Direct gene transfer into the ruminant mammary gland
First Claim
1. A method for producing, in a ruminant mammal, milk containing a heterologous protein, said method comprising:
- (a) providing a genetic construct including DNA encoding said heterologous protein and a signal peptide,(b) mixing said construct with a liquid carrier to form a liquid complex,(c) infusing said liquid complex into a ductal tree of said mammal at an age of said mammal earlier than sexual maturity and after development of a functional streak canal leading to said ductal tree,(d) raising said mammal so that the mammal lactates and the heterologous protein encoded by said construct is expressed and secreted into the milk produced by said ductal tree and mammary alveolar cells, and(e) obtaining milk from said mammal.
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Abstract
Disclosed is a method for expressing a heterologous gene in the mammary gland of a ruminant mammal. The invention is useful for producing a heterologous protein(s) in the milk of the mammal, for producing a heterologous enzyme(s) which is tethered to the mammary epithelial cell membrane and which can act on a component of the milk of the mammal, and for providing an antisense oligonucleotide(s) or ribozyme(s) which inhibits expression of a gene to a mammary epithelial cell. The method involves infusing a liquid complex including a genetic construct into a ductal tree of the mammal. The liquid complex can be infused before the mammal reaches sexual maturity and after the mammal develops a functional streak canal. Alternatively, a liquid complex that is free of live retroviruses can be infused. If desired, the infused genetic construct can be treated with a polycationic compound and/or a lipid to improve the efficiency with which it is taken up by an epithelial cell of the mammary gland.
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Citations
41 Claims
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1. A method for producing, in a ruminant mammal, milk containing a heterologous protein, said method comprising:
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(a) providing a genetic construct including DNA encoding said heterologous protein and a signal peptide, (b) mixing said construct with a liquid carrier to form a liquid complex, (c) infusing said liquid complex into a ductal tree of said mammal at an age of said mammal earlier than sexual maturity and after development of a functional streak canal leading to said ductal tree, (d) raising said mammal so that the mammal lactates and the heterologous protein encoded by said construct is expressed and secreted into the milk produced by said ductal tree and mammary alveolar cells, and (e) obtaining milk from said mammal. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A method of producing, in a ruminant mammal, milk containing a heterologous protein, said method comprising:
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(a) providing a genetic construct including DNA encoding said heterologous protein and a signal peptide, (b) mixing said construct with a liquid carrier to form a liquid complex free of live retroviruses, (c) infusing said liquid complex into a ductal tree of said mammal, (d) raising said mammal so that the mammal lactates and the heterologous protein encoded by said construct is expressed and secreted into the milk produced by said ductal tree and mammary alveolar cells, and (e) obtaining said milk from said mammal. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29)
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30. A method for expressing, in the mammary gland of a ruminant mammal, a heterologous enzyme which is tethered to the membrane of an epithelial cell of said mammary gland, said method comprising:
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(a) providing a genetic construct encoding said heterologous enzyme and a membrane-associated polypeptide, (b) mixing said construct with a liquid carrier to form a liquid complex, (c) infusing said liquid complex into a ductal tree of said mammal at an age of said mammal earlier than sexual maturity and after development of a functional streak canal leading to said ductal tree, (d) raising said mammal so that said heterologous enzyme is expressed and becomes associated with said membrane. - View Dependent Claims (31, 32, 33, 34, 35, 36)
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37. A method for determining whether a heterologous gene is expressed in the mammary gland of a ruminant mammal, said method comprising:
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(a) providing a genetic construct including said heterologous gene, (b) mixing said construct with a liquid carrier to form a liquid complex, (c) infusing said liquid complex into a ductal tree of said mammal at an age of said mammal earlier than sexual maturity and after development of a functional streak canal leading to said ductal tree, and (d) assaying for expression of said heterologous gene. - View Dependent Claims (38, 39, 40, 41)
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Specification