The transgene to obtain the recombinant polypeptide in the milk of transgenic cows, a method of obtaining a transgenic cows (options), milk from transgenic cows, food composition

 

(57) Abstract:

Usage: the invention relates to the production of recombinant polypeptides in transgenic cows and methods of producing transgenic cows. The inventive transgene for producing the recombinant polypeptide from the milk of transgenic cows of different breeds includes at least one sequence of the regulation of expression, secretory DNA sequence encoding a secretory signal, the current in the secretory cells of the mammary glands of the cow, and recombinant DNA sequence encoding a recombinant polypeptide. A method of obtaining a transgenic cows includes the introduction of the above transgene in embryonic the target cell cows, transplantation formed by such transgenic embryonic target cells of the parent cow-recipient and the identification of at least one born heifers, capable of producing a recombinant polypeptide with his milk. Milk from transgenic cows containing recombinant polypeptide produced transgenic cow, obtained according to the above method. Food composition, including transgenic milk containing recombinant is the production of recombinant polypeptides in transgenic cows and to methods of producing transgenic mammals (except humans), with the target phenotype.

There is a vast literature related to the expression of heterologous genes in lower organisms such as unicellular bacteria, yeast and filamentous fungi, and cells of higher type, such as mammalian cells. There are also numerous reports about the creation of transgenic animals, most of which relates to the creation of transgenic mice. Cm. for example, U.S. patent N 4736866 (transgenic mice with activated oncogene); Andres A. et al, 1987, Proc. Natl. Acad. Sci. USA, 84, 1299 1303 (HA-RAS oncogene under the control of the whey acidic protein promoter); Schoenberger C. A. et al. 1987, Experientia, 43, 644 and (1988) EMBO J., 1, 169 175 (C-myc oncogene under control of the whey acidic protein promoter); and Muller W. J. et al. 1988, Cell, 54, 105 115 (C-myc oncogene under the control of the viral promoter of breast cancer mouse). Some laboratories have reported the production of transgenic pigs (Miller K. F. et al, 1989, J. Endocrin, 120, 481

488 (gene expression of human or bovine growth hormone in transgenic pig); Vize, P. D. et al. 1988, J. Cell. Sci, 90, 295 300 (fused gene of porcine growth hormone in transgenic pigs) and K. Ebert et al. 1988, Mol. Endocrin. 2, 277 283 (fused gene somatotropin MMLV-rats in transgenic pigs), transgenic sheep (Nancarrow et al. 1987, Theriogenology, 27(human factor IX and -1 antitrypsin CONA in sheep's body) and rabbits (Hanover S. V. et al. 1987, Deutsche Tierarztliche Wochenschrift, 94, 476 478 (creation of transgenic rabbits by injection of a uterine-globingo-j-CAT built into fertilized oocytes rabbit). In several reports have also suggested the creation of cattle (Wagner et al. 1984, Theriogenology, 21, 29 and 44), one of which was reported some progress in the methods of microinjection (Lohse J. K. et al. 1985, Theriogenology, 23, 205). However, in obtaining transgenic cows was achieved little success. Scientific articles, which clearly shows the actual creation of transgenic cows capable of producing heterologous protein is currently unknown. And this is despite the claim that one transgenic cow was created in Canada and this cow is expressed in human b-interferon (Van Brunt, J. 1988, Bio/Technology, 6, 1149 - 1155), and that in one case achieved transient expression of human a-fetoprotein in liver and blood (Church, R. B. 1986, Biotechnology News Watch, 6, (15), 4). In one of the links has been reported that the virus bovine papilloma was probably integrated, but not expressed in transgenic cow (Roschlau et al. 1988, Arch. Tierz. Berlin, 31, 3 8). In a recent article summarized the successes of genetic engineering for livestock (Pursel, V. G. et al. 1989, Science, 244, 1281 1288).

Next lab is ucirvine different proteins in the milk of transgenic mice and sheep. For example, in the work of J. P. Simmons et al. 1987, Nature, 328, 530 532 reported microinjection genomic fragment 16.2 kb, encoding b-lactoglobulin (BLG), including 5'-sequence of 4 kb, the transcription link BLG 4.9 kb and flanking 3'-sequence 7.3 kb in fertilized mouse eggs. According to the authors, BUL expressively in the mammary tissue, but producyrovtsa BLG with milk of transgenic mice at a concentration of 3 to 23 mg/ml. However, in the case of embedding in the untranslated 5'-region of the BLG gene cDNA coding for human factor IX or human a1-antitripsin, and microinjection of this design (Simmons J. P. et al. 1988, Bio/Technology, 6, 179 183), the production of factor IX or 1-antitrypsin deficiency was significantly lower (25 ng/ml of factor IX and 10 mg/ml for 1-antitrypsin; see Clark et al, 1989, Bio/Technology, 7, 487 492).

It was reported (Lee et al. 1988, Nucl. Acids Res. 16, 1027 1041) microinjections in a similar way in fertilized oocytes of the mouse genomic clone 14 kb, consisting of the full rat-casein 7.5 kb flanking 5'-DNA 3.5 kb and flanking 3'-DNA. In this case, according to the report, the level of expression of rat b-transgene in the lactating mammary gland of transgenic mice was in the range from 0.01 to 1% of the endogenous gene of murine b-azelastine (t-PA) at the level of 0.2 to 0.4 µg/ml for the expression of cDNA encoding human t-PA, together with secreting endogenous sequence under the control of the 5'-sequence of the 2.6 kb of the gene of the mouse whey acidic protein (Gordon K. et al. 1987, Bio/Technology, 5, 1183 1187). Subsequent experiments using the same or similar constructions, as reported, showed the production of t-PA in mice of different lines ranging from less than 20 ng t-PA per ml of milk to about 50 μg/ml (Pittius, C. W. et al. 1988, Proc. Natl. The Aca. Sci. USA, 85, 5874 5878).

In U.S. patent N 4873316 (issued October 10, 1989) discloses the use of 5'sequences of the gene of bovine aS1-casein comprising the signal peptide of casein and several codons casein, merged with Mature t-PA sequence. Transgenic mouse created using this construct, according to the report, was produced in their milk 0.2 to 0.5 μg/ml t-PA fused protein.

In addition, in a number of patent publications reported the directed production of specific proteins in milk of transgenic mice and sheep (see. for example, European patent N 0264166, publ. April 20, 1988) (surface antigen of hepatitis b and the t-PA gene under the control of the promoter, whey acidic protein for specific expression in the mammary gland of the mouse PCT publicname cells, containing a recombinant expression system comprising a gene bovine a-lactalbumin, merged with interleukin-2); European Pat. N 0279582, publ. August 24, 1988 (the tissue-specific expression chloramphenicolchloramphenicol under the control of the rat b-casein promoter in transgenic mice) and PCT publication N WO 88/10118, publ. on December 29, 1988 (transgenic mice and sheep, having a transgene encoding bovine aS1-casein promoter and signal sequence, fused with t-PA).

When considering the state of the question in the field of transgenic technology is a clear need for methods that allow to efficiently generate transgenic mammal, particularly a transgenic mammal, non-transgenic mice.

In addition, a clear need in the way of creating transgenic cows that are capable of producing recombinant polypeptides, such as human milk proteins and human serum proteins in milk.

Besides the technical result of the invention consists in the creation of transgenic cows capable of producing recombinant polypeptides that remain inside the cells or are secreted extracellular and cows, spasoje serum proteins.

In addition, the invention allows to obtain from transgenic cows milk containing recombinant polypeptides, and create food compositions with the addition of recombinant polypeptides from milk of transgenic cows, such as baby food with the addition of human lactoferrin.

In the application described the creation of transgenes, which in the milk of transgenic cows produced recombinant polypeptides.

