PRODUCTION OF FIBRINOGEN IN TRANSGENIC ANIMALS

Description

Background of the invention

[0001] The final step in the blood coagulation cascade is the thrombin-catalyzed conversion of the soluble plasma protein fibrinogen to insoluble fibrin. Thrombin cleaves a small peptide (fibrinopeptide A) from one of the three component chains (the Aα-chain) of fibrinogen. Fibrin monomers subsequently polymerize and are cross-linked by activated factor XIII to form a stable clot.

[0002] Fibrinogen is a key component of biological tissue glues (see, e.g., U.S. Pat. Nos. 4,377,572 and 4,442,655), which mimic the formation of natural blood clots to promote hemostasis and repair damaged tissue. Tissue glues provide an adjunct or alternative to sutures, staples and other mechanical means for wound closure. However, the principal ingredients of these products (fibrinogen, factor XIII and thrombin) are prepared from pooled human plasma by cryoprecipitation (e.g. U.S. Pat. Nos. 4,377,572; 4,362,567; 4,909,251) or ethanol precipitation (e.g. U.S. Pat. No. 4,442,655) or from single donor plasma (e.g. U.S. Pat. No. 4,627,879; Spotnitz et al., Am. Surg. 55: 166-168, 1989). The resultant fibrinogen/factor XIII preparation is mixed with bovine thrombin immediately before use to convert the fibrinogen to fibrin and activate the factor XIII, thus initiating coagulation of the adhesive.

[0003] Commercially available adhesives are of pooled plasma origin. Because blood-derived products have been associated with the transmission of human immunodeficiency virus (HIV), hepatitis virus and other etiologic agents, the acceptance and availability of such adhesives is limited. At present they are not approved for use in the United States.

[0004] While the use of autologous plasma reduces the risk of disease transmission, autologous adhesives can only be used in elective surgery when the patient is able to donate the necessary blood in advance.

[0005] As noted above, fibrinogen consists of three polypeptide chains, each of which is present in two copies in the assembled molecule. These chains, designated the Aα, Bβ and γ-chains, are coordinately expressed, assembled and secreted by the liver. While it might be expected that recombinant DNA technology could provide an alternative to the isolation of fibrinogen from plasma, this goal has proven to be elusive. The three fibrinogen chains have been individually expressed in E. coli (Lord, DNA 4: 33-38, 1985; Bolyard and Lord, Gene 66: 183-192, 1988; Bolyard and Lord, Blood 73: 1202-1206), but functional fibrinogen has not been produced in a prokaryotic system. Expression of biologically competent fibrinogen in yeast has not been reported. Cultured transfected mammalian cells have been used to express biologically active fibrinogen (Farrell et al., Blood 74: 55a, 1989; Hartwig and Danishefsky, J. Biol. Chem. 266: 6578-6585, 1991; Farrell et al., Biochemistry 30: 9414-9420, 1991), but expression levels have been so low that production of recombinant fibrinogen in commercial quantities is not feasible. Experimental evidence suggests that lower transcription rates in cultured cells as compared to liver may be a factor in the low expression rates achieved to date, but increasing the amount of fibrinogen chain mRNA in transfected BHK cells did not produce corresponding increases in fibrinogen protein secretion (Prunkard and Foster, XIV Congress of the international Society on Thrombosis and Haemostasis, 1993). These latter results suggest that proper assembly and processing of fibrinogen involves tissue-specific mechanisms not present in common laboratory cell lines.

[0006] There remains a need in the art for methods of producing large quantities of high quality fibrinogen for use in tissue adhesives and other applications. There is a further need for fibrinogen that is free of blood-borne pathogens. The present invention fulfills these needs and provides other, related advantages.

Summary of the Invention

[0007] It is an object of the present invention to provide commercially useful quantities of recombinant fibrinogen, particularly recombinant human fibrinogen. It is a further object of the invention to provide materials and methods for expressing fibrinogen In the mammary tissue of transgenic animals, particularly livestock animals such as cattle, sheep, pigs and goats.

[0008] Within one aspect, the present invention provides a method for producing fibrinogen comprising (a) providing a first DNA segment encoding a secretion signal operably linked to a fibrinogen Aα chain, a second DNA segment encoding a secretion signal operably linked to a fibrinogen Bβ chain, and a third DNA segment encoding a secretion signal operably linked to a fibrinogen γ chain, wherein each of the 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 and third segments are linked in a single expression unit, (b) introducing the single expression unit into a fertilized egg of a non-human mammalian species; (c) inserting the egg into an oviduct or uterus of a female of the species to obtain offspring carrying the DNA constructs; (d) breeding the offspring to produce female progeny that express the first, second and third DNA segments and produce milk containing biocompetent fibrinogen encoded by the segments; (e) collecting milk from the female progeny; and (f) recovering the fibrinogen from the milk. Within one embodiment, the egg containing the introduced segments is cultured for a period of time prior to insertion.

[0009] Within another aspect, the invention provides a method of producing fibrinogen comprising the steps of (a) incorporating a first DNA segment encoding a secretion signal operably linked to an Aα chain of fibrinogen into a Bβ - lactoglobulin gene to produce a first gene fusion; (b) incorporating a second DNA segment encoding a secretion signal operably linked to a Bβ chain of fibrinogen into a β-lactoglobulin gene to produce a second gene fusion; (c) incorporating a third DNA segment encoding a secretion signal operably linked to a γ chain of fibrinogen into a β-lactoglobulin gene to produce a third gene fusion; linking the first, second and third gene fusions in a single expression unit, (d) introducing the single expression unit into the germ line of a non-human mammal so that the DNA segments are expressed in a mammary gland of the mammal or its female progeny and biocompetent fibrinogen is secreted into milk of the mammal or its female progeny; (e) obtaining milk from the mammal or its female progeny; and (f) recovering the fibrinogen from the milk. Within preferred embodiments, the mammal is a sheep, pig, goat or bovine.

[0010] Within another aspect, the invention provides a method for producing fibrinogen comprising the steps of (a) providing a transgenic female non-human mammal carrying in its germline heterologous DNA segments encoding Aα., Bβ, and γ chains of fibrinogen, wherein the DNA segments are expressed in a mammary gland of the mammal and fibrinogen encoded by the DNA segments is secreted into milk of the mammal; (b) collecting milk from the mammal; and (c) recovering the fibrinogen from the milk, wherein said providing step comprising; incorporating into the germline of the female non-human mammal a first DNA segment encoding a secretion signal operably linked to a firbrinogen Aα chain, a second DNA segment encoding a secretion signal operably linked to a firbrinogen Bβ chain, and a third DNA segment encoding a secretion signal operably linked to a firbrinogen γ chain, 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, wherein said first, second and third segments are linked in a single expression unit.

[0011] Within another aspect, the invention provides a method for producing a transgenic offspring of a mammal comprising the steps of (a) providing a first DNA segment encoding a fibrinogen Aα chain, a second DNA segment encoding a fibrinogen Bβ chain, and a third DNA segment encoding a fibrinogen γ chain, wherein each of said first, second and third segments is operably linked to additional DNA segments required for its expression in a mammary gland of a host female mammal and secretion into milk of the host female mammal; linking the first, second and third segments in a single expression unit; (b) introducing the single expression unit into a fertilized egg of a mammal of a non-human species; (c) inserting the egg into an oviduct or uterus of a female of the non-human species to obtain an offspring carrying the first, second and third DNA segments. In a related aspect, the invention provides non-human mammals produced according to this process.

[0012] These and other aspects of the invention will become evident to the skilled practitioner upon reference to the following detailed description and the attached drawings.

Brief Description of the Drawings

[0013] 

Figure 1 illustrates the subcloning of a human fibrinogen A.alpha. chain DNA sequence.

Figure 2 is a partial restriction map of the vector Zem228. Symbols used are MT-1p, mouse metallothionein promoter; SV40t, SV40 terminator; and SV40p, SV40 promoter.

Figure 3 illustrates the subcloning of a human fibrinogen B.beta. chain DNA sequence.

Figure 4 illustrates the subcloning of a human fibrinogen .gamma. chain DNA sequence.

Figure 5 is a partial restriction map of the vector Zem219b. Symbols used are MT-1p, mouse metallothionein promoter; hGHt, human growth hormone terminator; SV40p, SV40 promoter; DHFR, dihydrofolate reductase gene; and SV40t, SV40 terminator.

Detailed Description of the Invention

[0014] Prior to setting forth the invention in detail, it will be helpful to define certain terms used herein:

As used herein, the term "biocompetent fibrinogen" is used to denote fibrinogen that polymerizes when treated with thrombin to form insoluble fibrin.

[0015] The term "egg" is used to denote an unfertilized ovum, a fertilized ovum prior to fusion of the pronuclei or an early stage embryo (fertilized ovum with fused pronuclei).

[0016] A "female mammal that produces milk containing biocompetent fibrinogen" is one that, following pregnancy and delivery, produces, during the lactation period, milk containing recoverable amounts of biocompetent fibrinogen. Those skilled in the art will recognized that such animals will produce milk, and therefore the fibrinogen, discontinuously.

[0017] The term "progeny" is used in its usual sense to include children and descendants.

[0018] The term "heterologous" is used to denote genetic material originating from a different species than that into which it has been introduced, or a protein produced from such genetic material.

[0019] Within the present invention, transgenic animal technology is employed to produce fibrinogen within the mammary glands of a host female mammal. Expression in the mammary gland and subsequent secretion of the protein of interest into the milk overcomes many difficulties encountered in isolating proteins from other sources. Milk is readily collected, available in large quantities, and well characterized biochemically. Furthermore, the major milk proteins are present in milk at high concentrations (from about 1 to 15 g/l).

