Production of fibrinogen in transgenic animals
AssigneesPharmaceutical Proteins Ltd., ZymoGenetics, Inc.
InventorsIan Garner, Michael L. Dalrymple, Donna E. Prunkard, Donald C. Foster
Life of this patent
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Abstract
Materials and methods for producing fibrinogen in transgenic non-human mammals are disclosed. DNA segments encoding A.alpha., B.beta. and .gamma. 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.
Claims33
A method for producing biocompetent fibrinogen comprising:
- providing a first DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen A.alpha. chain, a second DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen B.beta. chain, and a third DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen .gamma. 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;
- 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.
A method of producing biocompetent fibrinogen comprising:
- incorporating a first DNA segment encoding a secretion signal operably linked to an A.alpha. chain of fibrinogen into a .beta.-lactoglobulin gene to produce a first gene fusion comprising a .beta.-lactoglobulin promoter operably linked to the first DNA segment;
- incorporating a second DNA segment encoding a secretion signal operably linked to a B.beta. chain of fibrinogen into a .beta.-lactoglobulin gene to produce a second gene fusion comprising a .beta.-lactoglobulin promoter operably linked to the second DNA segment;
- incorporating a third DNA segment encoding a secretion signal operably linked to a .gamma. chain of fibrinogen into a .beta.-lactoglobulin gene to produce a third gene fusion comprising a .beta.-lactoglobulin promoter operably linked to the third DNA segment wherein each of said first, second and third segments are of the same species;
- 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.
A method for producing biocompetent fibrinogen comprising:
- providing a transgenic female non-human mammal carrying in its germline heterologous DNA segments encoding A.alpha., B.beta. and .gamma. chains of fibrinogen, 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.
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 A.alpha. chain,
- a second DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen B.beta. chain, and
- a third DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen .gamma. 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.
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 A.alpha. chain, a second DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen B.beta. chain, and a third DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen .gamma. 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;
- 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.
A non-human mammal carrying in its germline DNA segments encoding human A.alpha., B.beta. and .gamma. chains of fibrinogen, wherein female progeny of said mammal express said DNA segments in a mammary gland to produce biocompetent human fibrinogen.
Dependencies are inferred from the claim text.