Thus, the invention includes transgenic for the production of recombinant polypeptides in the milk of transgenic cows. The receipt of such milk of transgenic cows, containing one or more recombinant polypeptides, preferably because this recombinant polypeptides obtained in this way do not require purification for human consumption. The transgene includes a secretory DNA sequence encoding a secretory signal sequence operable in the secretory cells of the mammary gland of interest cows and recombinant DNA sequence encoding a recombinant polypeptide. These sequences are related to each other with the formation of the working sequence: secretory posledovatelbno connected to at least one regulatory sequence, ensuring the expression of the secretory cells of the mammary gland of cows. The transgene construct with secretory sequence, which is capable of directing DNA containing the transgene, in secretory cells of the mammary gland of cows. The result of this expression is formed from a recombinant form of the polypeptide that is secreted from the secretory cells of the mammary gland of cows in milk of transgenic cows.

The invention discloses methods of making such transgenic cows. The method includes the introduction of the above transgene in embryonic target cell cows, transplantation resulting transgenic embryonic target cells parent-recipient and the identification of offspring at least one of the heifers, and are able to produce in their milk recombinant polypeptide.

The invention also includes milk from such transgenic cows containing recombinant polypeptides, and food compositions containing transgenic milk in liquid or dry form, as well as nutritional formulations with the addition of one or more recombinant polypeptides from the indicated transgenic milk.

In addition to the above, the invention includes TRANS is aresults fact, what are the regulatory sequences of the expression that provides the expression of DNA that encodes a recombinant polypeptide in a particular cell type or tissue, for example, human serum albumin in the liver of transgenic cows. If recombinant polypeptide must secretariats of these target cells or tissues, then the secretory DNA sequence encoding a secretory signal sequence that is effective in specific target cells or tissues, binds to recombinant DNA sequence that encodes a recombinant polypeptide, for example, when the secretion of human serum albumin from the liver of cows in the cardiovascular system.

The application describes methods of creating transgenic mammals (except humans) with the target phenotype. The method consists in the initial methylation of the transgene capable of creating the target phenotype when introduced into cells of the transgenic animal, for example, the acceptable transform bacteria, such as E. coli MM 294 plasmid containing the transgene. Methylated transgene, then cut out and injected into fertilized oocytes of the animal with integration into the genome. The oocytes then virudunagar preimplantation embryo extract at least one cell, which process and allocate it contains DNA. Each selected DNA is then treated with restriction enzyme that can split methylated transgene, but are unable to split demetilirovanny form of transgene formed after integration into genomic DNA and its replication. Those preimplantation embryos that have integrated the transgene contain DNA is resistant to cleavage by the restriction enzyme in the area, including the transgene. Such resistance to hydrolysis, which can be detected by electrophoresis of the hydrolysis product after PCR amplification of DNA and hybridization with a labeled probe that facilitates the identification of successful transgenesis.

A specific embodiment of the above method of early detection of transgenesis is the method of obtaining a population of transgenic offspring with the same phenotype. In this way methylated transgene injected into fertilized oocytes grown to preimplantation embryos. Then each preimplantation embryo divide with the formation of the first and second polyembryony. Each first polyembryony analyze the above method for transgenesis. After successful detection Transylvania the transgene, clone with the formation of multiple transgenic clones blastocytes and pooblastitev, each of which is characterized by one and the same genotype. After that transgenic embryos transplanted to one or more female recipients from obtaining a population of transgenic animals with the same genotype.

In Fig. 1-7 presents DNA (PEFC. ID No. 1) and amino acid sequence (th. ID No. 2) clone human lactoferrin derived from cDNA library human mammary gland in the form in which it is given here, except that the sequence between nucleotides 1557-1791 and 2050-2129 corresponds to the previously published sequence (Rado et al. 1987, Blood, 70, 989-993); Fig. 8 9 - full DNA (PEFC. ID No. 3) and amino acid sequence (th. ID No. 4) human lactoferrin, including 5'- and 3'-noncoding sequence, as well as a complete signal sequence of human lactoferrin; Fig. 10 restriction map of clone 5'-flanking region of the gene bovine aS1-casein; Fig. 11 restriction map of clone 3'-flanking region of the gene bovine aS1-casein; Fig. 12 14 design pSI 3'5' CAT pSI and 5' CAT; Fig. 15 pMN 1; Fig. 16 21 the construction of expression vectors, soderjashie albumin, the fragments used to generate transgenic mice and contained in this genomic DNA and identification of the size of the fragment, which must be obtained by hydrolysis of genomic DNA from transgenic mouse DNA restriction enzymes BstE-II and Nco I or Nco I and Hindi-III of Fig. 23 is an alternative way of constructing a transgene of the invention encoding human lactoferrin; Fig. 24 construction of plasmids pPC containing a transgene encoding a protein C; Fig. 25 DNA sequence of hybrid Nitron used in the embodiment of the invention. Hybrid sequence comprises the 5'part of intron bovine aS1-casein and the 3'part of intron IgG. The place of junction of the 5'- and 3'-part is HindIII.

"Mammals (except humans)" means all mammals, except humans, are able to produce a "transgenic mammals (except humans)" who have "target phenotype". Such mammals include primates besides humans, mice of different lines of cows of different breeds of dogs of different breeds and so on. Featured animals are cows, pigs and sheep of different breeds, most preferably cows of different breeds.

The target for transgenic phenotypes mleok transgenic mammals (except humans), the production of animal models for studying diseases, the creation of animals with higher resistance to disease (e.g., breast diseases, such as mastitis) and the production of recombinant polypeptides in blood, urine or other acceptable biological fluids or tissues of the animal. In the recommended embodiments of the invention described transgenic cows capable of producing recombinant human lactoferrin, human serum albumin and human protein C in the milk of lactating females or human serum albumin in the liver of transgenic animal.

Transgenic mammals (except humans) are obtained by the introduction of a "transgene in embryonic target cell selected animal. In one aspect of the invention, the transgene is a DNA sequence capable of creating a specific phenotype when present in the cellular genome of the transgenic mammal (except man). In specific embodiments of the invention, the transgene is a "recombinant DNA sequence encoding a "recombinant polypeptide". In such cases, the transgene is able to Express the recombinant polypeptide.

In primenyaemaya "heterologous polypeptide", or "homologous polypeptide". Heterologous polypeptides refer to polypeptides, usually not produced transgenic animals. Examples of heterologous proteins include human milk proteins such as lactoferrin, secrete lysozyme, secreted immunoglobulins, lacto albumin-stimulated lipase, bile etc., human serum proteins, such as albumin, immunoglobulin, factor VIII, factor IX, protein C, etc., and industrial enzymes, such as proteases, lipases, chitinases and leginsy from prokaryotic and eukaryotic sources. The sequence of recombinant DNA comprises genomic or DNA sequence encoding a recombinant polypeptide.

When using recombinant DNA sequences encoding heterologous polypeptides, the transgene may be integrated into the genome used for transgenesis animals at random. As described in the examples, a transgene encoding human lactoferrin, human serum albumin and human protein C, in combination with secretory signal sequence aS1-casein under control sequences regulating the expression of aS1-casein, were created for the production and secretion >/P>As used here, the value of the homologous polypeptide is a polypeptide endogenous to a particular transgenic animal mind. Examples of endogenous polypeptides for different breeds of cows include cow's milk proteins, such as aS1, aS2, b - and k-casein, b-lactoglobulin, lactoferrin, secrete lysozyme, cholesterol hydrolase, serum proteins, such as serum albumin and protein hormones such as growth hormones. When using a DNA sequence that encodes a homologous polypeptide, the transgene is recommended to introduce into the genome used for animal transgenesis arbitrary way. Such disordered introduction gives a transgenic animal that has not only the transgene encoding the endogenous polypeptide, but also the corresponding endogenous genomic DNA sequence. Accordingly, such a transgenic mammal (except man) are easily characterized by an increased number of copies of genes encoding the endogenous polypeptide. In addition, the transgene, as a rule, will be located in a position different from the position of the endogenous gene.