[0020] From a commercial point of view, it is clearly preferable to use as the host a species that has a large milk yield. While smaller animals such as mice and rats can be used (and are preferred at the proof-of-concept stage), within the present invention it is preferred to use livestock mammals including, but not limited to, pigs, goats, sheep and cattle. Sheep are particularly preferred due to such factors as the previous history of transgenesis in this species, milk yield, cost and the ready availability of equipment for collecting sheep milk. See WO 88/00239 for a comparison of factors influencing the choice of host species. It is generally desirable to select a breed of host animal that has been bred for dairy use, such as East Friesland sheep, or to introduce dairy stock by breeding of the transgenic line at a later date. In any event, animals of known, good health status should be used.

[0021] Fibrinogen produced according to the present invention may be human fibrinogen or fibrinogen of a non-human animal. For medical uses, it is preferred to employ proteins native to the patient. The present invention thus provides fibrinogen for use in both human and veterinary medicine. Cloned DNA molecules encoding the component chains of human fibrinogen are disclosed by Rixon et al. (Biochem. 22: 3237, 1983), Chung et al. (Biochem. 22: 3244, 1983), Chung et al. (Biochem. 22: 3250, 1983), Chung et al. (Adv. Exp. Med. Biol. 281: 39-48,1990) and Chung et al. (Ann. NY Acad. Sci. 408: 449-456, 1983). Bovine fibrinogen clones are disclosed by Brown et al. (Nuc. Acids Res. 17: 6397, 1989) and Chung et al. (Proc. Natl. Acad. Sci. USA 78: 466-1470, 1981). Other mammalian fibrinogen clones are disclosed by Murakawa et al. (Thromb. Haemost. 69: 351-360, 1993). Representative sequences of human Aα., and .gamma. chain genes are shown in SEQ ID NOS: 1, 3 and 5, respectively. Those skilled in the art will recognize that allelic variants of these sequences will exist; that additional variants can be generated by amino acid substitution, deletion, or insertion; and that such variants are useful within the present invention. In general, it is preferred that any engineered variants comprise only a limited number of amino acid substitutions, deletions, or insertions, and that any substitutions are conservative. Thus, it is preferred to produce fibrinogen chain polypeptides that are at least 90%, preferably at least 95, and more preferably 99% or more identical in sequence to the corresponding native chains. The term "γ chain" is meant to include the alternatively spliced .gamma.' chain of fibrinogen (Chung et al., Biochem. 23: 4232-4236, 1984). A human γ-chain amino acid sequence is shown in SEQ ID NO: 6. The shorter γ-chain is produced by alternative splicing at nucleotides 9511 and 10054 of SEQ ID NO: 5, resulting in translation terminating after nucleotide 10065 of SEQ ID NO: 5.

[0022] To obtain expression in the mammary gland, a transcription promoter from a milk protein gene is used. Milk protein genes include those genes encoding caseins, β-lactoglobulin (BLG), α-lactalbumin, and whey acidic protein. The β -lactoglobulin promoter is preferred. In the case of the ovine β -lactoglobulin gene, a region of at least the proximal 406 bp of 5' flanking sequence of the ovine BLG gene (contained within nucleotides 3844 to 4257 of SEQ ID NO:7) will generally be used. Larger portions of the 5' flanking sequence, up to about 5 kbp, are preferred. A larger DNA segment encompassing the 5' flanking promoter region and the region encoding the 5' non-coding portion of the β-lactoglobulin gene (contained within nucleotides 1 to 4257 of SEQ ID NO:7) is particularly preferred. See Whitelaw et al., Biochem J. 28: 31-39,1992. Similar fragments of promoter DNA from other species are also suitable.

[0023] Other regions of the β -lactoglobulin gene may also be incorporated in constructs, as may genomic regions of the gene to be expressed. It is generally accepted in the art that constructs lacking introns, for example, express poorly in comparison with those that contain such DNA sequences (see Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836-840, 1988; Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478-482,1991; Whitelaw et al., Transgenic Res. 1: 3-13, 1991; WO 89101343; WO 91/02318). In this regard, it is generally preferred, where possible, to use genomic sequences containing all or some of the native introns of a gene encoding the protein or polypeptide of interest. Within certain embodiments of the invention, the further inclusion of at least some introns from the beta-lactoglobulin gene is preferred. One such region is a DNA segment which provides for intron splicing and RNA polyadenylation from the 3' non-coding region of the ovine beta-lactoglobulin gene. When substituted for the natural 3' non-coding sequences of a gene, this ovine beta-lactoglobulin segment can both enhance and stabilize expression levels of the protein or polypeptide of interest. Within other embodiments, the region surrounding the initiation ATG of one or more of the fibrinogen sequences is replaced with corresponding sequences from a milk specific protein gene. Such replacement provides a putative tissue-specific initiation environment to enhance expression. It is convenient to replace the entire fibrinogen chain pre-pro and 5' non-coding sequences with those of, for example, the BLG gene, although smaller regions may be replaced.

[0024] For expression of fibrinogen, DNA segments encoding each of the three component polypeptide chains of fibrinogen are operably linked to additional DNA segments required for their expression to produce expression units. Such additional segments include the above-mentioned milk protein gene promoter, as well as sequences which provide for termination of transcription and polyadenylation of mRNA. The expression units will further include a DNA segment encoding a secretion signal operably linked to the segment encoding the fibrinogen polypeptide chain. The secretion signal may be a native fibrinogen secretion signal or may be that of another protein, such as a milk protein. The term "secretion signal" is used herein to denote that portion of a protein that directs it through the secretory pathway of a cell to the outside. Secretion signals are most commonly found at the amino-termini of proteins. See, for example, von Heinje, Nuc. Acids Res. 14: 4683-4690, 1986; and Meade et al., U.S. Pat. No. 4,873,316Construction of expression units is conveniently carried out by inserting a fibrinogen chain sequence into a plasmid or phage vector containing the additional DNA segments, although the expression unit may be constructed by essentially any sequence of ligations. It is particularly convenient to provide a vector containing a DNA segment encoding a milk protein and to replace the coding sequence for the milk protein with that of a fibrinogen chain (including a secretion signal), thereby creating a gene fusion that includes the expression control sequences of the milk protein gene. In any event, cloning of the expression units in plasmids or other vectors facilitates the amplification of the fibrinogen sequences. Amplification is conveniently carried out in bacterial (e.g. E. coli) host cells, thus the vectors will typically include an origin of replication and a selectable marker functional in bacterial host cells.

[0025] In the methods of the present invention, the three expression units are linked in a single suitable vector, such as a yeast artificial chromosome or phage P1 clone. Coding sequences for the three chains can be combined in polycistronic expression units (see, e.g., Levinson et al., U.S. Pat. No. 4,713,339).

[0026] The single expression unit is then Introduced into fertilized eggs (including early-stage embryos) of the chosen host species. Introduction of heterologous DNA can be accomplished by one of several routes, including microinjection (e.g. U.S. Pat. No. 4,873,191), retroviral infection (Jaenisch, Science 240: 1468-1474, 1988) or site-directed integration using embryonic stem (ES) cells (reviewed by Bradley et al., Bio/Technology 10: 534-539, 1992). The eggs are then implanted into the oviducts or uteri of pseudopregnant females and allowed to develop to term. Offspring carrying the introduced DNA in their germ line can pass the DNA on to their progeny in the normal, Mendelian fashion, allowing the development of transgenic herds. General procedures for producing transgenic animals are known in the art. See, for example, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al., Bio/Technology 6: 179-183, 1988; Wall et al., Biol. Reprod. 32: 645-651, 1985; Buhler et al., Bio/Technology : 140-143, 1990; Ebert et al., Bio/Technology: 835-838, 1991; Krimpenfort et al., Bio/Technology 9: 844-847, 1991; Wall et al., J. Cell. Biochem. 49: 113-120, 1992; and WIPO publications WO 88/00239, WO 90/05188, WO 92/11757; and GB 87/00458, which are incorporated herein by reference. Techniques for introducing foreign DNA sequences into mammals and their germ cells were originally developed in the mouse. See, e.g., Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380-7384, 1980; Gordon and Ruddle, Science 214: 1244-1246, 1981; Palmiter and Brinster, Cell 41: 343-345, 1985; Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442, 1985; and Hogan et al. (ibid.). These techniques were subsequently adapted for use with larger animals, including livestock species (see e.g., WIPO publications WO 88/00239, WO 90/05188, and WO 92/11757; and Simons et al., Bio/Technology 6: 179-183, 1988). To summarize, in the most efficient route used to date in the generation of transgenic mice or livestock, several hundred linear molecules of the DNA of interest are injected Into one of the pro-nuclei of a fertilized egg. Injection of DNA into the cytoplasm of a zygote can also be employed.

[0027] To obtain a balanced expression of each fibrinogen chain to allow for efficient formation of the mature protein, the three expression units are on the same DNA molecule for introduction into eggs. This approach, however, may generate technical problems at, for example, the injection and manipulation stages. For example, the size of fibrinogen expression units may necessitate the use of yeast artificial chromosomes (YACs) or phage P1 to amplify and manipulate the DNA prior to Injection. If this approach is followed, segments of DNA to be injected, containing all three expression units, would be very large, thus requiring modification of the injection procedure using, for example, larger bore needles. In general, female animals are superovulated by treatment with follicle stimulating hormone, then mated. Fertilized eggs are collected, and the heterologous DNA is injected into the eggs using known methods. See, for example, U.S. Pat. No. 4,873,191; Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380-7384, 1980; Gordon and Ruddle, Science 214: 1244-1246, 1981; Palmiter and Brinster, Cell. 41: 343-345, 1985; Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442, 1985; Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al. Bio/Technology 6: 179-183, 1988; Wall et al., Biol. Reprod. 32: 645-651, 1985; Buhler et al., Bio/Technology 8: 140-143, 1990; Ebert et al., Bio/Technology 9: 835-838, 1991; Krimpenfort et al., Bio/Technology 9: 844-847, 1991; Wall et al., J. Cell. Biochem. 49: 113-120, 1992; WIPO publications WO 88/00239, WO 90/05118, and WO 92/11757; and GB 87/00458.

