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US 6,713,662 B1Granted
Production of collagen in the milk of transgenic mammals
AssigneesPharming Intellectual Property B.V., Cohesion Technologies, Inc.
InventorsCostas N. Karatzas, Frank Pieper, Ineke De Wit, Richard Berg, Gerard Platenburg, Paul David Toman
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Abstract
The invention provides transgenic nonhuman mammals capable secreting exogenous procollagen or collagen into their milk. The mammals are healthy and capable of producing procollagen or collagen at high levels, usually in trimeric form. Suitable transgenes for incorporation into the mammals are also provided.
Claims26
A transgenic nonhuman mammal having a transgene comprising:
- a mammary-gland specific promoter;
- a mammary-gland specific enhancer
- a secretory DNA segment encoding a signal peptide functional in mammary secretory cells of the transgenic nonhuman mammal;
- and
- a recombinant DNA segment encoding an exogenous procollagen polypeptide operably linked to the secretory DNA segment to form a secretory-recombinant DNA segment, the secretory-recombinant DNA segment being operably linked to the promoter and to the enhancer;
- wherein the transgene, in an adult form of the nonhuman mammal or a female descendant of the nonhuman mammal, expresses the secretory-recombinant DNA segment in the mammary secretory cells to produce a form of the exogenous procollagen polypeptide that is processed and secreted by the mammary secretory cells into milk as exogenous procollagen or collagen in a detectable amount;
- provided the recombinant DNA segment comprises a genomic DNA segment from the α1(I) collagen gene and is
- without nucleotides 1-114 from the first exon of the gene, and/or
- without a segment from the first intron of the gene.
6 more elements
A method for preparing procollagen or collagen, the method comprising:
- providing a transgenic nonhuman mammal having a transgene comprising:
- a mammary-gland specific promoter;
- a mammary-gland specific enhancer
- a secretory DNA segment encoding a signal peptide functional in mammary secretory cells of the transgenic nonhuman mammal;
- and
- a recombinant DNA segment encoding an exogenous procollagen polypeptide operably linked to the secretory DNA segment to form a secretory-recombinant DNA segment, the secretory-recombinant DNA segment being operably linked to the promoter and to the enhancer;
- wherein the transgene, in an adult form of the nonhuman mammal or a female descendant of the nonhuman mammal, expresses the secretory-recombinant DNA segment in the mammary secretory cells to produce a form of the exogenous procollagen polypeptide that is processed and secreted by the mammary secretory cells into milk as exogenous procollagen or collagen in a detectable amount;
- supplementing the diet of the transgenic nonhuman mammal or its female descendant with Vitamin c at a level of 50-1000 mg/kg food;
- recovering milk from the adult form of the transgenic nonhuman mammal or its female descendant, wherein the milk comprises exogenous procollagen or collagen in a recoverable amount;
- and
- purifying procollagen or collagen from the milk.
8 more elements
A transgene for expressing procollagen or collagen, the transgene comprising:
- a casein promoter;
- a casein enhancer;
- a genomic DNA segment comprising a segment from a signal peptide coding sequence to a 3′ flanking region of a procollagen α2(I) gene, operably linked to the promoter and the enhancer.
A stable mammary-gland derived cell line, having a transgene comprising:
- a mammary-gland specific promoter;
- a mammary-gland specific enhancer;
- a secretory DNA segment encoding a signal peptide functional in the cell line;
- and
- a recombinant DNA segment encoding an exogenous procollagen polypeptide operably linked to the secretory DNA segment to form a secretory-recombinant DNA segment, the secretory-recombinant DNA segment being operably linked to the promoter and to the enhancer;
- wherein the cell line is induced by a lactogenic hormone to express the transgene to produce a form of the exogenous procollagen polypeptide that is processed and secreted by the cell lines as exogenous procollagen or collagen in trimeric form;
- provided the recombinant DNA segment comprises a genomic DNA segment from the α1(I) collagen gene and is
- without nucleotides 1-114 from the first exon of the gene, and/or
- without a segment from the first intron of the gene.
6 more elements
Dependencies are inferred from the claim text.