Method of producing a transgenic bovine or transgenic bovine embryo

• US 5,633,076 A
• Filed: 11/16/1993
• Issued: 05/27/1997
• 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: Lee, Sang H., Pieper, Frank, Heyneker, Herbert L., Platenburg, Gerard, DeBoer, Herman A., Strijker, Rein, Krimpenfort, Paul J. A.
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US08/154,019
• Time in Patent Office: 1,288 Days
• Field of Search: 800/2, 800/DIG. 1, 435/192.3, 435/317.1, 935/11, 935/53, 935/111
• US Class Current: 800/25
• 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 method is disclosed for the production of a transgenic bovine or a transgenic bovine embryo comprising obtaining an ovum from bovine ovaries, maturing the ovum in vitro, fertilizing the mature ovum or ova in vitro to form a zygote, introducing a transgene into the zygote in vitro and maturing the zygote to a preimplantation stage embryo in vitro. To produce the transgenic bovine, the embryo is transplanted into a recipient female bovine, wherein the female bovine gestates the embryo to produce a transgenic bovine.

Claims

1. A method of producing a transgenic bovine, the method comprising:

obtaining an ovum from bovine ovaries;

maturing the ovum in vitro;

fertilizing the mature ovum in vitro to form a zygote;

introducing a transgene into the zygote in vitro;

maturing the zygote to a preimplantation stage embryo in vitro; and

transplanting the embryo into a recipient female bovine, wherein the female bovine gestates the embryo to produce a transgenic bovine.

2. The method of claim 1, wherein the transgene is introduced into the zygote by microinjection.

3. The method of claim 1, wherein a plurality of ova are obtained from bovine ovaries, matured and fertilized in vitro to form a plurality of zygotes, and wherein in said introducing step said zygotes are substantially synchronous.

4. The method of claim 1, wherein the transgene is a human genomic fragment encoding a human polypeptide.

5. The method of claim 4, wherein the human polypeptide is lactoferrin.

6. The method of claim 1, further comprising mating said transgenic bovine to produce a transgenic bovine progeny.

7. The method of claim 1 or 6, 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 secretory cells; and

a DNA sequence encoding a polypeptide of interest;

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

8. The method of claim 7, wherein the promoter is

a bovine α

-s1 casein promoter; and

the enhancer is a a bovine α

-s1 casein enhancer.

9. The method of claim 8, wherein the transgene further comprises

an α

-s1 casein 3'"'"' untranslated sequence.

10. The method of claim 9, wherein the transgene further comprises an α

-s1 casein 3'"'"' flanking sequence.

11. The method of claim 10, wherein the α

-s1 casein 3'"'"' flanking sequence has a length of at least 2 kb.

12. The method of claim 11, wherein the transgene further comprises at least 16 kb of α

-s1 casein 5'"'"' flanking sequence.

13. The method of claim 12, wherein the transgene further comprises an intronic sequence.

14. The method of claim 13, wherein the intronic sequence is a hybrid intronic sequence.

15. The method of claim 14, wherein the hybrid intronic sequence comprises a 5'"'"' portion of a bovine α

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

16. The method of claim 15 wherein the 3'"'"' portion is a 3'"'"' splice signal sequence associated with the J-C segment rearrangement of an IgG heavy chain.

17. The method of claim 16, wherein the polypeptide is a homologous polypeptide from the bovine.

18. The method of claim 16, wherein the polypeptide is a heterologous polypeptide.

19. The method of claim 18, wherein the heterologous polypeptide is selected from the group consisting of human milk proteins, human serum proteins, and industrial enzymes.

20. The method of claim 19, wherein the heterologous polypeptide is a human milk protein.

21. The method of claim 20, wherein the human milk protein is selected from the group consisting of secretory immunoglobulins, lysozyme, lactoferrin, lactoglobulin, α

-lactalbumin and bile salt-stimulated lipase.

22. The method of claim 21, wherein the milk protein is lactoferrin or lysozyme.

23. The method of claim 18, wherein the heterologous polypeptide is a human serum protein.

24. The method of claim 23, wherein the human serum protein is selected from the group consisting of albumin, immunoglobulin, Factor VIII, Factor IX and Protein C.

25. The method of claim 24, wherein the serum protein is albumin.

26. The method of claim 18, wherein the heterologous polypeptide is an industrial enzyme selected from the group consisting of proteases, lipases, chitinases and ligninases.

27. The method of claim 16, wherein the polypeptide is a wild-type polypeptide or an allelic variant thereof.

28. The method of claim 8, wherein the transgene is the 26 kb NotI fragment of plasmid p26,8hlF4.

29. A method of producing a transgenic bovine embryo, the method comprising:

obtaining an ovum from bovine ovaries;

maturing the ovum in vitro;

fertilizing the mature ovum in vitro to form a zygote; and

introducing a transgene into the zygote, wherein the transgene integrates into the genome of the zygote to form the transgenic bovine embryo.