[0028] For injection into fertilized eggs, the expression units are removed from their respective vectors by digestion with appropriate restriction enzymes. For convenience, it is preferred to design the vectors so that the expression units are removed by cleavage with enzymes that do not cut either within the expression units or elsewhere in the vectors. The expression units are recovered by conventional methods, such as electro-elution followed by phenol extraction and ethanol precipitation, sucrose density gradient centrifugation, or combinations of these approaches.

[0029] DNA is injected into eggs essentially as described In Hogan et al., ibid. In a typical injection, eggs in a dish of an embryo culture medium are located using a stereo zoom microscope (x50 or x63 magnification preferred). Suitable media include Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid) or bicarbonate buffered media such as M2 or M16 (available from Sigma Chemical Co., St. Louis, USA) or synthetic oviduct medium (disclosed below). The eggs are secured and transferred to the center of a glass slide on an injection rig using, for example, a drummond pipette complete with capillary tube. Viewing at lower (e.g. x4) magnification is used at this stage. Using the holding pipette of the injection rig, the eggs are positioned centrally on the slide. Individual eggs are sequentially secured to the holding pipette for injection. For each injection process, the holding pipette/egg is positioned in the center of the viewing field. The injection needle is then positioned directly below the egg. Preferably using x40 Nomarski objectives, both manipulator heights are adjusted to focus both the egg and the needle. The pronuclei are located by rotating the egg and adjusting the holding pipette assembly as necessary. Once the pronucleus has been located, the height of the manipulator is altered to focus the pronuclear membrane. The injection needle is positioned below the egg such that the needle tip is in a position below the center of the pronucleus. The position of the needle is then altered using the injection manipulator assembly to bring the needle and the pronucleus into the same focal plane. The needle is moved, via the joy stick on the injection manipulator assembly, to a position to the right of the egg. With a short, continuous jabbing movement, the pronuclear membrane is pierced to leave the needle tip inside the pronucleus. Pressure is applied to the injection needle via the glass syringe until the pronucleus swells to approximately twice its volume. At this point, the needle is slowly removed. Reverting to lower (e.g. x4) magnification, the injected egg is moved to a different area of the slide, and the process is repeated with another egg.

[0030] After the DNA is injected, the eggs may be cultured to allow the pronuclei to fuse, producing one-cell or later stage embryos. In general, the eggs are cultured at approximately the body temperature of the species used in a buffered medium containing balanced salts and serum. Surviving embryos are then transferred to pseudopregnant recipient females, typically by inserting them into the oviduct or uterus, and allowed to develop to term. During embryogenesis, the injected DNA integrates in a random fashion in the genomes of a small number of the developing embryos.

[0031] Potential transgenic offspring are screened via blood samples and/or tissue biopsies. DNA is prepared from these samples and examined for the presence of the injected construct by techniques such as polymerase chain reaction (PCR; see Mullis, U.S. Pat. No. 4,683,202) and Southern blotting (Southern, J. Mol. Biol. 98:503, 1975; Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982). Founder transgenic animals, or G0s, may be wholly transgenic, having transgenes in all of their cells, or mosaic, having transgenes in only a subset of cells (see, for example, Wilkie et al., Develop. Biol. 118: 9-18, 1986). In the latter case, groups of germ cells may be wholly or partially transgenic. In the latter case, the number of transgenic progeny from a founder animal will be less than the expected 50% predicted from Mendelian principles. Founder G0 animals are grown to sexual maturity and mated to obtain offspring, or G1s. The G1s are also examined for the presence of the transgene to demonstrate transmission from founder G0 animals. In the case of male G0s, these may be mated with several non-transgenic females to generate many offspring. This increases the chances of observing transgene transmission. Female G0 founders may be mated naturally, artificially inseminated or superovulated to obtain many eggs which are transferred to surrogate mothers. The latter course gives the best chance of observing transmission in animals having a limited number of young. The above-described breeding procedures are used to obtain animals that can pass the DNA on to subsequent generations of offspring in the normal, Mendelian fashion, allowing the development of, for example, colonies (mice), flocks (sheep), or herds (pigs, goats and cattle) of transgenic animals.

[0032] The milk from lactating G0 and G1 females is examined for the expression of the heterologous protein using Immunological techniques such as ELISA (see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and Western blotting (Towbin et al., Proc. Natl. Acad. Sci. USA 76: 4350-4354, 1979). For a variety of reasons known in the art, expression levels of the heterologous protein will be expected to differ between individuals.

[0033] A satisfactory family of animals should satisfy three criteria: they should be derived from the same founder G0 animal; they should exhibit stable transmission of the transgene; and they should exhibit stable expression levels from generation to generation and from lactation to lactation of individual animals. These principles have been demonstrated and discussed (Carver et al., Bio/Technology 11: 1263-1270, 1993). Animals from such a suitable family are referred to as a "line." Initially, male animals, G0 or G1, are used to derive a flock or herd of producer animals by natural or artificial insemination. In this way, many female animals containing the same transgene integration event can be quickly generated from which a supply of milk can be obtained.

[0034] The fibrinogen is recovered from milk using standard practices such as skimming, precipitation, filtration and protein chromatography techniques.

[0035] Fibrinogen produced according to the present invention is useful within human and veterinary medicine, such as in the formulation of surgical adhesives. Adhesives of this type are known in the art. See, for example, U.S. Pat. Nos. 4,377,572; 4,442,655; 4,462,567; and 4,627,879. In general, fibrinogen and factor XIII are combined to form a first component that is mixed just prior to use with a second component containing thrombin. The thrombin converts the fibrinogen to fibrin, causing the mixture to gel, and activates the factor XIII. The activated factor XIII cross links the fibrin to strengthen and stabilize the adhesive matrix. Such adhesives typically contain from about 30 mg/ml to about 100 mg/ml fibrinogen and from about 50 .mu.g/ml to about 500 .mu.g/ml factor XIII. They may also contain additional ingredients, such as aprotinin, albumin, fibronectin, bulking agents, and solubilizers. Methods for producing factor XIII are known in the art. See, for example, U.S. Pat. No. 5,204,447. The fibrinogen is also useful for coating surfaces of polymeric articles, e.g. synthetic vascular grafts, as disclosed in U.S. Pat. No. 5,272,074.

[0036] The invention is further Illustrated by the following non-limiting examples. These examples are only part of the present invention insofar as they fall within the scope of the claims. Those examples or parts of examples that fall outside of the scope of the claims are merely provided by way of background information.

Examples

Example I

[0037] The multiple cloning site of the vector pUC18 (Yanisch-Perron et al., Gene 33:103-119, 1985) was removed and replaced with a synthetic double stranded oligonucleotide (the strands of which are shown in SEQ ID NO: 8 and SEQ ID NO: 27) containing the restriction sites Pvu I/Mlu I/Eco RV/Xba I/Pvu I/Mlu I, and flanked by 5' overhangs compatible with the restriction sites Eco RI and Hind III. pUC18 was cleaved with both Eco RI and Hind III, the 5' terminal phosphate groups were removed with calf intestinal phosphatase, and the oligonucleotide was ligated into the vector backbone. The DNA sequence across the junction was confirmed by sequencing, and the new plasmid was called pUCPM.

[0038] The β-lactoglobulin (BLG) gene sequences from pSS1tgXS (disclosed in WIPO publication WO 88/00239) were excised as a Sal I-Xba I fragment and recloned into the vector pUCPM that had been cut with Sal I and Xba I to construct vector pUCXS. pUCXS is thus a pUC18 derivative containing the entire BLG gene from the Sal I site to the Xba I site of phage SS1 (Ali and Clark, J. Mol. Biol. 199: 415-426, 1988).

[0039] The plasmid pSS1tgSE (disclosed in WIPO publication WO 88/00239) contains a 1290 bp BLG fragment flanked by Sph I and EcoR I restriction sites, a region spanning a unique Not I site and a single Pvu II site which lies In the 5' untranslated leader of the BLG mRNA. Into this Pvu II site was ligated a double stranded, 8 bp DNA linker (5'-GGATATCC-3') encoding the recognition site for the enzyme Eco RV. This plasmid was called pSS1tgSE/RV. DNA sequences bounded by Sph I and Not I restriction sites In pSS1tgSE/RV were excised by enzymatic digestion and used to replace the equivalent fragment in pUCXS. The resulting plasmid was called pUCXSRV. The sequence of the BLG insert in pUCSXRV is shown in SEQ ID NO: 7, with the unique Eco RV site at nucleotide 4245 in the 5' untranslated leader region of the BLG gene. This site allows insertion of any additional DNA sequences under the control of the BLG promoter 3' to the transcription initiation site.

[0040] Using the primers BLGAMP3 (5'-TGG ATC CCC TGC CGG TGC CTC TGG-3'; SEQ ID NO: 9) and BLGAMP4 (5'-AAC GCG TCA TCC TCT GTG AGC CAG-3'; SEQ ID NO: 10) a PCR fragment of approximately 650 bp was produced from sequences immediately 3' to the stop codon of the BLG gene in pUCXSRV. The PCR fragment was engineered to have a BamH I site at its 5' end and an Mlu I site at its 3' end and was cloned as such into BamH I and Mlu I cut pGEM7zf(+) (Promega) to give pDAM200(+).

[0041] pUCXSRV was digested with Kpn I, and the largest, vector containing band was gel purified. This band contained the entire pUC plasmid sequences and some 3' non-coding sequences from the BLG gene. Into this backbone was ligated the small Kpn I fragment from pDAM200(+) which, in the correct orientation, effectively engineered a BamH I site at the extreme 5' end of the 2.6 Kbp of the BLG 3' flanking region. This plasmid was called pBLAC200. A 2.6 Kbp Cla l-Xba I fragment from pBLAC200 was ligated into Cla l-Xba I cut pSP72 vector (Promega), thus placing an EcoR V site immediately upstream of the BLG sequences. This plasmid was called pBLAC2-10.

