Expression of a heterologous polypeptide in mammary tissue of transgenic non-human mammals using a long whey acidic protein promoter
First Claim
1. A transgenic non-human mammal containing a DNA sequence stably integrated in its genome, wherein said exogenous DNA sequence comprises the 5'"'"' 4.2 kb Sau3A-Kpn1 promoter of the mouse whey acidic protein gene, operably linked to a DNA sequence encoding heterologous protein C and a signal peptide, wherein said whey acidic protein promoter is specifically active in mammary cells and said signal peptide is effective in directing the secretion of said protein C into the milk of said transgenic mammal, wherein the activated form of said secreted protein C has an enzymatic activity of at least 50% as plasma-derived protein C, and wherein said transgenic mammal is selected from the group consisting of mice, rats, rabbits, pigs, sheep, goats and cows.
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Abstract
Heterologous polypeptides are produced in the milk of transgenic non-human mammals by the expression of a stably integrated DNA sequence containing the long whey acidic protein promoter operably linked to a DNA sequence encoding a heterologous polypeptide and a signal sequence. The transgenic non-human mammals of the present invention are produced by introducing this DNA sequence such that the DNA sequence is stably integrated into the DNA of germ line cells of the mature mammal and inherited in normal Mendelian fashion. A representative heterologous polypeptide is protein C.
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Citations
20 Claims
- 1. A transgenic non-human mammal containing a DNA sequence stably integrated in its genome, wherein said exogenous DNA sequence comprises the 5'"'"' 4.2 kb Sau3A-Kpn1 promoter of the mouse whey acidic protein gene, operably linked to a DNA sequence encoding heterologous protein C and a signal peptide, wherein said whey acidic protein promoter is specifically active in mammary cells and said signal peptide is effective in directing the secretion of said protein C into the milk of said transgenic mammal, wherein the activated form of said secreted protein C has an enzymatic activity of at least 50% as plasma-derived protein C, and wherein said transgenic mammal is selected from the group consisting of mice, rats, rabbits, pigs, sheep, goats and cows.
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5. A process for the production of heterologous protein C, comprising the steps of:
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(A) providing a non-human transgenic mammal whose genome comprises a stably integrated DNA sequence comprising the 5'"'"' 4.2 kb Sau3A-Kpn1 promoter of the mouse whey acidic protein gene, operably linked to a DNA sequence encoding a heterologous protein C and a signal peptide, said promoter being specifically active in mammary cells and said signal peptide being effective in directing the secretion of said protein C into the milk of said transgenic mammal, and wherein said transgenic mammal is selected from the group consisting of mice, rats, rabbits, pigs, sheep, goats and cows; (B) producing milk from said transgenic mammal, wherein said milk contains said secreted protein C, and wherein the activated form of said protein C has an enzymatic activity of at least 50% as plasma-derived protein C; (C) collecting said milk; and (D) isolating said protein C from said milk. - View Dependent Claims (6, 7, 8)
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9. A process for producing non-human transgenic mammals, comprising the steps of (A) providing a mixture containing a double-stranded DNA;
- (B) subjecting said mixture to anion-exchange high performance liquid chromatography to obtain purified double-stranded DNA; and
thereafter (C) microinjecting an aqueous buffer solution containing said purified double-stranded DNA into an animal embryo, wherein said double-stranded DNA is selected from the group consisting of a double-stranded DNA comprising the 5'"'"' 4.2 kb Sau3A-Kpn1 promoter fragment of the mouse whey acidic protein promoter, a double-stranded DNA comprising a fragment of the human protein C gene beginning 21 basepairs upstream of the protein C start codon and ending at the NheI site in the 3'"'"' end of the protein C gene, and a double-stranded DNA comprising a DNA sequence comprising the 5'"'"' 4.2 kb Sau3A-Kpn1 promoter fragment of the mouse whey acidic protein promoter ligated directly or by a linker to a fragment of the human protein C gene beginning 21 basepairs upstream of the protein C start codon and ending at the NheI site in the 3'"'"' end of the protein C gene, wherein the activated form of protein C encoded by said double-stranded DNA has an enzymatic activity of at least 50% as plasma-derived protein C, and wherein said transgenic mammal is selected from the group consisting of mice, rats, rabbits, pigs, sheep, goats and cows.
- (B) subjecting said mixture to anion-exchange high performance liquid chromatography to obtain purified double-stranded DNA; and
- 10. A transgenic non-human mammal containing a DNA sequence stably integrated in its genome, wherein said DNA sequence comprises the 5'"'"' 4.2 kb Sau3A-Kpn1 promoter fragment of the mouse whey acidic protein promoter, operably linked to a DNA sequence encoding a heterologous polypeptide whereby said polypeptide is expressed specifically in mammary cells of said transgenic mammal and said polypeptide comprises a signal peptide, said signal peptide being effective in directing the secretion of said polypeptide into the milk of said mammal.
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15. A process for the production of a heterologous polypeptide in the milk of a transgenic non-human mammal, comprising the steps of:
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(A) providing a non-human transgenic mammal whose genome comprises a stably integrated DNA sequence comprising the 5'"'"' 4.2 kb Sau3A-Kpn1 promoter of the mouse whey acidic protein gene, operably linked to a DNA sequence encoding a heterologous polypeptide and a signal peptide, said promoter being specifically active in mammary cells and said signal peptide being effective in directing the secretion of said polypeptide into the milk of said transgenic mammal; (B) producing milk from said transgenic mammal, wherein said milk contains said polypeptide; (C) collecting said milk; and (D) isolating said polypeptide from said milk. - View Dependent Claims (16, 17, 18, 19)
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20. An isolated DNA molecule which regulates the expression of a heterologous gene, wherein said DNA molecule consists of the 5'"'"' 4.2 kb Sau3A-Kpn1 promoter of the mouse whey acidic protein gene.
Specification