Production of fibrinogen in transgenic animals

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.

1. 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. 
 
     
     
       2. A method according to  claim 1  wherein said species into which said DNA segments are introduced is selected from the group consisting of sheep, pigs, goats, and cattle. 
     
     
       3. A method according to  claim 1  wherein each of said first, second and third DNA segments comprises an intron. 
     
     
       4. A method according to  claim 1  wherein the molar ratio of said first, second and third DNA segments is within the range of 0.5-1:0.5-1:0.5-1. 
     
     
       5. A method according to  claim 1  wherein each of said first, second and third DNA segments is operably linked to a transcription promoter selected from the group consisting of casein, β-lactoglobulin, α-lactalbumin and whey acidic protein gene promoters. 
     
     
       6. A method according to  claim 1  wherein said first, second and third DNA segments are expressed under the control of a β-lactoglobulin promoter. 
     
     
       7. A method according to  claim 1  wherein said introducing step comprises injecting said first, second and third DNA segments into a pronucleus of said fertilized egg. 
     
     
       8. A method according to  claim 1  wherein said fibrinogen is human fibrinogen. 
     
     
       9. A method according to  claim 1  wherein said second DNA segment comprises a sequence of nucleotides as shown in SEQ ID NO: 3 from nucleotide 470 to nucleotide 8100. 
     
     
       10. A method according to  claim 1  wherein said second DNA segment comprises a sequence of nucleotides as shown in SEQ ID NO: 3 from nucleotide 512 to nucleotide 8100. 
     
     
       11. A method according to  claim 1  wherein said species into which said DNA segments is introduced is sheep. 
     
     
       12. 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. 
 
     
     
       13. A method according to  claim 12  wherein said mammal is a sheep, pig, goat or cow. 
     
     
       14. A method according to  claim 12  wherein each of said first, second and third gene fusions comprises an intron. 
     
     
       15. A method according to  claim 12  wherein the molar ratio of said first, second and third gene fusions introduced is within the range of 0.5-1:0.5-1:0.5-1. 
     
     
       16. A method according to  claim 12  wherein said introducing step comprises injecting said first, second and third gene fusions into a pronucleus of a fertilized egg and inserting said egg into an oviduct of a pseudopregnant female to produce female offspring carrying said gene fusions in the germ line, wherein said egg and said pseudopregnant female are of the same species. 
     
     
       17. A method according to  claim 12  wherein said mammal is a sheep. 
     
     
       18. 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.   
     
     
       19. A method according to  claim 18  wherein said mammal is a sheep, pig, goat or cow. 
     
     
       20. A method according to  claim 18  wherein said mammal is a sheep. 
     
     
       21. 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. 
     
     
       22. A mammal according to  claim 21  wherein said mammal is a sheep. 
     
     
       23. 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. 
 
     
     
       24. A process according to  claim 23  wherein said offspring is female. 
     
     
       25. A process according to  claim 23  wherein said offspring is male. 
     
     
       26. A non-human mammal produced according to the process of  claim 23 . 
     
     
       27. A non-human mammal according to  claim 26  wherein said mammal is female. 
     
     
       28. A non-human female mammal according to  claim 27  that produces milk containing biocompetent fibrinogen encoded by said DNA segments. 
     
     
       29. A non-human mammal according to  claim 26  wherein said mammal is male. 
     
     
       30. 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. 
     
     
       31. A mammal non-human according to  claim 30  wherein said mammal is female. 
     
     
       32. A mammal non-human according to  claim 30  wherein said mammal is male. 
     
     
       33. A mammal according to  claim 30 , wherein said mammal is a sheep. 
     
     
       34. 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.