[0042] The 2.6 Kbp Eco RV-Xba I fragment from pBLAC210 was ligated into Eco RV-Xba I cut pUCXSRV to form pMAD6. This, in effect, excised all coding and intron sequences from pUCXSRV, forming a BLG minigene consisting of 4.3 Kbp of 5' promoter and 2.6 Kbp of 3' downstream sequences flanking a unique EcoR V site. An oligonucleotide linker (ZC6839: ACTACGTAGT; SEQ ID NO: 11) was inserted into the Eco RV site of pMAD6. This modification destroyed the Eco RV site and created a Sna Bl site to be used for cloning purposes. The vector was designated pMAD6-Sna. Messenger RNA initiates upstream of the Sna Bl site and terminates downstream of the Sna BI site. The precursor transcript will encode a single BLG-derived intron, intron 6, which is entirely within the 3' untranslated region of the gene.

Example II

[0043] Clones encoding the individual fibrinogen chains were obtained from the laboratory of Dr. Earl W. Davle, University of Washington, Seattle. A genomic fibrinogen Aα.-chain clone (Chung et al., 1990, ibid.) was obtained from the plasmid BS4. This plasmid contains the Aα. clone inserted into the Sal I and Bam HI sites of the vector pUC18, but lacks the coding sequence for the first four amino acids of the Aα chain. A genomic Bβ.-chain DNA (Chung et al., ibid.) was isolated from a lambda Charon 4A phage clone (designated β.lambda.4) as two EcoRI fragments of ca. 5.6 Kbp each. The two fragments were cloned separately into pUC19 that had been digested with Eco RI and treated with calf intestinal phosphatase. The resulting clones were screened by digestion with the restriction enzyme Pvu II to distinguish plasmids with the 5' and 3' Bβ inserts (designated Beta5'RI/puc and Beta3'RI/puc, respectively). Genomic .gamma.-chain clones were isolated as described by Rixon et al. (Biochemistry 24: 2077-2086, 1985). Clone p.gamma.12A9 comprises 5' non-coding sequences and approximately 4535 bp of γ-chain coding sequence. Clone p.gamma.12F3 comprises the remaining coding sequence and 3' non-coding nucleotides. Both are pBR322-based plasmids with the fibrinogen sequences inserted at the EcoRI site. These plasmids were used as templates for the respective PCR reactions.

[0044] The fibrinogen chain coding sequences were tailored for Insertion into expression vectors using the polymerase chain reaction (PCR) as generally described by Mullis (U.S. Pat. No. 4,683,202). This procedure removed native 5' and 3' untranslated sequences, added a 9 base sequence (CCT GCA GCC) upstream of the first ATG of each coding sequence, supplied the first four codons for the Aα-chain sequence, removed an internal Mlu l site in the Aα sequence and added restriction sites to facilitate subsequent cloning steps.

[0045] Referring to FIG. 1, the 5' end of the Aα coding sequence was tailored in a PCR reaction containing 20 pmole for each of primers ZC6632 (SEQ ID NO: 12) and ZC6627 (SEQ ID NO: 13), approximately 10 ng of plasmid BS4 template DNA, 10 .mu.l of a mix containing 2.5 mM each dNTP, 7.5 .mu.l 10.times. Pyrococcus furiosus (Pfu) DNA polymerase buffer #1 (200 mM Tris-HCl, pH 8.2, 100 mM KCI, 60 mM (NH4)2 SO4, 20 mM MgCl2, 1% Triton X-100, 100 .mu.g/ml nuclease free bovine serum albumin)(Stratagene, La Jolla, Calif.), and water to 75 .mu.l. The mixture was heated to 94 DEG C. in a DNA thermal cycler (Perkin-Elmer Corp., Norwalk, Conn.). To the heated mixture was added 25 .mu.l of a mixture containing 2.5 .mu.l 10.times. Pfu buffer #1, 22 .mu.l H2 O and 1 .mu.l 2.5 units/.mu.l Pfu DNA polymerase (Stratagene). The reactions were run in a DNA thermal cycler (Perkin-Elmer) for five cycles of 94 DEG , 45 seconds; 40 DEG , 90 seconds; 72 DEG , 120 seconds; 20 cycles of 94 DEG , 45 seconds; 45 DEG , 90 seconds; 72 DEG , 120 seconds; then incubated at 72 DEG for 7 minutes. The 5' PCR-generated fragment was digested with Bam HI and Hind III, and the Bam HI-Hind III fragment was then ligated to an internal 2.91 Kbp Hind III-Xba I fragment and Bam HI, Xba I-digested pUC18. PCR-generated exon sequences were sequenced.

[0046] Referring again to FIG. 1, the 3' end of the Aα coding sequence was tailored in a series of steps in which the Mlu I site 563 bases upstream from the stop codon of the Aα sequence was mutated using an overlap extension PCR reaction (Ho et al., Gene 77: 51-59, 1989). In the first reaction 40 pmole of each of primers ZC6521 (SEQ ID NO: 14) and ZC6520 (SEQ ID NO: 15) were combined with approximately 10 ng of plasmid BS4 template DNA In a reaction mixture as described above. The reaction was run for 5 cycles of 94 DEG , 45 seconds; 40 DEG , 60 seconds; 72 DEG , 120 seconds; 15 cycles of 94 DEG , 45 seconds; 45 DEG , 60 seconds; 72 DEG , 120 seconds; then incubated at 72 DEG for 7 minutes. A second reaction was carried out in the same manner using 40 pmole of each of primers ZC6519 (SEQ ID NO: 16) and ZC6518 (SEQ ID NO: 17) and BS4 as template. The PCR-generated DNA fragments from the first and second reactions were isolated by gel electrophoresis and elution from the gel. Approximately 1/10 of each recovered reaction product was combined with 40 pmole of each of primers ZC6521 (SEQ ID NO: 14) and ZC6518 (SEQ ID NO: 17) in a PCR reaction in which the complementary 3' ends of each fragment (containing the single base change) annealed and served as a primer for the 3' extension of the complementary strand. PCR was carried out using the same reaction conditions as in the first and second 3' PCR steps. The reaction product was then digested with Xba l and Bam Hl, and the Xba l-Bam Hl fragment was cloned into Xba l, Bam Hl-digested pUC18. PCR-generated exons were sequenced.

[0047] As shown in FIG. 1, the 5' Bam HI-Xba I fragment (3.9 Kbp) and the 3' Xba I-Bam HI fragment (1.3 Kbp) were inserted into the Bam HI site of the vector Zem228. Zem228 is a pUC18 derivative comprising a Bam HI cloning site between a mouse MT-1 promoter and SV40 terminator, and a neomycin resistance marker flanked by SV40 promoter and terminator sequences. See European Patent Office Publication EP 319,944 and FIG. 2. The entire A.alpha. coding sequence was isolated from the Zem228 vector as an Sna BI fragment, which was inserted into the Sna BI site of the plasmid pMAD6-Sna.

[0048] Referring to FIG. 3, the 5' end of the Bβ -chain was tailored by PCR using the oligonucleotides ZC6629 (SEQ ID NO: 18), ZC6630 (SEQ ID NO: 19) and ZC6625 (SEQ ID NO: 20). These primers were used in pairwise combinations (ZC6629+ZC6625 or ZC6630+ZC6625) to generate Bβ coding sequences beginning at the first ATG codon (position 470 in SEQ ID NO: 3)(designated N1-Beta) or the third ATG codon (position 512 in SEQ ID NO: 3)(designated N3-Beta). Approximately 5 ng of Beta5'RI/puc template DNA was combined with 20 pmole of each of the primers (N1-Beta:ZC6629, SEQ ID NO: 18+ZC6625, SEQ ID NO: 20; or N3-Beta:ZC6630, SEQ ID NO: 19+ZC6625, SEQ ID NO: 20) in a reaction mixture as described above. The mixtures were incubated for 5 cycles of 94 DEG , 45 seconds; 40 DEG , 120 seconds; (N1-Beta) or 90 seconds (N3-Beta); 72 DEG , 120 seconds; 20 cycles of 94 DEG , 45 seconds; 45 DEG , 120 seconds; (N1-Beta) or 90 seconds (N3-Beta); 72 DEG , 120 seconds; then incubated at 72 DEG for 7 minutes. The two reaction products N1, 555 bp or N3, 510 bp) were each digested with Eco RI and Bgl ll, and the fragments were ligated to the internal Bgl ll-Xba I fragment and Eco RI+Xba I-digested pUC19. The 3' end of the Bβ sequence was tailored in a reaction mixture as described above using the oligonucleotide primers ZC6626 (SEQ ID NO: 21) and ZC6624 (SEQ ID NO: 22) and approximately 5 ng of Beta3'RI/puc template. The mixtures were incubated for 5 cycles of 94 DEG , 45 seconds; 40 DEG , 90 seconds; 72 DEG , 120 seconds; 15 cycles of 94 DEG , 45 seconds; 45 DEG , 90 seconds; 72 DEG , 120 seconds; then incubated at 72 DEG for 7 minutes. A 990 bp Bgl ll-Eco RI fragment was isolated. This 3' fragment was ligated to the adjacent coding fragment (340 bp, Sphl-Bgl II) and Sph I+Eco RI-digested pUC19. The 3' and 5' PCR-generated exons were sequenced. A third intermediate vector was constructed by combining two internal fragments (4285 bp Xba I-Eco RI and 383 kb Eco RI-Sph I) In Xba I+Sph I-digested pUC19. The entire Bβ coding sequence (two forms) was then assembled by ligating one of the 5' Eco RI-Xba I fragments, the internal Xba I-Sph I fragment, the 3' Sph I-Eco RI fragment and Eco RI-digested vector pUC19. The Bβ sequence was then isolated as a 7.6 Kbp Sna BI fragment and inserted into the Sna BI site of pMAD6-Sna.