When the expression of DNA that encodes a polypeptide, for example, in cows of different breeds transgenic animal is different povyshyeniya, and/or the presence of a homologous protein in the tissue and/or biological fluid, in which homologous polypeptide or not usually contains or is produced at a much lower level.

For example, cow cholesterol hydrolase usually present in colostrum within the first 15 to 20 days of lactation. This natural endogenous protein increases the weight of the calf. However, this protein is also homologous to a polypeptide where, for example, its expression in the secreting cells of the mammary gland by using sequences of gene regulation bovine casein, which promote the expression of homologous polypeptide and beyond normal lactation. Thus, according to one aspect of the invention, expression of bovine cholesterol-hydrolases in the milk of transgenic cows is recombinant DNA (or cDNA or genomic DNA) cholesterol-hydrolases under control sequences regulating the expression of cow aSl-casein. When using genomic recombinant DNA create it so that it has a reasonable restriction sites (for example, ClaI and SalI) at the 5' and 3' end of the structural gene so that it can be embedded in the ACC will owu cholesterol-hydrolase and originating from cDNA, embed control sequence regulating the expression of cow aSl-casein by replacing sequences of human lactoferrin in this plasmid, p 16, 8 HLF 3 (includes hybrid intron) or p16, 8 HLF 4 (contains homologous intron aSl-casein). When using these specific plasmids the cDNA clone create so that it is acceptable ClaI and SalI restriction sites at both ends of recombinant DNA.

And another example. Bovine lactoferrin is usually present in cow's milk only in trace quantities. However, if the cow lactoferrin is expressed under the control of other regulatory sequences, for example derived from a gene aSl-casein, in milk of transgenic cows lactoferrin is present in large quantities. In another example, the transgene comprising DNA encoding the homologous bovine growth hormone injected into bovine genome for making transgenic animal higher growth characteristics. In other cases, homologous polypeptides include, for example, the polypeptide, which is usually in a particular animal species is contained within the cells, but which is secreted in milk or other extracellular space transgenic animals, for example in the agreement for him by the sequence of amino acids and nucleic acids. You must specify that such sequences include natural allelic variations and variants derived recombinant methods in which such nucleic acid sequences and polypeptide sequences are modified by substitution, implementation and/or deletions of one or more nucleotides in such nucleic acids to cause the substitution, insertion or deletion of one or several amino acid residues in the recombinant polypeptide.

When the expression of the DNA of the transgene is required to produce the target phenotype, for example for producing a recombinant polypeptide, in this case, the transgene typically includes at least 5, but preferably more, and 3' sequences regulating the expression" each of which is effectively connected with or recombinant secretory-recombinant DNA defined below. This sequence expression regulation, in addition to regulating transcription, contribute to the stability and processing of RNA, at least to the extent in which they are transcribed.

Such sequences regulating the expression choose to create tissue-specific or the delan choice of tissue or cell type for expression, pick the 5'- and possibly the 3'- sequence expression regulation. Typically, such sequences regulating the expression come from genes that are expressed in a selected tissue or cell type. It is recommended that the genes which received such sequences regulating the expression, expressionlist only essentially in the desired tissues or cell types, although the secondary expression in other tissues and/or cells of another type is also acceptable, if this expression does not adversely affect the transgenic animal. Especially it is recommended to use the sequence expression regulation, which is endogenous to being processed animal species. However, it can also be used and the sequence regulating the expression of other types, for example from human genes. In some cases, sequences regulating the expression and recombinant DNA sequences (genomic or cDNA) derived from the same source, such as both come from cows or humans. In such cases, consistency of regulation of expression and recombinant DNA sequence homologous with respect to each other. Or posledovat, for example, a sequence regulating the expression from the cow, and recombinant DNA sequence from the human. In this case, the sequence regulating the expression and recombinant DNA sequence of heterologic in relation to each other. Below follows the definition of the sequence regulating the expression of endogenous genes. Made definition also applies to sequences regulating the expression of endogenic heterologous genes.

In General, the 5'sequence of the regulation of expression includes the transcribed portion of the endogenous gene in an upward direction from the sequence of initiation of translation (5' retranslation region or 5' UTR) and those rising from her flanking sequences that contain a functional promoter. In applied here in the sense of "functional promoter" includes the optional transcribed DNA sequences that provide for the binding of RNA polymerase with the endogenous gene with the promotion of transcription. Such sequences usually consist of a TATA sequence or frame Hogness, usually located within a 25 - to 30 nucleotides from the site of transcription initiation. Frame Hogness several distal factors, located in the upstream direction from the proximal factor (frame Hogness) and is required for transcription initiation. Such sequences of promoter, as a rule, are contained within the first 100 to 200 nucleotides located upstream from the site of transcription initiation, but may extend up to 500 to 600 nucleotides from the site of transcription initiation. Such sequences are either self-evident to a specialist, or are readily determined by standard methods. Similar to the sequence of the promoter by themselves or in combination with 5' untranslated region are referred to here as "proximal 5'- sequence expression regulation".

In addition to these proximal 5'-sequences regulating the expression of it is also recommended that the transgene were included additional flanking the 5'-sequence (hereinafter referred to as "distal 5'-sequences regulating the expression"). Such distal 5'-sequence expression regulation, believed to contain one or more enhancers and/or other sequences that facilitate expression of the endogenous gene and as a consequence the expression of recombinant or secretory-recombinant polii expression. The size of the distal 5'-sequence regulating the expression depends on endogenous gene, which is a sequence expression regulation. However, typically, such sequences include flanking the 5'-region of about 1 p. O. more preferably 16 p. O., and most preferably about 30 p. O. Optimal size of the distal 5'-sequence expression regulation, taken from a specific endogenous gene, is easily determined by changing the size of the distal 5'-sequence expression regulation with maximum expression. In General, the distal 5'-sequence regulating the expression is not very large, so as not to enter the adjacent gene, and will not include DNA sequences that adversely affect the level of transgenic expression.

In addition, it is recommended to enhance the tissue-specific or specific to the cell type expression also include 3'-sequence expression regulation. Such 3'-sequence expression regulation include 3'-proximal and 3'-distal sequence regulating the expression of the corresponding endogenous gene. The proximal 3'-sequence regulating the expression of the recombinant DNA sequence (also called the 3' untranslated region or 3' utrs). Such sequences usually end the polyadenylation sequence (either from an endogenous gene or from another source, such as SV 40) and sequences that can affect the stability of the RNA. Typically, the 3' UTR consists of 100 to 500 nucleotides downstream of the translational stop signal in the gene from which regulation 3'-sequence occurs. The distal 3'-sequence expression regulation includes flanking DNA sequences in the downstream direction from the proximal 3'- sequence expression regulation. Some of these distal sequences are transcribed, but do not form part of the mRNA, while the other sequences in this distal 3'sequences regulating the expression is not transcribed at all. Similar to the distal 3'sequences regulating the expression is considered to contain enhancer and/or other amplifying the expression of the sequence. I believe that such sequences required for efficient polyadenylation and contain sequences of the transcription has been completed. It is recommended that this sequence was approximately 2 kb, more predpochetaet the use of both 5'- and 3'-sequences regulating the expression, however, in some embodiments of the invention the endogenous 3'-sequence expression regulation does not apply. In such cases, to ensure polyadenylation usually applied proximal to the 3'sequence regulating the expression normally associated with genomic DNA, encoded by the recombinant DNA sequence. In addition, can also be used distal regulatory 3'sequences from genomic DNA that encodes a recombinant polypeptide, preferably the same size as that of the above endogenous 3'-sequence expression regulation. You must specify that in such cases, encoded by the transgene recombinant polypeptide can include either genomic DNA or originating from cDNA Dunaeva DNA. As in the case of 5'sequences regulating the expression of the optimal size of 3'-sequences regulating the expression can be easily determined by changing the size of the flanking 3'sequences to achieve the maximum expression of the recombinant polypeptide. In General, distal regulatory 3'-sequence occurring or endogenous gene, or heterologous gene, will not extend into the adjacent gene, from Sodeistvie on the level of transgenic expression.

