence associated with the J-C segment rearrangement of an IgG heavy chain gene.

13. The transgenic bovine of claim 8, wherein the bovine expression regulation sequence and the 3'"'"' flanking sequence are from the same bovine gene.

14. The transgenic bovine of claim 8, wherein the bovine expression regulation sequence, the 3'"'"' untranslated sequence and the 3'"'"' flanking sequence are from the same bovine gene.

15. The transgenic bovine of claim 13 or claim 14, wherein the bovine gene is the α

-S₁ -casein gene.

16. The transgenic bovine of claim 15, wherein the bovine expression regulation sequence comprises about a 30 kb 5'"'"'-flanking region of the bovine α

-S₁ -casein gene and the 3'"'"' flanking sequence comprises about a 15 kb 3'"'"'-flanking region of the bovine α

-S₁ -casein gene.

17. The transgenic bovine of claim 8, wherein the milk comprises greater than 50 micrograms of the recombinant polypeptide per milliliter.

18. The transgenic bovine of claim 1, wherein the promoter is within a 5'"'"' expression regulation sequence from a gene of interest, and wherein the transgene further comprises a 3'"'"' untranslated sequence from a gene of interest and a 3'"'"' flanking sequence from a human gene.

19. The transgenic bovine of claim 18, wherein the 5'"'"' expression regulation sequence is a bovine sequence.

20. The transgenic bovine of claim 18, wherein the 3'"'"' flanking sequence is from the human lactoferrin gene.

21. The transgenic bovine of claim 20, wherein the 3'"'"' flanking sequence is 9 kilobase pairs in length.

22. The transgenic bovine of claim 20, wherein the polypeptide is human lactoferrin.

23. The transgenic bovine of claim 18, wherein the 5'"'"' expression regulatory sequence, the signal sequence, the DNA sequence encoding a heterologous polypeptide, the 3'"'"' untranslated region and the 3'"'"' flanking sequence are each from a human gene.

24. The transgenic bovine of claim 23, wherein the human gene is lactoferrin.

25. The transgenic bovine of claim 1, wherein the promoter is within a 5'"'"' expression regulation sequence from a first milk protein gene, wherein the DNA sequence encoding a heterologous polypeptide is from second milk protein gene, and wherein the DNA sequence encoding the signal sequence is from either the first or second milk protein gene, and wherein the transgene further comprises a 3'"'"' untranslated sequence and a 3'"'"' flanking sequence each from either the first or second milk protein gene.

26. The transgenic bovine of claim 25, wherein the transgene further comprises a 5'"'"' untranslated sequence from either the first or second milk protein gene.

27. The transgenic bovine of claim 26, where in the DNA sequence encoding a heterologous polypeptide is a genomic sequence comprising at least one intronic sequence.

28. The transgenic bovine of claim 27, wherein the first milk protein gene is the α

-S₁ -casein gene.

29. The transgenic bovine of claim 27, wherein the first milk protein gene is the β

-lactoglobulin gene.

30. The transgenic bovine of claim 28, wherein the polypeptide is human lactoferrin.

31. The transgenic bovine of claim 29, wherein the polypeptide is human lactoferrin.

32. The transgenic bovine of claim 28, wherein the polypeptide is human lysozyme.

33. The transgenic bovine of claim 30, wherein the 3'"'"' untranslated sequence and the 3'"'"' flanking sequence are from the second milk protein gene.

34. The transgenic bovine of claim 30, wherein the 3'"'"' untranslated sequence and the 3'"'"' flanking sequence are from the first milk protein gene.

35. The transgenic bovine of claim 31, wherein the 3'"'"' untranslated sequence and the 3'"'"' flanking sequence are from the second milk protein gene.

36. The transgenic bovine of claim 32, wherein the 3'"'"' untranslated sequence and the 3'"'"' flanking sequence are from the first milk protein gene.