[0049] Referring to FIG. 4, the 5' end of the gamma chain sequence was tailored by PCR using the oligonucleotide primers ZC6514 (SEQ ID NO: 23) and ZC6517 (SEQ ID NO: 24) and approximately 50 ng of p.gamma.12A9 as template. The PCR reaction was run as described above using 40 pM of each primer. The reaction was run for 5 cycles of 94 DEG , 45 seconds; 40 DEG , 60 seconds, 72 DEG , 120 seconds, followed by 15 cycles of 94 DEG , 45 seconds; 45 DEG , 60 seconds; 72 DEG , 120 seconds. The resulting 213 bp fragment was digested with Bam HI and Spe I, and the resulting restriction fragment was ligated with the adjacent downstream 4.4 kb Spe I-Eco RI fragment and Bam HI+Eco RI digested pUC19. The 3' end of the gamma chain sequence was tailored using oligonucleotide primers ZC6516 (SEQ ID NO: 25) and ZC6515 (SEQ ID NO: 26) using 40 pM of each primer, approximately 50 ng of p.gamma.12F3 template and the same thermal cycling schedule as used for the 5' fragment. The resulting 500 bp fragment was digested with Spe I and Bam HI, and the resulting restriction fragment was ligated with the upstream 2.77 kb Eco RI-Spe I fragment and Eco RI+Bam HI-digested pUC19. All PCR-generated exons were sequenced. The entire γ'-chain coding sequence was then assembled by ligating a 4.5 Kbp Bam HI-Eco RI 5' fragment, a 1.1 Kbp Eco RI-Pst I internal fragment and a 2.14 Kbp Pst I-Xba I 3' fragment in Bam HI+Xba I-digested Zem219b. Zem219b is a pUC18-derived vector containing a mouse metallothionein promoter and a DHFR selectable marker operably linked to an SV40 promoter (FIG. 5). Plasmid Zem219b has been deposited with American Type Culture Collection as an E. coli XL1-blue transformant under Accession No. 68979. The entire γ'-chain coding sequence was then isolated as a 7.8 Kbp Sna B1 fragment and inserted into the Sna BI site of pMAD6-Sna.

Example III

[0050] Mice for initial breeding stocks (C57BL6J, CBACA) were obtained from Harlan Olac Ltd. (Bicester, UK). These were mated in pairs to produce F1 hybrid cross (B6CBAF1) for recipient female, superovulated females, stud males and vasectomized males. All animals were kept on a 14 hour light/10 hour dark cycle and fed water and food (Special Diet Services RM3, Edinburgh, Scotland) ad libitum.

[0051] Transgenic mice were generated essentially as described in Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986, which is incorporated herein by reference in its entirety. Female B6CBAF1 animals were superovulated at 4-5 weeks of age by an i.p. injection of pregnant mares' serum gonadotrophin (FOLLIGON, Vet-Drug, Falkirk, Scotland) (5 iu) followed by an i.p. injection of human chorionic gonadotrophin (CHORULON, Vet-Drug, Falkirk, Scotland) (5 iu) 45 hours later. They were then mated with a stud male overnight. Such females were next examined for copulation plugs. Those that had mated were sacrificed, and their eggs were collected for microinjection.

[0052] DNA was injected into the fertilized eggs as described in Hogan et al. (ibid.) Briefly, each of the vectors containing the A.alpha., B.beta. and .gamma. expression units was digested with Mlu I, and the expression units were isolated by sucrose gradient centrifugation. All chemicals used were reagent grade (Sigma Chemical Co., St. Louis, Mo., U.S.A.), and all solutions were sterile and nuclease-free. Solutions of 20% and 40% sucrose in 1 M NaCl , 20 mM Tris pH 8.0, 5 mM EDTA were prepared using UHP water and filter sterilized. A 30% sucrose solution was prepared by mixing equal volumes of the 20% and 40% solutions. A gradient was prepared by layering 0.5 ml steps of the 40%, 30% and 20% sucrose solutions Into a 2 ml polyallomer tube and allowed to stand for one hour. 100 .mu.l of DNA solution (max. 8 .mu.g DNA) was loaded onto the top of the gradient, and the gradient was centrifuged for 17-20 hours at 26,000 rpm, 15 DEG C. in a Beckman TL100 ultracentrifuge using a TLS-55 rotor (Beckman instruments, Fullerton, Calif., USA). Gradients were fractionated by puncturing the tube bottom with a 20 ga. needle and collecting drops in a 96 well microtiter plate. 3 .mu.l aliquots were analyzed on a 1% agarose mini-gel. Fractions containing the desired DNA fragment were pooled and ethanol precipitated overnight at -20 DEG C. in 0.3M sodium acetate. DNA pellets were resuspended In 50-100 .mu.l UHP water and quantitated by fluorimetry. The expression units were diluted in Dulbecco's phosphate buffered saline without calcium and magnesium (containing, per liter, 0.2 g KCl, 0.2 g KH2 PO4, 8.0 g NaCl, 1.15 g Na2 HPO4), mixed (using either the N1-Beta or N3-Beta expression unit) in a 1:1:1 molar ratio, concentration adjusted to about 6 .mu.g/ml, and injected into the eggs (.about.2 pi total DNA solution per egg).

[0053] Recipient females of 6-8 weeks of age are prepared by mating B6CBAF1 females In natural estrus with vasectomized males. Females possessing copulation plugs are then kept for transfer of microinjected eggs.

[0054] Following birth of potential transgenic animals, tail biopsies are taken, under anesthesia, at four weeks of age. Tissue samples are placed in 2 ml of tail buffer (0.3M Na acetate, 50 mM HCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 8.5, 0.5% NP40, 0.5% Tween 20) containing 200 .mu.g/ml proteinase K (Boehringer Mannheim, Mannheim, Germany) and vortexed. The samples are shaken (250 rpm) at 55 DEG -60 DEG for 3 hours to overnight. DNA prepared from biopsy samples Is examined for the presence of the injected constructs by PCR and Southern blotting. The digested tissue is vigorously vortexed, and 5 .mu.l aliquots are placed in 0.5 ml microcentrifuge tubes. Positive and negative tail samples are included as controls. Forty .mu.l of silicone oil (BDH, Poole, UK) is added to each tube, and the tubes are briefly centrifuged. The tubes are incubated in the heating block of a thermal cycler (e.g. Omni-gene, Hybaid, Teddington, UK) to 95 DEG C. for 10 minutes. Following this, each tube has a 45 .mu.l aliquot of PCR mix added such that the final composition of each reaction mix is: 50 mM KCl; 2 mM MgCl2 ; 10 mM Tris-HCl (pH 8.3); 0.01 % gelatin; 0.1% NP40, 10% DMSO; 500 nM each primer, 200 .mu.M dNTPs; 0.02 U/.mu.L Taq polymerase (Boehringer Mannheim, Mannheim, Germany). The tubes are then cycled through 30 repeated temperature changes as required by the particular primers used. The primers may be varied but in all cases must target the BLG promoter region. This is specific for the injected DNA fragments because the mouse does not have a BLG gene. Twelve .mu.l of 5.times. loading buffer containing Orange G marker dye (0.25% Orange G [Sigma] 15% Ficoll type 400 [Pharmacia Biosystems Ltd., Milton Keynes, UK]) is then added to each tube, and the reaction mixtures are electrophoresed on a 1.6% agarose gel containing ethidium bromide (Sigma) until the marker dye has migrated 2/3 of the length of the gel. The gel is visualized with a UV light source emitting a wavelength of 254 nm. Transgenic mice having one or more of the injected DNA fragments are identified by this approach.

[0055] Positive tail samples are processed to obtain pure DNA. The DNA samples are screened by Southern blotting using a BLG promoter probe (nucleotides 2523-4253 of SEQ ID NO: 7). Specific cleavages with appropriate restriction enzymes (e.g. Eco RI) allow the distinction of the three constructs containing the Aα, Bβ and γ sequences.

[0056] Southern blot analysis of transgenic mice prepared essentially as described above demonstrated that more than 50% of progeny contained all three fibrinogen sequences. Examination of milk from positive animals by reducing SDS polyacrylamide gel electrophoresis demonstrated the presence of all three protein chains at concentrations up to 1 mg/ml. The amount of fully assembled fibrinogen was related to the ratios of individual subunits present in the milk. No apparent phenotype was associated with high concentrations of human fibrinogen in mouse milk.

Example IV

[0057] Donor ewes are treated with an intravaginal progesterone-impregnated sponge (CHRONOGEST Goat Sponge, Intervet, Cambridge, UK) on day 0. Sponges are left in situ for ten or twelve days.

[0058] Superovulation is induced by treatment of donor ewes with a total of one unit of ovine follicle stimulating hormone (OFSH) (OVAGEN, Horizon Animal Reproduction Technology Pty. Ltd., New Zealand) administered in eight intramuscular injections of 0.125 units per injection starting at 5:00 pm on day -4 and ending at 8:00 am on day 0. Donors are injected intramuscularly with 0.5 ml of a luteolytic agent (ESTRUMATE, Vet-Drug) on day -4 to cause regression of the corpus luteum, to allow return to estrus and ovulation. To synchronize ovulation, the donor animals are injected intramuscularly with 2 ml of a synthetic releasing hormone analog (RECEPTAL, Vet-Drug) at 5:00 pm on day 0.

[0059] Donors are starved of food and water for at least 12 hours before artificial insemination (A.I.). The animals are artificially inseminated by intrauterine laparoscopy under sedation and local anesthesia on day 1. Either xylazine (ROMPUN, Vet-Drug) at a dose rate of 0.05-0.1 ml per 10 kg bodyweight or ACP injection 10 mg/ml (Vet-Drug) at a dose rate of 0.1 ml per 10 kg bodyweight is injected intramuscularly approximately fifteen minutes before A.I. to provide sedation. A.I. is carried out using freshly collected semen from a Poll Dorset ram. Semen is diluted with equal parts of filtered phosphate buffered saline, and 0.2 ml of the diluted semen is injected per uterine horn. Immediately pre- or post-A.I., donors are given an intramuscular injection of AMOXYPEN (Vet-Drug).