Examples of sequences regulating the expression given in table. 1.

It is recommended that in addition to the 5'- and 3'-sequences regulating the expression and recombinant DNA (either genomic or originating from cDNA) transgene of the invention also contain recombinant intron, which interrupts transcribed, but not translated 5'-region of the transgene. Such introns can occur, for example, from bovine aS1-casein from human lactoferrin. Such applied here sequences are "homologous recombinant introns in 5'- and 3'- RNA splice signals in such recombinant introns are the same as those usually found in the intron of an endogenous or heterologous gene. Recombinant introns, however, can also be a hybrid intron". Such hybrid introns include the 5' RNA splice signal and 3' RNA splice signal from introns of different sources. In some aspects of the invention such hybrid introns include at least one "resolution RNA splice sequence. As used here, the value of the signal, allowing the RNA splicing, means RNA splice signal sequence, preferably the 3' RNA splice signal from the Intro of differentiation. Examples of such gene sets include: immunoglobulin supergene family, including immunoglobulins and T-cell antigen receptors, as well as a set of genes of major histocompatibility complex (MHC) and others. It is especially recommended sequences permitting splicing, are those derived from immunoglobulin set, preferably of the IgG class, and more preferably the 3' splice signal sequences that are associated with the J C segment rearrangement of the heavy and light chain Ig, most preferably the heavy chain. Especially recommended sequence, resolution, splicing, consists of that part of the sequence, which in Fig. 25 shows in a downward direction from the Hind III site. Featured hybrid intron consists of the complete sequence shown in Fig. 25, and includes the 5'portion of the intron of the bovine aS1-casein and the 3'portion of the sequence of intron heavy chain of IgG.

Such hybrid introns containing signals that allow the RNA splicing, it is recommended if recombinant DNA corresponds to the cDNA sequence. As shown in the examples, when using a 5'-sequence regulation expressively, effectively associated with secretory signal sequence aS1-casein, transgenic mouse producing approximately 1330 ág/ml CLP in transgenic milk. Given the amount of recombinant polypeptide is significantly higher than previously reported quantity production of various proteins in the milk of transgenic mice, which were usually less than 10 µg/ml, and in one case, about 50 μg/ml And is also higher than the maximum of 8 µg/ml CLF, which is produced in our case, using the same transgene containing the homologous bovine intron, and not a hybrid intron.

However, such hybrid introns are not limited transgenes using cDNA sequences. Moreover, hybrid introns applicable also when the recombinant polypeptide is encoded by the genomic sequence. Based on the results obtained from recombinant DNA on the basis of the cDNA and the General assumption that genomic DNA sequence is expressed at a higher level than originating from cDNA sequences, it is expected that such hybrid introns used in combination with genomic recombinant DNA further enhance the level of expression in comparison with the foregoing, it is obvious that featured transgenes include a large portion of the 5'- and 3'-sequences regulating the expression. Moreover, recombinant DNA, it is recommended to get from genomic clones, which may be a length of tens to hundreds of cilaisnowbuy. Based on the existing technology of cloning and processing of DNA, design and the microinjection of transgenes practically limited to the linear form of DNA of length no more than about 50 kb. However, the transgenes of the invention, particularly those longer than about 50 kb, can be easily created by the introduction of two or more overlapping fragments of the target transgene in embryonic the target cell. After the introduction of overlapping fragments undergo homologous recombination, which leads to the integration completely rebuilt the transgene into the genome of target cells. In General, it is recommended that such overlapping fragments of the transgene had 100% homologically in areas that overlap. However, there is a lower gomologichnosti sequences provided efficient homologous recombination. If the parts between homologous sequence still exists ahomologically, recomend discrete areas. Although only 14 of the base pairs at 100% gomologichnosti sufficient for homologous recombination in mammalian cells (Rubnitz J. and Subramani S. 1984, Mol. Cell. Biol. 4, 2253 - 2258), however, recommended a longer part of the homologous sequences, for example, 500 p. O. more preferably 1000 p. O. more preferably 2000 p. O., and most preferably more than 2000 p. O. for each part of the homologous sequence.

As shown in the examples, three overlapping fragment of human serum albumin were microinjection approximately equal molar portions into the pronucleus of mouse zygotes. These fragments successfully recombinable and were integrated into the genome of the mouse, which is confirmed by analysis of the integrated DNA by southern blot-hybridization, detection of the RNA transcript and human serum albumin in the serum of transgenic mice. Although created as a result of the transgene has length 38 kb, virtually no known limit to the size of the transgene that can be formed using larger and/or more overlapping fragments of the transgene. In particular, it is expected that this approach may be formed transgenes length of now, expected to be fruitful in creating larger transgenic animals such as transgenic cows of different breeds, with transgenes carrying the recombinant DNA, including genomic DNA, which in no other way can not be entered into the pronucleus with the formation of the transgenic animal. It is expected that such genomic transgenes will provide higher levels of expression in transgenic cows compared with transgenes encoding the recombinant cDNA.

When this recombinant polypeptide must secretariats, and in this case, "secretory DNA sequence encoding a signal peptide functional secretion must be operable linked to the transgene to direct the secretion of one or more cell types of transgenic animal. Secretory DNA sequence, usually originate from genes encoding secreted proteins of the same species of animal. It is recommended that such secretory DNA sequence was derived from genes encoding polypeptides that are excreted from the cell type, aimed at the tissue-specific expression, such as the secretion of milk proteins in the cells of the breast. Odaku used secretory DNA sequence of proteins secreted by other cell types within a species transgenic animal. For example, the native signal sequence homologous to the gene encoding a protein secretory optional in the mammary glands. In addition, can also be used "heterologous DNA sequence encoding a signal peptide secretion from species other than transgenic animals, such as human t-PA, human lacto albumin and signals the secretion of microbial genes encoding secreted polypeptides, for example, from yeast, filamentous fungi and bacteria. In General secretory DNA sequence may be functionally defined as any DNA sequence that is operable when the binding of recombinant DNA encodes a signal peptide capable of inducing the secretion of the recombinant polypeptide.

In one of the recommended embodiments of the invention secretory DNA sequence encoding a secretory signal sequence operable in the secretory cells of the mammary glands of cows, is used to trigger the secretion of the recombinant polypeptide in the cells of the mammary gland of the cow. Secretory DNA sequence effectively connected is posledovatelnosti, encoding the signal sequence of bovine aS1-casein, mouse lactoferrin and human transferrin. It featured secretory DNA sequence is a sequence encoding a secretory sequence aS1-casein cows of different breeds. The application of this secretory DNA sequence in more detail in the examples.

"Operable linked" in the context of binding secretory DNA sequences from recombinant DNA sequence means that the secretory DNA sequence comprising codons encoding the sequence of the secretory signal peptide) covalently linked to a recombinant DNA sequence so that the resulting secretory-recombinant DNA sequence in the direction 5'-3' region encodes the secretory signal sequence and the recombinant polypeptide. Accordingly, the frame readout for the secretory sequences and recombinant DNA sequence must be covalently linked in such a way that there is an open reading frame from the 5'-end of mRNA sequences formed after transcription and processing of the primary P is priori signal peptide, and part of the 3'-sequence that encodes a recombinant polypeptide. With this design is a recombinant polypeptide produced in the expression of secretory-recombinant DNA sequence is in a form capable of secretariats of target cells expressing a DNA sequence. The signal peptide, as a rule, is removed in vivo during secretion by producing extracellular forms of the recombinant polypeptide.

