Production of membrane proteins in the milk of transgenic mammals

• US 6,472,584 B1
• Filed: 07/28/2000
• Issued: 10/29/2002
• Est. Priority Date: 10/02/1991
• Status: Expired due to Fees

• Current Assignee: Genzyme Corporation (Sanofi )
• Original Assignee: Genzyme Corporation (Sanofi )
• Inventors: Smith, Alan E.
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US09/627,290
• Time in Patent Office: 823 Days
• Field of Search: 435/320.1, 435/328, 800/7, 800/14, 800/15, 800/16, 800/18, 436/23
• 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 2227/10 Mammal
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic

Abstract

Production of proteins not normally secreted through conventional pathways such as membrane proteins including, for example, CFTR associated with cystic fibrosis, is now made possible by collection of such protein from the milk of lactating transgenic animals.

Claims

1. A DNA construct comprising:

as operably linked components, a DNA sequence encoding a cystic fibrosis transmembrane conductance regulator protein and a mammary gland specific promoter sequence wherein, upon expression of the DNA sequence, the cystic fibrosis transmembrane conductance regulator inserts into an apical membrane of a mammary cell so that said cystic fibrosis transmembrane conductance regulator protein is secreted in a milk fat globule.

2. The DNA construct of claim 1, wherein said mammary gland specific promoter is a beta lactoglobulin promoter.

3. The DNA construct according to claim 1, wherein said mammary gland specific promoter is an alpha-casein promoter.

4. The DNA construct according to claim 3, wherein said mammary gland specific promoter is a whey acid protein promoter.

5. A DNA construct comprising, as operably linked components, a DNA sequence encoding a cystic fibrosis transmembrane conductance regulator protein and a beta-casein promoter.

6. The DNA construct according to claim 5, wherein said beta casein promoter is a goat beta-casein promoter.

7. A method of producing a cystic fibrosis transmembrane conductance regulator protein in milk, said method comprising:

providing a transgenic non-human female mammal which contains within its genome a DNA construct comprising, as operably linked components, a DNA sequence encoding said a cystic fibrosis transmembrane conductance regulator protein and a mammary gland specific promoter sequence, wherein, upon expression of the DNA sequence, said cystic fibrosis transmembrane conductance regulator protein inserts into an apical membrane of mammary epithelial cell of said female mammal, thereby producing said cystic fibrosis transmembrane conductance regulator protein in the milk of said female mammal.

8. The method according to claim 7, wherein said cystic fibrosis transmembrane conductance regulator protein is mature and fully glycosylated.

9. The method according to claim 7, wherein the mammary gland specific promoter is selected from the group consisting of a beta-cascin promotor, a beta lactoglobulin promoter, an alpha-casein promoter, and a whey acid protein promoter.

10. A milk fat globule membrane comprising a cystic fibrosis transmembrane conductance regulator protein wherein said milk fat globule membrane is from a transgenic non-human female mammal.

11. A method of producing a cystic fibrosis transmembrane conductance regulator protein in milk, said method comprising:

providing a transgenic non-human female mammal containing within its genome a DNA construct comprising, as operably linked components, a DNA sequence encoding a cystic fibrosis transmembrane conductance regulator protein and a beta-casein promoter sequence, wherein, upon expression of said DNA sequence, said cystic fibrosis transmembrane conductance regulator protein inserts into an apical membrane of a mammary epithelial cell of the female mammal thereby producing said cystic fibrosis transmembrane conductance regulator protein in the milk of said female mammal.

12. A DNA construct comprising:

as operably linked components, a DNA sequence encoding a membrane protein and a mammary specific promoter sequence wherein, upon expression of the DNA sequence, the membrane protein inserts into an apical membrane of a mammary epithelial cell so that said membrane protein is secreted in a milk fat globule of a transgenic non-human female mammal.

13. The DNA construct of claim 12, wherein said mammary gland specific promoter is a beta lactoglobulin promoter.

14. The DNA construct according to claim 12, wherein said mammary gland specific promoter is an alpha-casein promoter.

15. The DNA construct according to claim 12, wherein said mammary gland specific promoter is a whey acid protein promoter.

16. The DNA construct according to claim 12, wherein said mammary gland specific promoter is a beta-casein promotor.

17. The DNA construct according to claim 12, wherein the membrane protein is a transmembrane ion channel protein.

18. The DNA construct according to claim 12, wherein the membrane protein is selected from the group consisting of a transmembrane ion channel, a hormone receptor protein, a thyroid stimulating hormone receptor, a cytokine receptor protein, and a viral glycoprotein.

19. A method of producing a membrane protein in milk, said method comprising:

providing a transgenic non-human female mammal which contains within its genome a DNA construct comprising, as operably linked components, a DNA sequence encoding a membrane protein and a mammary gland specific promoter sequence, wherein, upon expression of the DNA sequence, said membrane protein inserts into an apical membrane of a mammary epithelial cell of said female mammal, thereby producing said membrane protein in the milk of said female mammal.

20. The method according to claim 19, wherein the mammary gland specific promoter is selected from the group consisting of a beta-casein promoter, a beta lactoglobulin promoter, an alpha-casein promoter, and a whey acid protein promoter.

21. The method of claim 19, wherein the membrane protein is a transmembrane ion channel protein.

22. The method of claim 19, wherein the membrane protein is selected from the group consisting of a transmembrane ion channel, a hormone receptor protein, a thyroid stimulating hormone receptor, a cytokine receptor protein, and a viral glycoprotein.

23. A method of producing a membrane protein in milk, said method comprising:

providing a transgenic non-human female mammal which contains within its genome a DNA construct comprising, as operably linked components, a DNA sequence encoding a membrane protein and a beta casein promoter sequence, wherein, upon expression of said DNA sequence, said membrane protein inserts into an apical membrane of a mammary epithelial cell of the female mammal, thereby producing said membrane protein in the milk of said female mammal.

24. A milk fat globule membrane comprising a membrane protein wherein said membrane protein is other than a native membrane protein associated with a milk fat globule membrane and wherein said milk fat globule membrane is from a transgenic non-human female mammal.