[0060] Fertilized eggs are recovered on day 2 following starvation of donors of food and water from 5:00 pm on day 1. Recovery is carried out under general anesthesia induced by an intravenous injection of 5% thiopentone sodium (INTRAVAL SODIUM, Vet-Drug) at a dose rate of 3 ml per 10 kg bodyweight. Anesthesia is maintained by inhalation of 1-2% Halothane/O2 /N2 O after intubation. To recover the fertilized eggs, a laparotomy incision is made, and the uterus is exteriorized. The eggs are recovered by retrograde flushing of the oviducts with Ovum Culture Medium (Advanced Protein Products, Brierly Hill, West Midlands, UK) supplemented with bovine serum albumin of New Zealand origin. After flushing, the uterus is returned to the abdomen, and the incision is closed. Donors are allowed to recover post-operatlvely or are euthanized. Donors that are allowed to recover are given an intramuscular injection of Amoxypen L.A. at the manufacturer's recommended dose rate immediately pre- or post-operatively.

[0061] Plasmids containing the three fibrinogen chain expression units are digested with Mlu I, and the expression unit fragments are recovered and purified on sucrose density gradients. The fragment concentrations are determined by fluorimetry and diluted in Dulbecco's phosphate buffered saline without calcium and magnesium as described above. The concentration is adjusted to 6 .mu.g/ml and approximately 2 pl of the mixture is microinjected into one pronucleus of each fertilized eggs with visible pronuclei.

[0062] All fertilized eggs surviving pronuclear microinjection are cultured in vitro at 38.5 DEG C. in an atmosphere of 5% CO2 :5% 02 :90% N2 and about .about.100% humidity in a bicarbonate buffered synthetic oviduct medium (see Table) supplemented with 20% v/v vasectomized ram serum. The serum may be heat inactivated at 58 DEG C. for 30 minutes and stored frozen at -20 DEG C. prior to use. The fertilized eggs are cultured for a suitable period of time to allow early embryo mortality (caused by the manipulation techniques) to occur. These dead or arrested embryos are discarded. Embryos having developed to 5 or 6 cell divisions are transferred to synchronized recipient ewes.

Table

Synthetic Oviduct Medium
Stock A (Lasts 3 Months)
NaCl	6.28 g
KCl	0.534 g
KH2PO4	0.162 g
MgSO4.7H2O	0.182 g
Penicillin	0.06g
Sodium Lactate 60% syrup	0.6 mls
Super H2O	99.4 mls

Stock B (Lasts 2 weeks)
NaHCO3	0.21 g
Phenol red	0.001 g
Super H2O	10 mls

Stock C (Lasts 2 weeks)
Sodium Pyruvate	0.051g
Super H2O	10 mls

Stock D (Lasts 3 months)
CaCl2.2H2O	0.262 g
Super H2O	10 mls

Stock E (Lasts 3 months)
Hopes	0.651 g
Phenol red	0.001 g
Super H2O	10 mls
To make up 10mls of Bicarbonate Buffered Medium
STOCK A	1 ml
STOCK B	1 ml
STOCK C	0.07 ml
STOCK D	0.1 ml
Super H2O	7.83 ml
Osmolarity should be 265 - 285 mOsm.
Add 2.5 ml of heat inactivated sheep serum and filter sterilize.
To make up 10 mls of HEPES Buffered Medium
STOCK A	1 ml
STOCK B	0.2 ml
STOCK C	0.07 ml
STOCK D	0.1 ml
STOCK E	0.8 ml
Super H20	7.83 ml
Osmolarity should be 265 - 285 mOsm
Add 2.5 ml of heat inactivated sheep serum and filter sterilize.

[0063] Recipient ewes are treated with an intravaginal progesterone-Impregnated sponge (Chronogest Ewe Sponge or Chronogest Ewe-Lamb Sponge, intervet) left in situ for 10 or 12 days. The ewes are injected intramuscularly with 1.5 ml (300 lu) of a follicle stimulating hormone substitute (P.M.S.G., Intervet) and with 0.5 ml of a luteolytic agent (Estrumate, Coopers Pitman-Moore) at sponge removal on day -1. The ewes are tested for estrus with a vasectomized ram between 8:00 am and 5:00 pm on days 0 and 1.

[0064] Embryos surviving in vitro culture are returned to recipients (starved from 5:00 pm on day 5 or 6) on day 6 or 7. Embryo transfer is carried out under general anesthesia as described above. The uterus is exteriorized via a laparotomy incision with or without laparoscopy. Embryos are returned to one or both uterine horns only in ewes with at least one suitable corpora lutea. After replacement of the uterus, the abdomen is closed, and the recipients are allowed to recover. The animals are given an intramuscular injection of Amoxypen L.A. at the manufacturer's recommended dose rate immediately pre- or post-operatively.

[0065] Lambs are identified by ear tags and left with their dams for rearing. Ewes and lambs are either housed and fed complete diet concentrates and other supplements and or ad lib. hay, or are let out to grass.

[0066] Within the first week of life (or as soon thereafter as possible without prejudicing heath), each lamb is tested for the presence of the heterologous DNA by two sampling procedures. A 10 ml blood sample is taken from the jugular vein into an EDTA vacutainer. If fit enough, the lambs also have a second 10 ml blood sample taken within one week of the first. Tissue samples are taken by tail biopsy as soon as possible after the tail has become desensitized after the application of a rubber elastrator ring to its proximal third (usually within 200 minutes after "talling"). The tissue is placed immediately in a solution of tail buffer. Tail samples are kept at room temperature and analyzed on the day of collection. All lambs are given an intramuscular injection of Amoxypen L.A. at the manufacturer's recommended dose rate immediately post-biopsy, and the cut end of the tail is sprayed with an antibiotic spray.

[0067] DNA is extracted from sheep blood by first separating white blood cells. A 10 ml sample of blood is diluted in 20 ml of Hank's buffered saline (HBS; obtained from Sigma Chemical Co.). Ten ml of the diluted blood is layered over 5 ml of Histopaque (Sigma) in each of two 15 ml screw-capped tubes. The tubes are centrifuged at 3000 rpm (2000.times. g max), low brake for 15 minutes at room temperature. White cell interfaces are removed to a clean 15 ml tube and diluted to 15 ml in HBS. The diluted cells are spun at 3000 rpm for 10 minutes at room temperature, and the cell pellet is recovered and resuspended in 2-5 ml of tail buffer.

[0068] To extract DNA from the white cells, 10% SDS is added to the resuspended cells to a final concentration of 1%, and the tube is inverted to mix the solution. One mg of fresh proteinase K solution is added, and the mixture is incubated overnight at 45 DEG C. DNA is extracted using an equal volume of phenol/chloroform (.times.3) and chloroform/isoamyl alcohol (.times.1). The DNA is then precipitated by adding 0.1 volume of 3M NaOAc and 2 volumes of ethanol, and the tube is inverted to mix. The precipitated DNA is spooled out using a clean glass rod with a sealed end. The spool is washed in 70% ethanol, and the DNA is allowed to partially dry, then is redissolved in TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.4).

[0069] DNA samples from blood and tail are analyzed by Southern blotting using probes for the BLG promoter region and the fibrinogen chain coding regions.

[0070] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made. Accordingly, the invention is not limited except as by the appended claims.

SEQUENCE LISTING

[0071] 

(1) GENERAL INFORMATION:

(i) APPLICANT: ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle, Washington 98102
United States of America
Pharmaceutical Proteins Ltd.
Roslin
Edinburgh
Midlothian, Scotland EH25 9PP

(ii) TITLE OF INVENTION: Production of Fibrinogen in Transgenic Animals

(iii) NUMBER OF SEQUENCES: 27

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: ZymoGenetics, Inc.

(B) STREET: 1201 Eastlake Avenue East

(C) CITY: Seattle

(D) STATE: WA

(E) COUNTRY: USA

(F) ZIP: 98102

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Parker, Gary E

(B) REGISTRATION NUMBER: 31-648

(C) REFERENCE/DOCKET NUMBER: 93-15PC

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 206-442-6673

(B) TELEFAX: 206-442-6678

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5943 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Human Fibrinogen A-alpha chain

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: join(31..84, 1154..1279, 1739..1922, 3055..3200, 3786..5210)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 644 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8878 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: human fibrinogen B-beta chain

(ix) FEATURE:

(A) NAME/KEY: misc_RNA

(B) LOCATION: 1..469

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 470..583

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 584..3257

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 3258..3449

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 3450..3938

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 3939..4122

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 4123..5042

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 5043..5270

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 5271..5830

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 5831..5944

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 5945..6632

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 6633..6758

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 6759..6966

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 6967..7252

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 7253..7870

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 7871..8102

(ix) FEATURE:

(A) NAME/KEY: 3'UTR

(B) LOCATION: 8103..8537

(ix) FEATURE:

(A) NAME/KEY: misc_RNA

(B) LOCATION: 8538..8878

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: join(470..583, 3258..3449, 3939..4122, 5043..5270, 5831..5944, 6633..6758, 6967..7252, 7871..8102)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 491 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10564 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: human fibrinogen gamma chain

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: join(1799..1876, 1973..2017, 2207..2390, 2510 ..2603, 4211..4341, 4645..4778, 5758..5942, 7426 ..7703, 9342..9571)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 453 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10807 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ovine beta-lactoglobulin

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
AATTCCGATC GACGCGTCGA CGATATACTC TAGACGATCG ACGCGTA    47

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: BLGAMP3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
TGGATCCCCT GCCGGTGCCT CTGG   24

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: BLGAMP4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
AACGCGTCAT CCTCTGTGAG CCAG    24

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6839

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
ACTACGTAGT    10

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6632

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CGACGCGGAT CCTACGTACC TGCAGCCATG TTTTCCATGA GG    42

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6627

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
AGGGCTTCGG CAAGCTTCAG G    21

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6521

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GCCAAAGACT TACTTCCCTC TAGA    24

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6520

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GCATGAACGT CGCGTGGTGG TTGTGCTACC    30

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6519

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
ACCACGCGAC GTTCATGCTC TAAAACCGTT    30

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6518

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GCTGCGGGAT CCTACGTACT AGGGGGACAG GGAAGG    36

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6629

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
CGACGCGAAT TCTACGTACC TGCAGCCATG AAAAGGATGG TTTCT    45

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6630

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
CGACGCGAAT TCTACGTACC TGCAGCCATG AAACATCTAT TATTG    45

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6625

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GTGAGATTTT CAGATCTTGT C    21

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6626

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
AAGAATTACT GTGGCCTACC A    21

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6624

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
GCTGCGGAAT TCTACGTACT ATTGCTGTGG GAA    33

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6514

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CGACGCGGAT CCTACGTACC TGCAGCCATG AGTTGGTCCT TGCAC    45

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6517

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GTCTCTGGTA GCAACATACT A    21

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6516

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
GGGTTTCTAG CCCTACTAGT AG    22

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZC6515

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
GGGTTTCTAG CCCTACTAGT AG    22

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
AAGCTACGCG TCGATCGTCT AGAGTATATC GTCGACGCGT CGATCGG

Claims

1. A method for producing fibrinogen comprising:

providing a first DNA segment encoding a secretion signal operably linked to a fibrinogen Aα chain, a second DNA segment encoding a secretion signal operably linked to a fibrinogen Bβ chain, and a third DNA segment encoding a secretion signal operably linked to a fibrinogen γ chain, 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 and third segments are linked in a single expression unit;

introducing said single expression unit into a fertilized egg of a non-human mammalian species;

inserting said egg into an oviduct or uterus of a female of said 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

recovering the fibrinogen from the milk.