In the preferred embodiment of the invention secretory-recombinant DNA sequence is expressed predominantly in the secretory cells of the mammary glands of transgenic cows. Such tissue-specific expression is achieved by operable binding DNA sequences that regulate specific expression in the mammary glands, with the specified secretory-recombinant DNA sequence. Such DNA sequences that regulate specific expression in the mammary glands associated with the specified secretory-recombinant DNA sequence, include the above-mentioned regulatory sequences contained in the various genes cows, predominantly expressed in the secretory cells of the mo is Aina, K-casein, a-lactalbumin and b-lactoglobulin. Recommended sequence regulating the expression originating from a S1-casein, as discussed more fully stated in the examples.

Typically, the transgenes of the invention, designed for secretion of the recombinant polypeptide in the milk of transgenic cows that can cause such a secretion level, significantly higher compared with what was previously reported for transgenic mice and sheep. When encoding the recombinant polypeptide of recombinant DNA corresponding to or originating from cDNA, the molar concentration of the recombinant polypeptide preferably greater than 1 μm, more preferably higher than about 100 microns, and most preferably greater than 100 μm. If we look at the perspective level present in the transgenic milk recombinant polypeptide, the amount of recombinant polypeptide must be greater than 50 μg/ml, more preferably more than 500 μg/ml and most preferably more than 1000 µg/ml (1 mg/ml).

If the transgene of the invention encodes a recombinant polypeptide encoded by the recombinant DNA occurring or the corresponding genomic DNA (or consisting essentially of such ganong, encoding the recombinant polypeptide, originate from genomic sequences), in this case, the molar concentration and the protein content in the milk of transgenic cows are the same as for cDNA or higher. Usually the molar concentration of the recombinant polypeptide in such transgenic milk preferably greater than 50 microns, more preferably greater than 150 microns and most preferably above 500 microns. If you consider the protein content in transgenic milk, it preferably exceeds 10 mg/ml, more preferably greater than 2.5 mg/ml and most preferably greater than 5 mg/ml

The above molar concentration and the protein content in the milk of transgenic cows will vary depending on the molecular weight of the specific recombinant polypeptide. A particular advantage of producing a recombinant polypeptide from the milk of transgenic cows is that it can be produced polypeptides with a relatively large molecular weight, which can hardly be produced in large quantities in other systems, such as a prokaryotic expression system. Although, according to the invention, any of the recombinant polypeptide can be produced with molokovyksa about 10,000 daltons. However, other recombinant polypeptides with molecular masses above 15000, above 20000 and above 60000 daltons can be also expressed in milk of transgenic cows. For example, according to the invention, in the milk of transgenic cows can be easily produced human secrete lysozyme with a molecular mass of 17,000 daltons and lactoferrin with a molecular mass 79000 daltons. Thus, recombinant polypeptides of the invention are characterized by a wide range of molecular masses.

As a consequence of the above molecular concentrations of recombinant polypeptides is determined during the production of recombinant polypeptides of higher molecular weight. Such an installation is performed by translation of the molar concentrations in the amount of produced protein and the regulation of molar concentrations so that the content of the recombinant protein is covered by the following preferred concentrations.

A large part of the previous messages related to the production of transgenic milk, concerned transgenic mice. The mouse, however, usually produces 55-80 mg per ml of milk. The cow, on the other hand, typically produces 30-34 mg per ml Because excessively high level product is lichnogo protein and/or adversely affect the secretory mammary glands, it is recommended that the concentration of the recombinant polypeptide was 3-50% of the normal concentration of cow's milk protein (for example, 1-17 mg recombinant polypeptide per ml transgenic milk), more preferably 10-20% (e.g., 3-7 mg / ml and most preferably 10-15% (e.g., 3-5 mg / ml) from the normal amount of protein produced from cow's milk. Such preferred intervals determine the recommended maximum limit for the above level of protein produced from the milk of transgenic cows.

The above binding of different DNA sequences with the formation of the transgene of the invention carried out by standard methods known in the art, or as described here. After the design of the transgene or overlapping fragments encoding the transgene, they are used to create transgenic animals.

Methods of introducing transgenes or overlapping fragments of the transgene in embryonic target cells include microinjection of the transgene into the pronucleus of fertilized oocytes or kernel ES-cells of the animal. Such methods are well known in the art for mice. Or transgene, may be in komendealii way is to microinjection of fertilized oocytes. In the proposed method, the fertilized oocytes initially microinjection standard methods. After that they are grown in vitro until a "pre-implantation embryo". It is recommended that such preimplantation embryo contained 16-150 cells. The stage of the embryo in the 16-32 cell is usually called the morula. Preimplantation embryos, containing more than 32 cells, usually referred to as a blastocyst. Blastocyst generally characterized as demonstrating the creation of blastocele cavity, usually at the stage in 64 cells. Processes for the production of fertilized oocytes to the preimplantation stage include different methods [Gordon et al. 1984, Methods in Enzymology, 101, 414; Hogan et al. 1986, in Manipulating the Mouse Embryo, Cold Spring Harbor, N. Y. (mouse embryo); Hammer et al. 1985, Nature, 315. 680 (for embryos rabbit and pig); Gandolfi et al. 1987, J. Reprod. Pert. 81, 23-28; Rexroad et al. 1988, J. Anim. Sci, 66, 947-953 (for sheep embryo); Eyestone, W. h. et al. 1989, J. Reprod. Pert. 85, 715-720; Camous et al. 1984, J. Reprod. Pert. 72, 779-785; Heyman Y. et al. 1987, Theriogenology, 27, 5968 (for embryo cow)] Such pre-implantation embryo is then transplanted corresponding female standard methods, which gives rise to a transgenic or chimeric animal depending on the stage of development at the time of introduction of the transgene. As is well known, can>As the rate of introduction of the transgene is often low, it is highly desirable detection of integration of the transgene in the preimplantation embryo. The application discloses methods of identifying embryos that happened transgenesis and implantation which leads to the creation of transgenic animals. In this method of preimplantation embryo delete one or more cells. When using equal division, it is recommended not to grow the embryo after the morula stage (32 cells). The division of the preimplantation embryo (Williams et al. 1986, Theriogenology, 22, 521-531) leads to two "polyembryony" (palmarola or Polyplastic), one of which is capable of subsequent development after implantation of the corresponding female development in utero. Although it is recommended that an equal division of the preimplantation embryo, it is necessary to specify that the embryo intentionally or unintentionally can be divided into unequal parts on two polyembryony, optional composed of an equal number of cells. All that is necessary is that one of the embryos not subject to analysis in the following way, had a sufficient number of cells for normal development in utero. Not subjected to analysis polyembryony, which is shown trance the ions, formed by dividing the preimplantation embryo, analyze to determine, whether integrated the transgene into the genome of the organism. Every other polyembryony make up for the subsequent implantation of the female recipient of this species. The recommended method of detection of transgenesis at this early stage of embryo development using the received polyembryony in combination with the unique feature of the restriction enzyme Dpn I. This enzyme recognizes the sequence GATC in double DNA, but only when the adenine in each circuit within the specified sequence are methylated at N-6. When using this method, the transgene containing the sequence GATC, was identified in before microinjection by transfer of the transgene on the corresponding plasmid with the participation DAM+the strain of microorganism, such as E. coli MM 294, or direct methylation of the transgene dam by metrazol. Methylated transgene (preferably without any exogenous sequences, such as a plasmid vector) then microinjected in fertilized oocytes (approximately 10-500 copies on the pronucleus, more preferably 50-100 copies on the pronucleus). The resulting fertilized oocytes expressed platinums. Accordingly, those copies of methylated transgene integrated into the genome of the fertilized oocyte, after replication become neetilirovannyj, while any reintegrirovat transgenes that can still exist after replication, remain methylated (Lacks S. et al. 1977, J. Mol. Biol. 114, 153). Such differentiated nature of the methylation of integrated compared to neintegriruemykh the transgenes allows you to identify which of fertilized oocytes has transgene integrated into the genome.

