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US RE42,704 E1Granted
Production of fibrinogen in transgenic animals
AssigneePharming Intellectual Property B.V.
InventorsDonna E. Prunkard, Donald C. Foster
Life of this patent
Estimating the term…
Abstract
Materials and methods for producing fibrinogen in transgenic non-human mammals are disclosed. DNA segments encoding Aα, Bβ and γ chains of fibrinogen are introduced into the germ line of a non-human mammal, and the mammal or its female progeny produces milk containing fibrinogen expressed from the introduced DNA segments. Non-human mammalian embryos and transgenic non-human mammals carrying DNA segments encoding heterologous fibrinogen polypeptide chains are also disclosed.
Claims34
A method for producing biocompetent fibrinogen comprising:
- providing a first DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen Aα chain, the DNA segment comprising genomic DNA encoding the Aα chain, a second DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen Bβ chain, the DNA segment comprising genomic DNA encoding the Bβ chain, and a third DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen γ chain, the DNA segment comprising genomic DNA encoding the γ chain, wherein each chain is from the same species, and wherein each of said first, second and third segments is operably linked to additional DNA segments required for its expression in the mammary gland of a host female mammal and the first, second, third segments are linked in a single vector;
- introducing said DNA segments into a fertilized egg of a non-human mammalian species heterologous to the species of origin of said fibrinogen chains;
- inserting said egg into an oviduct or uterus of a female of said mammalian species to obtain offspring carrying said DNA segments;
- breeding said offspring to produce female progeny that express said first, second and third DNA segments and produce milk containing biocompetent fibrinogen encoded by said segments;
- collecting milk from said female progeny;
- and
- and recovering the biocompetent fibrinogen from the milk.
4 more elements
A method of producing biocompetent fibrinogen comprising:
- incorporating into operable linkage a first DNA segment encoding a secretion signal operably linked to, a genomic DNA segment encoding an Aα chain of fibrinogen into a β-lactoglobulin gene and an additional segment required for expression of the Aα chain in the mammary gland of a mammal to produce a first gene fusioncomprising a β-lactoglobulin promoter operably linked to the first DNA segment;
- incorporating into operable linkage a second DNA segment encoding a secretion signal operably linked to, a genomic DNA segment encoding a Bβ chain of fibrinogen into a β-lactoglobulin gene and an additional segment required for expression of the Bβ chain to produce a second gene fusion comprising a β-lactoglobulin promoter operably linked to the second DNA segment;
- incorporating into operable linkage a third DNA segment encoding a secretion signal operably linked to, a genomic DNA segment encoding a γ chain of fibrinogen and an additional segment required for expression of the γ chain into a β-lactoglobulin gene to produce a third gene fusion, comprising a β-lactoglobulin promoter operably linked to the third DNA segment wherein each of said first, second and third segments are of the same species;
- linking the first, second and third gene fusions in a single vector;
- introducing said first, second and third gene fusions into the germ line of a non-human mammal so that said DNA segments are expressed in a mammary gland of said mammal or its female progeny and biocompetent fibrinogen is secreted into milk of said mammal or its female progeny;
- obtaining milk from said mammal or its female progeny;
- and
- recovering said fibrinogen from said milk.
5 more elements
A method for producing biocompetent fibrinogen comprising:
- providing a transgenic female non-human mammal carrying in its germline heterologous DNA segments encoding Aα, Bβ and γ chains of flbrinogen, wherein said segments are expressed in a mammary gland of said mammal and biocompetent fibrinogen encoded by said segments is secreted into milk of said mammal;
- collecting milk from said mammal;
- and
- recovering said biocompetent fibrinogen from said milk.
1 more element
A transgenic non-human female mammal that produces recoverable amounts of biocompetent human fibrinogen in its milk, wherein said mammal comprises:
- a first DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen Aα chain,
- a second DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen Bβ chain, and
- a third DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen γ chain, and
- further wherein each chain is derived from the same species and is operably linked to additional DNA segments required for its expression in the mammary gland of a host female mammal.
1 more element
A process for producing a transgenic offspring of a mammal comprising:
- providing a first DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen Aα chain, the DNA segment comprising genomic DNA encoding the Aα chain;
- a second DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen Bβ chain, the DNA segment comprising genomic DNA encoding the Bβ chain;
- and a third DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen γ chain, the DNA segment comprising genomic DNA encoding the γ chain;
- wherein each chain is derived from the same species, and wherein each of said first, second and third segments is operably linked to additional DNA segments required for its expression in the mammary gland of a host female mammal;
- linking the first, second and third segments in a single vector;
- introducing said DNA segments into a fertilized egg of a non-human mammalian species heterologous to the species of origin of said fibrinogen chains;
- inserting said fertilized egg into an oviduct or uterus of a female of said mammalian species;
- and
- allowing said fertilized egg to develop thereby producing transgenic offspring carrying said first, second and third DNA segments, wherein female progeny of said mammal express said DNA segments in a mammary gland to produce biocompetent fibrinogen.
6 more elements
A non-human mammal carrying in its germline DNA segments encoding human Aα, Bβ and γ chains of fibrinogen, wherein female progeny of said mammal express said DNA segments in a mammary gland to produce biocompetent human fibrinogen.
A set of DNA sequences comprising:
- a first DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen Aα chain, the DNA segment comprising genomic DNA encoding the Aα chain;
- a second DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen Bβ chain, the DNA segment comprising genomic DNA encoding the Bβ chain;
- and
- a third DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen γ chain, the DNA segment comprising genomic DNA encoding the γ chain, wherein each chain is from the same species, and wherein each of said first, second and third segments is operably linked to additional DNA segments required for its expression in the mammary gland of a host female mammal;
- and the first, second, third segments are linked in a single vector.
2 more elements
Dependencies are inferred from the claim text.