2. A method according to claim 1 wherein said species 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 is operably linked to a transcription promoter selected from the group consisting of casein, β-lactoglobulin, α-lactalbumin and whey acidic protein gene promoters.

4. A method according to claim 1 wherein said first, second and third DNA segments are expressed under the control of a β-lactoglobulin promoter.

5. A method according to claim 1 wherein said introducing step comprises injecting said single expression unit into a pronucleus of said fertilized egg.

6. A method according to claim 1 wherein said fibrinogen is human fibrinogen.

7. 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.

8. 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.

9. A method of producing fibrinogen comprising:

incorporating a first DNA segment encoding a secretion signal operably linked to an Aα chain of fibrinogen into a β-lactoglobulin gene to produce a first gene fusion;

incorporating a second DNA segment encoding a secretion signal operably linked to a Bβ chain of fibrinogen into a β-lactoglobulin gene to produce a second gene fusion;

incorporating a third DNA segment encoding a secretion signal operably linked to a γ chain of fibrinogen into a β-lactoglobulin gene to produce a third gene fusion;

linking the first, second and third gene fusions in a single expression unit;

introducing said single expression unit 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.

10. A method according to claim 9 wherein said mammal is a sheep, pig, goat or bovine.

11. A method according to claim 9 wherein said introducing step comprises injecting said single expression unit 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.

12. A process for producing a transgenic offspring of a mammal comprising:

providing a first DNA segment encoding a fibrinogen Aα chain, a second DNA segment encoding a fibrinogen Bβ chain, and a third DNA segment encoding a fibrinogen γ chain, 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 secretion into milk of said host female mammal;

linking the first, second and third segments in a single expression unit;

introducing said single expression unit into a fertilized egg of a mammal of a non-human species;

inserting said egg into an oviduct or uterus of a female of said non-human species to obtain an offspring carrying said first, second and third DNA segments.

13. A process according to claim 12 wherein said offspring is female.

14. A process according to claim 12 wherein said offspring is male.

15. A non-human mammal produced according to the process of claim 12.

16. A non-human mammal according to claim 15 wherein said mammal is female.

17. A female mammal according to claim 16 that produces milk containing biocompetent fibrinogen encoded by said DNA segments.

18. A non-human mammal according to claim 15 wherein said mammal is male.

19. A method for producing fibrinogen comprising:

providing a transgenic female non-human mammal carrying in its germline heterologous DNA segments encoding Aα, Bβ and γ chains of fibrinogen, wherein said segments are expressed in a mammary gland of said mammal and fibrinogen encoded by said segments is secreted into milk of said mammal;

collecting milk from said mammal; and

recovering said fibrinogen from said milk

wherein the providing step comprises:

incorporating into the germline of the female non-human mammal a first DNA segment encoding a secretion signal operably linked to a fibrinogen Aα chain, a second DNA segment encoding a secretion signal operably linked to a fibrinogen Bβ chain, and a third DNA segment encoding a secretion signal operably linked to a fibrinogen γ chain;

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;

wherein the first, second and third DNA segments are linked in a single expression unit.

Ansprüche

1. Verfahren zur Fibrinogenproduktion, welches enthält:

Bereitstellen eines ersten DNA-Segmentes, das ein Sekretionssignal codiert, welches mit einer Fibrinogen-Aα-Kette funktional verbunden ist, eines zweiten DNA-Segmentes, das ein Sekretionssignal codiert, welches mit einer Fibrinogen-Bβ-Kette funktional verbunden ist, und eines dritten DNA-Segmentes, das ein Sekretionssignal codiert, welches mit einer Fibrinogen-γ-Kette funktional verbunden ist, wobei jedes der ersten, zweiten und dritten Segmente funktional mit zusätzlichen DNA-Segmenten, die für deren Expression in der Brustdrüse eines weiblichen Wirtssäugetieres notwendig sind, verbunden sind;

und wobei die ersten, zweiten und dritten Segmente in einer einzelnen Expressionseinheit verbunden sind;

Einfügen der einzelnen Expressionseinheit in ein befruchtetes Ei einer nichtmenschlichen Säugetierspezies;

Einführen dieses Eies in den Eileiter oder den Uterus eines Weibchens dieser Spezies, um Nachwuchs zu erhalten, der diese DNA-Segmente trägt;

Züchten des Nachwuchses, um weibliche Nachkommen zu erzeugen, die die ersten, zweiten und dritten DNA-Segmente exprimieren und Milch produzieren, die durch die genannten Segmente codiertes biokompetentes Fibrinogen enthält;

Auffangen der Milch der weiblichen Nachkommen;

und Gewinnen des Fibrinogens aus dieser Milch.

2. Verfahren nach Anspruch 1, wobei genannte Spezies aus der Gruppe von Schafen, Schweinen, Ziegen und Kühen ausgewählt ist.

3. Verfahren nach Anspruch 1, wobei jedes der ersten, zweiten und dritten DNA-Segmente funktional mit einem Transkriptionspromotor verbunden ist, der aus der Gruppe ausgewählt ist, die aus Casein-, β-Lactoglobulin-, α-Lactalbumin- und whey acidic protein-Genpromotoren besteht.

4. Verfahren nach Anspruch 1, wobei die ersten, zweiten und dritten DNA-Segmente unter der Kontrolle eines β-Lactoglobulin-Promotors exprimiert werden.

5. Verfahren nach Anspruch 1, wobei der Einfügungsschritt die Injektion der einzelnen Expressionseinheit in einen Pronucleus des befruchteten Eies aufweist.

6. Verfahren nach Anspruch 1, wobei das Fibrinogen Humanfibrinogen ist.

7. Verfahren nach Anspruch 1, wobei das zweite DNA-Segment eine NucleotidSequenz wie in SEQ ID NO: 3 von Nucleotid 470 bis Nucleotid 8100 enthält.

8. Verfahren nach Anspruch 1, wobei das zweite DNA-Segment eine NucleotidSequenz wie in SEQ ID NO: 3 von Nucleotid 512 bis Nucleotid 8100 wiedergegeben enthält.

9. Verfahren zur Fibrinogenproduktion, welches enthält:

Einbauen eines ersten DNA-Segmentes, das ein Sekretionssignal codiert, das funktional mit einer Fibrinogen-Aα-Kette verbunden ist, in ein β-Lactoglobulin-Gen, um eine erste Genfusion zu erzeugen;

Einbauen eines zweiten DNA-Segmentes, das ein Sekretionssignal codiert, das funktional mit einer Fibrinogen-Bβ-Kette verbunden ist, in ein β-Lactoglobulin-Gen, um eine zweite Genfusion zu erzeugen;

Einbauen eines dritten DNA-Segmentes, das ein Sekretionssignal codiert, das mit einer Fibrinogen-γ-Kette funktional verbunden ist, in ein β-Lactoglobulin-Gen, um eine dritte Genfusion zu erzeugen;

Verbinden der ersten, zweiten und dritten Genfusionen in einer einzelnen Expressionseinheit;

Einführen der einzelnen Expressionseinheit in die Keimbahn eines nichtmenschlichen Säugetieres, so dass die DNA-Segmente in der Brustdrüse des Säugetieres oder dessen weiblicher Nachkommen exprimiert werden und biokompetentes Fibrinogen in die Milch des Säugetieres oder dessen weiblicher Nachkommen sekretiert wird;

Auffangen der Milch des Säugetieres oder dessen weiblicher Nachkommen; und

Gewinnen des Fibrinogens aus dieser Milch.

10. Verfahren nach Anspruch 9, wobei das Säugetier ein Schaf, Schwein, eine Ziege oder Kuh ist.

11. Verfahren nach Anspruch 9, wobei der Einführungsschritt die Injektion der einzelnen Expressionseinheit in den Pronucleus des befruchteten Eies und das Einführen dieses Eies in den Eileiter eines pseudograviden Weibchens aufweist, um weiblichen Nachwuchs zu erhalten, der die Genfusionen in seiner Keimbahn trägt.

12. Verfahren zur Produktion von transgenen Nachkommen eines Säugetieres, welches enthält:

Bereitstellen eines ersten, Fibrinogen-Aα-Kette-codierenden DNA-Segmentes, eines zweiten. Fibrinogen-Bβ-Kette-codierenden DNA-Segmentes und eines dritten, Fibrinogen-γ-Kette-codierenden DNA-Segmentes, wobei jedes der ersten, zweiten und dritten Segmente funktional mit einem zusätzlichen DNA-Segment verbunden ist, welches für die Expression in der Brustdrüse eines weiblichen Wirtssäugetieres und die Sekretion in die Milch dieses weiblichen Wirtssäugetieres notwendig ist;

Verbinden der ersten, zweiten und dritten Segmente in einer einzelnen Expressionseinheit;

Einfügen der einzelnen Expressionseinheit in ein befruchtetes Ei einer nichtmenschlichen Säugetierspezies;

Einführen dieses Eies in den Eileiter oder den Uterus eines Weibchens der nichtmenschlichen Spezies, um Nachwuchs zu erhalten, der die ersten, zweiten und dritten DNA-Segmente trägt.