Identification of the preimplantation embryo, containing the integrated transgene is achieved by analysis of DNA from each polyembryony. This DNA is usually obtained by lysis of polyembryony and analysis are identified in the DNA (Ninomiy T. et al. 1989, Molecular Reproduction and Development, 1, 242-248). Each DNA sample is treated with enzyme Dpn I. then conduct the polymerization reaction chain using polymerase (Saiki et al. 1985, Science, 230, 1350-1354) with the amplification of all or part of the transgene. When the full amplification of the transgene amplification using two primers elongation, each complementary to opposite strands at different ends of the transgene. However, if amplifier the site in the transgene. If removal of Dpn I not occurred, PCR amplification leads to amplified sequences of a given size, while the primer elongation for those of transgenes that were not split does not result in exponential amplification. As a rule, Dpn I/PCR amplified DNA from polyembryony subjected to electrophoresis followed by hybridization with a labeled probe complementary to a region of the transgene between the two primers elongation. This facilitates determination of the amount of the amplified DNA sequences, if any, and indicates whether the transgene is integrated in the preimplantation embryo, from which polyembryony (hereinafter referred to as "transgenic polyembryony"). In the case of integration of the transgene remaining untreated transgenic polyembryony transplanted parent-recipient. After development in utero transgenic animal with the target phenotype, which is attached to the integrated transgene, identify the appropriate method in utro or after birth. Of course, in the above method can be used and other restriction enzyme, is able to cleave methylated DNA sequences, but is unable to cleave the non-is of Buet presence in interest transgene sequence GATC. In the case when such a sequence is missing, it can be easily introduced in the transgene, site-directed mutagenesis (Kunkel, T. A. 1985, Proc. Nail. Acad. Sci. 82, 488) or cassette mutagenesis (Wells, J. A. et al. 1985, Gene, 34, 315), provided that this mutation does not alter the amino acid sequence encoded by the transgene (and does not cause undue changes in amino acid sequence) and that any resulting codons operate in the interest of the transgenic animal.

The above methods of detection of transgenesis in preimplantation embryos represent a cost-effective and efficient methods of creating transgenic animals, because their use reduces the number of pregnancies that are required for producing transgenic animal, and significantly increases the likelihood that the implanted embryo will lead to the birth of the transgenic animal. Such techniques are especially important for those animals that have a low frequency of transgenesis, for example for cows of different breeds.

In an alternative embodiment of the invention the above method of detection of transgenesis in preimplantation embryos combined with the stages of AMB shall be implanted females-recipients with the production of a population of clones of transgenic animals with the same genotype. In this connection it is necessary to indicate that transgenic embryos and/or transgenic animals with the same genotype" means essentially the identity of the genomic DNA of individual embryos in a population or of individuals in animal populations. However, it should be noted that during mitosis can occur in a variety of mutations, which can occur variations in the genotype of one or more cells and/or animals. Thus, in a population with the same genotype can show the individual or subpopulation variations.

After identification of polyembryony as transgenic embryo, his clone. Embryonic cloning can be done in various ways. In one of the methods cloning transgenic polyembryony grown in the same or the same environment as used for the cultivation of individual oocytes to preimplantation stage. Educated in the "transgenic embryo" (preferably the transgenic morula) then divide by "transgenic polyembryony", which are then implanted the female recipient with the formation of a population of two clones of transgenic animals. Or both of the received polyembryony m is the Rion. This procedure is repeated until then, until the desired number of clones of transgenic embryos with the same genotype. Transgenic embryos can then be implanted females-recipients with the production of a population of clones of transgenic animals.

In the recommended method of cloning transgenic embryo clone nuclear transfer (Prather et al. 1988, Biol. Reprod. 37, 59 86; Roble et al. 1987, J. Anim. Sci. 64. 642-664). According to this method, the kernel of transgenic embryos transplanted in enucleation oocytes, each of which then grow to the blastocyst stage. At this point transgenic embryos may be subjected to a new round of cloning by nuclear transplantation or can be transferred to the parent-recipient for producing transgenic offspring with the same genotype.

In addition to the above methods of early detection of transgenesis, for detection of transgenesis can be used and other methods. Such methods include in utero and post oartum analysis of tissue. Analysis of in utero carried out by several methods. In one of them spend transvaginal puncture of a cavity of the amnion under ultrasound control (Bowgso et al. 1975, Bet. Res. 96, 124-127; Rumsey et al. Nem pregnancy. In the specified volume of the amniotic fluid contains 1000 12000 cells per ml originating from the urogenital tract, skin, and possibly the lungs of the developing embryo. Most of these cells are dead. However, these cells contain genomic DNA, which is subjected to PCR analysis for the transgene as an indicator of successful transgenesis. Or fetal cells can be extracted Kurinnoy puncture. This method can be carried out transvaginally and under the control of the sonogram. In this method, to puncture the placenta animal-recipient used a needle, in particular, for puncturing placentally structures, fixed relative to the walls of the vagina. For cows, this selection of the sample can be carried out within 60 days of pregnancy. The cells of the chorion, if necessary, separated from the maternal tissue and subjected to PCR analysis for the transgene, as an indicator of successful transgenesis.

Transgenesis can be detected after birth. In such cases, the integration of the transgene can be detected by biopsy acceptable tissue, for example, from the ear or tail may transgenic animal. Select one to two inches of the tail, or five to ten square millimeters ear followed the southern batirovna with tenantry polypeptide is expressed and secreted into the milk of transgenic cows, the resulting transgenic milk can be used as such or can be subjected to additional processing for purification of recombinant polypeptide. This partly depends on contained in the transgenic milk recombinant polypeptide and the final application of the protein. So, if the recombinant polypeptide is secreted in transgenic milk to improve the nutritional value of cow's milk, no additional purification, as a rule, is not required. An example of this situation can serve as an example, when cow's milk is produced for human lactoferrin as an additive to control infections in the intestines of a newborn human and to improve the absorption of iron. In other situations, it may be desirable partial purification of emitting a specific polypeptide because of its nutritional value. So, for example, human lactoferrin obtained in the milk of transgenic cows, may be partially purified by acidification of milk to pH 4-5 with precipitation of Caseins. Soluble fraction (whey) contains human lactoferrin, which is partially cleared.

Recombinant polypeptide found in the milk of transgenic cows, can also be used in the one or more recombinant polypeptides of the transgenic cow's milk, which one or more nutrients, or have other valuable properties. For example, baby food containing human lactoferrin, has a bacteriostatic action, which helps in the fight against diarrhea in infants. Similarly, recombinant polypeptides, such as human casein and human secrete lysozyme, can be also formed in transgenic cow's milk to give it nutritional value. In table. 2 shows the components of a conventional baby food. As shown in the table, the protein content varies from 1.8 to 4.5 g per 100 kcal diet. Thus, the complete protein, including recombinant polypeptide should be covered values, based on existing U.S. requirements (see tab. 2). The number of complete protein, including recombinant polypeptide may differ from the above values depending on local facilities where specific power will be used.

In addition to baby food recombinant polypeptide from the transgenic cow's milk can be added to other food compositions. Such recombinant polypeptides can be used as additives to conventional diet compositions.

If recombi the cleaning responding to such use. Such cleaning methods depend on the particular subjected to the purification of the recombinant polypeptide, and they are well known in the art. Methods include partial purification by fractionation of casein followed by chromatography of the appropriate fractions containing the recombinant polypeptide. Chromatographic methods include affinity chromatography, ion exchange chromatography, gelfiltration and HPLC.

