Transgenic bovines and milk from transgenic bovines

• US 6,013,857 A
• Filed: 06/05/1995
• Issued: 01/11/2000
• 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: Heyneker, Herbert L., Deboer, Herman A., Krimpenfort, Paul J. A., Pieper, Frank, Platenburg, Gerard, Lee, Sang He, Strijker, Rein
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US08/464,167
• Time in Patent Office: 1,681 Days
• Field of Search: 435/69.1, 435/172.3, 435/3, 800/2, 800/7, 800/15, 800/4, 800/5, 800/13, 426/556, 426/580, 426/587, 426/588, 424/535
• US Class Current: 800/15
• 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

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.

Claims

1. Milk produced by a transgenic bovine whose genome comprises a transgene comprising a DNA sequence encoding a recombinant polypeptide heterologous to the milk of the bovine species operably linked to a mammary gland promoter, wherein expression of said DNA sequence results in the production of a detectable level of said polypeptide in said milk.

2. The milk of claim 1 wherein said s a bovine protein not found in the milk of nontransgenic bovines.

3. The milk of claim 1 wherein said recombinant polypeptide is from a species other than bovine.

4. The milk of claim 3 wherein said polypeptide is selected from the group consisting of human milk proteins, human serum proteins, and industrial enzymes.

5. The milk of claim 4 wherein said polypeptide is a human milk protein.

6. The milk of claim 5 wherein said human milk protein is selected from the group consisting of secretory immunoglobulins, lysozyme, lactoferrin, lactoglobulin, α

-lactalbumin and bile salt-stimulated lipase.

7. The milk of claim 6 wherein said milk protein is lactoferrin or lysozyme.

8. The milk of claim 3 wherein said polypeptide is a human serum protein.

9. The milk of claim 8 wherein said human serum protein is selected from the group consisting of albumin, immunoglobulin, Factor VIII, Factor IX and Protein C.

10. The milk of claim 9 wherein said serum protein is albumin.

11. The milk of claim 4, wherein the polypeptide is an industrial enzyme.

12. The milk of claim 11, wherein the polypeptide is selected from the group consisting of proteases, lipases, chattiers, and liginases.

13. A food formulation comprising (a) milk produced by a transgenic bovine whose genome comprises a transgene comprising a DNA sequence encoding a recombinant polypeptide heterologous to the milk of the bovine species operably linked to a mammary gland promoter, wherein expression of said DNA sequence results in the production of a detectable level of said polypeptide in said milk;

and (b) other ingredients.

14. The food formulation of claim 13 wherein said recombinant polypeptide is at least partially purified from said transgenic milk.

15. The food formulation of claim 13 formulated with nutrients appropriate for an infant formula.

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

a regulatory sequence from a gene which is preferentially expressed in the mammary gland over other tissues;

a DNA sequence coding 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 bovine is disposed to express 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 when in a lactating state; and

wherein the DNA sequence encodes a naturally occurring heterologous polypeptide.

17. A method of producing a polypeptide, comprising recovering milk from the transgenic bovine or female descendant of claim 16, wherein the milk contains the polypeptide.

18. The method of claim 17, further comprising purifying the polypeptide from the milk.

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

a regulatory sequence from a gene which is preferentially expressed in the mammary gland over other tissues;

a DNA sequence coding 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 bovine is disposed to express 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 when in a lactating state; and

wherein the polypeptide is a bovine protein undetectable in milk of a nontransgenic bovine, or a heterologous polypeptide.

20. A method of producing a polypeptide, comprising recovering milk from the transgenic bovine or female descendant of claim 19, wherein the milk contains the polypeptide.

21. The method of claim 20, further comprising purifying the polypeptide from the milk.

22. Semen of a transgenic bovine whose genome comprises a transgene comprising a DNA sequence encoding a recombinant polypeptide heterologous to the milk of the bovine species operably linked to a mammary gland promoter, wherein progeny produced using said semen are disposed to express said DNA sequence sufficiently to result in the production of a detectable level of said polypeptide in milk of said progeny.