13. Verfahren nach Anspruch 12, wobei der Nachwuchs weiblich ist.

14. Verfahren nach Anspruch 12, wobei der Nachwuchs männlich ist.

15. Nichtmenschliches Säugetier, das gemäß dem Verfahren unter Anspruch 12 hergestellt ist.

16. Nichtmenschliches Säugetier nach Anspruch 15, wobei das Säugetier weiblich ist.

17. Weibliches Säugetier nach Anspruch 16, das Milch produziert, die durch die DNA-Segmente codiertes biokompetentes Fibrinogen enthält.

18. Nichtmenschliches Säugetier nach Anspruch 15, wobei das Säugetier männlich ist.

19. Verfahren zur Fibrinogenproduktion, welches enthält:

Bereitstellen eines transgenen, weiblichen, nicht menschlichen Säugetiers, welches in seiner Keimbahn heterologe, Aα-Bβ-und γ-Fibrinogenketten-codierende DNA-Segmente trägt, wobei diese Segmente in einer Brustdrüse dieses Säugetieres exprimiert werden, und wobei von diesen Segmenten kodiertes Fibrinogen in Milch dieses Säugetiers sekretiert wird;

Auffangen der Milch des Säugetieres; und

Gewinnen des Fibrinogens aus dieser Milch

wobei der Schritt des Bereitstellens enthält:

Einbauen in die Keimban des weiblichen, nicht menschlichen Säugetiers eines ersten DNA-Segmentes, das ein Sekretionssignal codiert, das funktional mit einer Fibrinogen-Aα-Kette verbunden ist, eines zweiten DNA-Segmentes, das ein Sekretionssignal codiert, das funktional mit einer Fibrinogen-Bβ-Kette verbunden ist, und eines dritten DNA-Segmentes, das ein Sekretionssignal codiert, das mit einer Fibrinogen-γ-Kette funktional verbunden ist;

wobei jedes dieser ersten, zweiten und dritten Segmente mit zusätzlichen DNA-Segmenten, die für deren Expression in der Brustdrüse eines weiblichen Wirtssäugetieres notwendig sind, funktional verbunden ist;

wobei die ersten, zweiten und dritten DNA-Segmente in einer einzelnen Expressionseinheit verbunden sind.

Revendications

1. Procédé de production de fibrogène, comprenant :

la mise à disposition d'un premier segment d'ADN codant un signal de sécrétion lié de manière fonctionnelle à une chaîne Aα de fibrogène, d'un deuxième segment d'ADN codant un signal de sécrétion lié de manière fonctionnelle à une chaîne Bβ de fibrogène et d'un troisième segment d'ADN codant un signal de sécrétion lié de manière fonctionnelle à une chaîne γ de fibrogène, chacun desdits premier, deuxième et troisième segments étant lié de manière fonctionnelle à des segments d'ADN supplémentaires nécessaires pour son expression dans la glande mammaire d'un mammifère femelle hôte ;

et le premier, deuxième et troisième segment sont liés dans un unique motif d'expression ;

l'introduction dudit unique motif d'expression dans un oeuf fécondé d'une espèce mammifère différente de l'homme ;

l'insertion dudit oeuf dans un oviducte ou un utérus de femelle de ladite espèce pour obtenir une descendance portant lesdits segments d'ADN ;

l'élevage de ladite descendance dans le but de produire une progéniture femelle qui exprime lesdits premier, deuxième et troisième segments d'ADN et produit du lait contenant un fibrinogène biologiquement compétent codé par lesdits segments ;

le fait de recueillir le lait de ladite progéniture femelle ; et la récupération du fibrinogène à partir du lait.

2. Procédé selon la revendication 1, dans lequel ladite espèce est choisie dans le groupe constitué par les moutons, les porcs, les chèvres et les bovins.

3. Procédé selon la revendication 1, dans lequel chacun desdits premier, deuxième et troisième segments d'ADN est lié de manière fonctionnelle à un promoteur de transcription choisi dans le groupe constitué par les promoteurs de la caséine, de la β-lactoglobuline, de l'α-lactalbumine et du gène de la protéine acide de lactosérum.

4. Procédé selon la revendication 1, dans lequel lesdits premier, deuxième et troisième segments d'ADN sont exprimés sous le contrôle d'un promoteur de β-lactoglobuline.

5. Procédé selon la revendication 1, dans lequel ladite étape d'introduction comprend l'injection dudit unique motif d'expression dans un pronucléus dudit oeuf fécondé.

6. Procédé selon la revendication 1, dans lequel ledit fibrinogène est le fibrinogène humain.

7. Procédé selon la revendication 1, dans lequel ledit deuxième segment d'ADN comprend une séquence de nucléotides représentée dans la SEQ ID No : 3, du nucléotide 470 au nucléotide 8100.

8. Procédé selon la revendication 1, dans lequel ledit deuxième segment d'ADN comprend une séquence de nucléotides représentée dans la SEQ ID No : 3, du nucléotide 512 au nucléotide 8100.

9. Procédé de production de fibrinogène, comprenant :

l'incorporation d'un premier segment d'ADN codant un signal de sécrétion lié de manière fonctionnelle à une chaîne Aα de fibrogène dans un gène de β-lactoglobuline pour produire une première fusion de gènes ;

l'incorporation d'un deuxième segment d'ADN codant un signal de sécrétion lié de manière fonctionnelle à une chaîne Bβ de fibrogène dans un gène de β-lactoglobuline pour produire une deuxième fusion de gènes ;

l'incorporation d'un troisième segment d'ADN codant un signal de sécrétion lié de manière fonctionnelle à une chaîne γ de fibrogène dans un gène de β-lactoglobuline pour produire une troisième fusion de gènes ;

la liaison des première, deuxième et troisième fusions de gènes dans un unique motif d'expression ;

l'introduction dudit unique motif d'expression dans la lignée germinale d'un mammifère différent de l'homme de manière à ce que lesdits segments d'ADN soient exprimés dans une glande mammaire dudit mammifère ou de sa progéniture femelle et que le fibrinogène biologiquement compétent soit sécrété dans le lait dudit mammifère ou de sa progéniture femelle ;

l'obtention du lait à partir dudit mammifère ou de sa progéniture femelle ; et

la récupération dudit fibrinogène à partir dudit lait.

10. Procédé selon la revendication 9, dans lequel ledit mammifère est un mouton, un porc, une chèvre ou un bovin.

11. Procédé selon la revendication 9, dans lequel ladite étape d'introduction comprend l'injection dudit unique motif d'expression dans un pronucléus dudit oeuf fécondé et l'insertion dudit oeuf dans un oviducte de femelle en pseudo-gestation pour produite une descendance femelle portant lesdites fusions de gènes dans la lignée germinale.

12. Procédé de production d'une descendance transgénique d'un mammifère comprenant :

la mise à disposition d'un premier segment d'ADN codant une chaîne Aα de fibrogène, d'un deuxième segment d'ADN codant une chaîne Bβ de fibrogène et d'un troisième segment d'ADN codant une chaîne γ de fibrogène, chacun desdits premier, deuxième et troisième segments étant lié de manière fonctionnelle à des segments d'ADN supplémentaires nécessaires pour son expression dans la glande mammaire d'un mammifère femelle hôte et la sécrétion dans le lait dudit mammifère femelle hôte ;

la liaison des premier, deuxième et troisième segments dans un unique motif d'expression ;

l'introduction dudit unique motif d'expression dans un oeuf fécondé de mammifère d'une espèce différente de l'homme ;

l'insertion dudit oeuf dans un oviducte ou un utérus de femelle de ladite espèce différente de l'homme pour obtenir une descendance portant lesdits premier, deuxième et troisième segments d'ADN.

13. Procédé selon la revendication 12, dans lequel ladite descendance est une femelle.

14. Procédé selon la revendication 12, dans lequel ladite descendance est un mâle.

15. Mammifère différent de l'homme produit selon le procédé de la revendication 12.

16. Mammifère différent de l'homme selon la revendication 15, dans lequel ledit mammifère est une femelle.

17. Mammifère femelle selon la revendication 16, qui produit du lait contenant un fibrinogène biologiquement compétent codé par lesdits segments d'ADN.

18. Mammifère différent de l'homme selon la revendication 15, dans lequel ledit mammifère est un mâle.

19. Procédé de production de fibrinogène, comprenant :

la mise à disposition d'un mammifère femelle transgénique différent de l'homme portant dans sa lignée germinale des segments d'ADN hétérologues codant les chaînes Aα, Bβ et γ de fibrinogène, lesdits segments étant exprimés dans une glande mammaire dudit mammifère et le fibrinogène codé par lesdits segments étant sécrété dans le lait dudit mammifère ;

le fait de recueillir le lait dudit mammifère ; et

la récupération dudit fibrinogène à partir dudit lait

dans lequel l'étape de mise à disposition comprend :

l'incorporation dans la lignée germinale du mammifère femelle différent de l'homme d'un premier segment d'ADN codant un signal de sécrétion liée de manière fonctionnelle à une chaîne Aα de fibrogène, d'un deuxième segment d'ADN codant un signal de sécrétion lié de manière fonctionnelle à une chaîne Bβ de fibrogène et d'un troisième segment d'ADN codant un signal de sécrétion lié de manière fonctionnelle à une chaîne γ de fibrogène ;

chacun desdits premier, deuxième et troisième segments étant lié de manière fonctionnelle à des segments d'ADN supplémentaires nécessaires pour son expression dans la glande mammaire d'un mammifère femelle hôte ;

les premier, deuxième et troisième segments d'ADN étant liés dans un unique motif d'expression.

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