In the description presents the transgene for producing human lactoferrin (CLF) in the milk of transgenic cows. Human lactoferrin is a single-chain glycoprotein that binds two iron ion (II). The secretory exocrine glands (Mason et al. 1978, J. Clin. Path. 31, 316-327; Tenovuo et al. 1986, Infect. Immun. 51, 49-53) and polymorphisim neutrophile granulocytes (Mason et al. 1969, J. Exp. Med. 130, 643-658), this protein functions as part of the nonspecific defense system of the host by inhibiting the growth of a broad spectrum of bacteria. CLF exerts bacteriostatic action by the formation of chelates with the existing environment, iron, making this important metal is not available for aggressive microorganisms (Bullen et al. 1972, Br.Med. J. 1, 69-75; Griffiths et al. 1977, Infect. Immun. 15, 396-401; Spik et al. 1978, researchers suggest, what CLF has a direct bactericidal effect (Arnold et al. 1980, Infect. Immun. 28, 893-898; Arnold et al. 1977, Science, 197. 263-265; Arnold et al. 1981, Infect. Immun. 32, 655-660; Arnold et al. 1982, Infect. Immun. 35, 792-797; Bortner et al. 1986, Infect. Immun. 51, 373-377). Bactericidal activity is also inhibited by saturating protein iron. No mechanism of bactericidal action CLF not been postulated, although it was shown that protein can damage the outer membrane and alter the permeability of the outer membrane of gram-negative bacteria (Ellison et al. 1988, Infect. Immun. 56, 2774-2781).

Lactoferrin is the major iron binding protein in the milk man (present at a concentration of 1.5-1.7 mg/ml) and may take part in the adsorption of iron the small intestine. All present in breast milk iron is thought to be due to CLF and absorbed with very high efficiency (Hide D. W. et al. 1981, Arch. Dis. Child. 56, 172). It was postulated that a high intake associated with CLF iron due to the presence of receptors in the small intestine. Data were present, which gives reason to assume the existence of these receptors in monkeys of resursov (Cox et al. 1979, Biochem. Biophys. Acta, 588, 120; Davidson L. A. et al. 1985, Fed. Proc. 18, 901). There is also evidence in favor of the specific lactoferrin receptors in the cells of clisis the ü on intestinal flora (Mevissen-Verhage et al. 1985, Eur. J. Clin. Environ. 4, 14). In faecal samples of breast-fed infants compared with infants fed cow's milk with the addition and without iron supplementation, as shown, is substantially reduced content of coliform, increased content of bifidobacteria and clostridia. In vitro studies of milk man showed his specific inhibitory effect on E. coli (Brock et al. 1983, Infect, and Immunit. 40, 453). Milk man also shows specific inhibitory effect on E. coli in the small intestine due to the high content of iron binding protein, mainly CLF (Bullen et al. 1972, British Med. J. i, 69).

Thus, the production of human lactoferrin in the milk of transgenic cows can serve as a source of human lactoferrin. Such lactoferrin can be isolated from transgenic milk for the preparation of the compositions. Or transgenic milk can be used in whole, preferably after pasteurization in liquid or dry form. In addition, the beneficial effect of human lactoferrin can be enhanced by mixing human lactoferrin or its containing transgenic milk with human secrete lysozyme. Human secrete lysozyme may be one is by creating transgenic cows, capable of producing more than one recombinant polypeptide in the transgenic milk. Or transgenes can be introduced cow series. If this is the case, create a transgenic cow with one of the transgenes. Then from transgenic females get embryonic cells, such as eggs and process with the introduction of a second transgene encoding a second polypeptide. Eggs are fertilized with the subsequent microinjection pronucleuses the resulting zygote. It must be emphasized that the above combination of more than two polypeptides in transgenic cow's milk is not limited to the above combination of human lactoferrin and lysozyme. Thus, the establishment of transgenic cows of different breeds and obtaining transgenic milk, and in transgenic animals transgenic milk is produced by more than one recombinant polypeptide.

Determined the complete amino acid sequence CLF (Metz-Boutiqye et al. 1984, Eur. J. Biochem. 1451. 659-676). CLF includes two domains, each of which has a binding site and N-linked glycosylation site. These domains are homologically in relation to each other, which indicates that hereditary dup is transferring (Metz-Boutique, supra, Pentecost et al. 1987, J. Biol. Chem. 262, 10134-10139). The location of amino acids in the binding sites of iron, determined by x-ray crystallography (Anderson et al. 1987, Proc. Natl. Acad. Sci. 84, 1769-1773). A partial cDNA sequence for neutrophilic CLF published Rado et al. 1987, Blood 70. 989 993. There is more than 98% correspondence between the amino acid sequence, derived on the basis of the cDNA, and the fact that it was determined by direct analysis of lactoferrin from milk man. Recently published data on the structure of the saturated iron lactoferrin and free from iron lactoferrin (Anderson et al. 1989, J. Mol. Biol. 209, 711-734: Anderson et al. 1990, Nature, 784 787).

As used here, the value of human lactoferrin" means a polypeptide with the amino acid sequence essentially the one given in the work of Metz-Boutique et al. 1984, Eur. J. Biochem. 1451, 659-676 and shown in Fig. 8-9. Note, however, that the previously determined partial amino acid sequence of human lactoferrin reveals a number of discrepancies between the published sequence and what is received. Namely, there are the following inconsistencies (number of amino acids corresponds to the sequence of Fig. 1-7, and in brackets the position of the DNA, CX sequences or recombinant variants of human lactoferrin, in which one or more amino acids are modified by substitution, insertion or deletion of one or more amino acid residues.

In applied here in the sense "DNA sequence of human lactoferrin" refers to a DNA sequence encoding human lactoferrin in the above value. Such DNA sequence of human lactoferrin can be obtained from a cDNA library human mammary gland or may come from the human genome. Example 2 describes the cloning and nucleotide sequence of human lactoferrin derived from cDNA library human mammary gland. The DNA sequence of human lactoferrin is shown in Fig. 1-9 and essentially corresponds to the sequence given in the work of Rado et al. 1987, Blood 70. 989 993. Construction of plasmids containing is able to be expressed the transgene encoding CLF described in the examples. One of these plasmids (cGP 1 HLF, sometimes also called 16,8 HLF 3) contains a transgene designed for tissue-specific expression in the secretory cells of the mammary gland of the cow.

In addition, in the application described the transgene to obtain chelovecheskom protein, contains 584 amino acid residue (Minghetti et al. 1986, J. Biol. Chem. 261, 6747). This is the most common protein of human serum, which performs two very important physiological functions. Serum albumin is responsible for about 80% of the total osmoticnosti blood, and in addition, carries fatty acids between the fat tissues.

Human serum albumin is used primarily to increase plasma volume by restoring osmotic pressure in the cardiovascular system. Currently, the fraction of CSA obtained by heat treatment of whey, pour in the majority of the victims of shock or injury, including the majority of patients subjected to complex surgical operations. Currently CSA derived from human blood plasma as a by-product of the fractionation of blood to obtain a rare blood proteins, such as factors VIII and IX.

In applied here in the sense of "human serum albumin" means the amino acid sequence matches that listed in the work Minghetti et al. ibid; Lawn et al. 1981, Nucl. Acids Res. 9, 6103. This concept also covers its variations, including recombinant variations of human serum Alba is or more amino acid residues (Minghetti et al. 1986, J. Biol. ChEm. 261. 6747-6757). In some cases, human serum albumin can be obtained from milk by expression of the transgene containing DNA encoding the secretory signal sequence CSA. Or human serum albumin can be produced and secreted by the liver cells of the transgenic animal with a heterologous transgene, including human genomic DNA encoding a 5'-sequence expression regulation, signal secretion of human serum albumin and the structural gene and the 3'sequence expression regulation. As shown in the examples containing such heterologous sequence of the transgene form of homologous recombination in vivo overlapping fragments of the transgene with the restoration of the gene CSA in the transgenic animal. Created by such transgenic animal producing human serum albumin in your cardiovascular system.

In applied here in the sense "DNA sequence of human serum albumin" means a DNA sequence encoding human serum albumin in the above definition. This DNA sequence che is 3244-3251 and Urano et al. 1984, Gene, 32, 255-261 and here in the examples.

