Production of recombinant polypeptides by bovine species and transgenic methods

• US 6,140,552 A
• Filed: 06/07/1995
• Issued: 10/31/2000
• 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: Deboer, Herman A., Pieper, Frank, Heyneker, Herbert L., Platenburg, Gerard, Lee, Sang He, Strijker, Rein, Krimpenfort, Paul J. A.
• Primary Examiner(s): HAUDA, KAREN MARIE
• Application Number:
  US08/476,798
• Time in Patent Office: 1,973 Days
• Field of Search: 435/69.1, 435/320.1, 435/172.3, 800/2, 800/15, 800/4, 800/3, 800/8, 800/7
• 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. A transgenic or chimeric bovine whose mammary gland cells contain a nucleic acid construct, wherein the construct comprises in operable association:

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

a DNA sequence encoding a polypeptide of interest; and

a regulatory sequence that promotes expression of the DNA sequence encoding the polypeptide of interest,

wherein the transgenic or chimeric bovine or a female descendant of the transgenic bovine is disposed to express the construct in mammary secretory cells such that the polypeptide of interest is detectable in milk produced by the transgenic or chimeric bovine or the female descendant of the transgenic bovine;

wherein the polypeptide is heterologous to the milk of a natural bovine.

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

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

4. The method of claim 3, wherein the polypeptide is an immunoglobulin.

5. A method of producing a chimeric 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 into the zygote in vitro a nucleic acid construct comprising in operable association a DNA sequence encoding a signal sequence functional in bovine mammary gland secretory cells, a DNA sequence encoding a polypeptide of interest, and a regulatory sequence that promotes expression of the DNA sequence encoding the polypeptide of interest;

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 chimeric bovine.

6. The method of claim 5, further comprising obtaining milk comprising the polypeptide of interest from the chimeric bovine or female descendant of the chimeric bovine.

7. A bovine whose mammary gland cells have a genome comprising in operable association:

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

a DNA sequence encoding a polypeptide of interest; and

a regulatory sequence that promotes expression of the DNA sequence encoding the polypeptide in the mammary gland;

wherein the 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 bovine or a female descendant of the bovine;

wherein the polypeptide is a heterologous polypeptide.