The DNA sequence of the clone by the method of example 10, and then treated with substitution in the gene of human lactoferrin, plasmid encoded cGP 1 HLF (also called p 16, 8 HLF 4). From this plasmid receive a transgene containing the 5'sequence of the regulation of gene expression of bovine aS1-casein in 16 kb DNA sequence of human serum albumin and flanking the 3'region of the gene bovine aS1-casein in 8 kb. This transgene is used to microinjection in fertilized oocytes cows of different breeds. After early detection of transgenesis blastocyte containing the transgene of CSA, implanted females-recipients and wait for delivery.

Example 1. The design of the probe, specific for sequences of bovine aS1-casein

A. Selection of chromosomal DNA

Placenta tissue are obtained from slaughterhouses. The surrounding connective tissue were removed and pieces about 30 g quickly frozen in liquid N2. Chromosomal DNA allocate as follows. Fabric (30 g) homogenized (on ice) with 35 ml of buffer 1 containing 300 mm sucrose, 60 mm KCl, 15 mm NaCl, 60 mm Tris-HCl (pH 8,2), 0.5 mm spermidine, 0.15 mm of spermine, 2 mm add, 0.5 mm EGTC. EXT is by 5 min at 3000 x g precipitate rinsed with buffer 1, containing 1% NP 40. After the redo phase centrifugation the precipitate is again suspended in 5 ml of buffer 1. Quickly add 5 ml of 0.5 M etc. The final volume is 15 ml Add 0.15 ml of a 10% aqueous solution of VAT. After stirring the PH kazoo and T1 to a final concentration of 0.4 mg/ml and 6 u/ml, respectively. After incubation for 3 h at 37oC add proteinase K to a final concentration of 0.1 mg/ml of the resulting mixture is incubated for 15 h at 37oC. the mixture is Then carefully extracted with phenol. The aqueous phase is separated and to it was added 1/30 volume of 3 M NaOAc (pH 5.2) and one volume of isopropyl alcohol. The precipitate (DNA), rinsed with 70% ethanol and slowly dissolve in the 40oC in 0.5 ml of 10 mm Tris-HCl (pH 8), 1 mm etc, 4oC.

B. Amplification of sequences from the 5'-flanking region of the gene aS1-casein

Based on the sequence published in the work of Yu-Lee et al. 1986, Nucl. Acids Res. 14, 1883-1902, synthesized two DNA primers. Primer 1 is located at position 681 relative to the primary site of transcription initiation and has the following sequence:

5'-TCC ATG GGG GTC ACA AAG AAC TGG AC-3' (Seg. ID NO. 5)

Primer 2 is located at position +164 relative to the primary site of transcription initiation and has the following consistently contain a Hind III restriction site to facilitate subsequent cloning. The resulting primers hybridized with chromosomal DNA and lengthened in the presence of deoxynucleotides using TAG polymerase. After 3 min the mixture was denatured for one minute at 92oC, again hybridized 1.5 min at 50oC and again incubated for 2 min at elevated temperature (68oC). This cycle is repeated 30 times. After the last cycle, DNA tested for the presence of the expected EcoRI sites. And the fragment size, and the presence of the EcoRI site you expected. Then the fragment is treated with enzyme maple for repair excess ends of the handle kinase to attach phosphate groups to the ends of the fragment, incubated 10 min at 65oC decontamination kinases and enzymes maple, and finally hydrolyzing Hind III. Then the fragment subcloning pUC 19 (Yanisch-Perron et al. 1985, Gene, 33, 103 109), hydrolyzing with Smal and Hind III. The formal proof of the identity of the received fragment obtained by sequencing parts of this subclone (after re-cloning in M 13 vector). Specific sequence was identical to the published sequence. This probe was then used for screening of the genomic library cows with the aim of obtaining clones specific for sequences flanking the 5'region of the gene is riment approach similar to the above. Based on the sequence published in the work of Stewart et al. 1984, Nucl. Acids Res. 12, 3895-3907, synthesize two primers. the 5'primer is located immediately downstream of the coding sequence beginning at position 713 cDNA sequences. Primer has the following sequence:

5'- GAG GGA CTC CAC AGT TAT GG-3' (Seq. ID No. 7).

The other primer is located at 1070 cDNA sequence and has the following sequence:

5'- GCA CAC AAT TAT TTG ATA TG-3' (Seq. N. 8).

The resulting primers hybridizing with the chromosomal DNA and the region between the two primers is amplified by the above method. The resulting fragment was about 900 p. O. longer than expected. The sequence analysis showed that between nucleotides 737 and 738 cDNA is present in the intron of the specified size. Amplificatory fragment is treated with a polymerase maple with reparation excess ends and handle kinase attach to the ends of the fragment phosphate groups. Then the fragment is inserted into the pUC 19, pretreated with Smal.

D. Selection ragovoy library cows for Flanking sequences aS1-casein

Bovine genomic atom. The titer of the bacteriophage particles identified in Escherichia coli 406 MB permissive strain-host (Stratagene Inc.). To do this, prepare several dilutions of phage in SM buffer (50 mm Tris-HCl with pH 7.5, 100 mm NaCl, 10 mm MgSO4of 0.01% gelatin) and mixed with 200 µl MB 406 (O. D.5500,9); after 20 min at 37oC cover 3 ml of agarose (environment Luria - Bertani, 0,8% agarose), a mixture of cover LB-plates and incubated for about days at 37oC.

Approximately 600,000 phage were then added to 400 ml MB 406. The next step is to transfer the phage on nitrocellulose filters. Plates incubated 1 h at 4oC. Nitrocellulose filters (S&S) placed on the top layer of agarose and mark the exact position. After lifting the filters are soaked in a period of (1) 30 min in denaturing buffer (1.5 M NaCl, 0.5 M NaOH), (2) 5 min in neutralization buffer (1.5 M NaCl, 0.5 M Tris-HCl pH 8). After rinsing 2xSSPE (360 mm NaCl, 20 mm NaH2PO42 mm etc) filters is sintered for 2 hours under vacuum at 80oC.

Prehybridization filters is carried out in buffer containing 50% formamide, 5x denhardt's solution (0.1% ficoll, 0.1% polyvinylpyrrolidone, 0.1% of bovine serum albumin), 5xSSPE, 0,1% VAT and 100 μg/ml DNA denatured salmon sperm for 2 h at 42oC. Hybridization assisted mark with a set of unordered tagging primer firm Boehringer Mannheim. After hybridization for about days the filters at room temperature three times washed with 2xSSC, 0,1% VAT.

Film Kodak XAR irradiated approximately day 70oC amplificating screens (Dupont). The assumed positive samples are removed from the plates and placed about a day in SM buffer at 4oC. the resulting product is applied on the plate as described above and DNA allocate according to the method of obtaining a lysate of the plate (Maniatis T. et al. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, N. Y.). To the top layer of agarose add 5 ml of SM buffer, after 2 hours of careful shaking the buffer is removed and centrifuged 10 min at 4000 rpm and 4oC. the Supernatant is transferred into a sterile tube, add Mkasa and Tenkasu (both to a final concentration of 1 μg/ml) and all is incubated for 30 min at 37oC. Add one volume of 20% peg, 2.5 M NaCl solution and incubated for 1 h on ice. By centrifugation for 30 min at 4oC and 4000 rpm receive precipitated particles of the bacteriophage. Particles suspended in 500 ml of SM buffer, add VAT (final concentration 0.1% ) and add (final concentration 5 mm) and all is incubated for 15 min at 68oC. Protein extract once by extraction with phenol and once with chloroform. OS and dissolved in 50 ml of buffer Tris-HCl (pH 7.5), 1 mm etc.

Restriction permantely analysis, electrophoresis in agarose gel, the DNA transfer from the gel to nitrocellulose filter and southern botirovna carried out by standard methods (Maniatis et al. 1982, Molecular Cloning: A Laboratory Manual). Hybridization with probes (see below) carried out by the same method that the above criteria.

E. Selection of clones containing flanking the 5'-region of the cow S1-casein

Three alleged clone identified by use of the probe and of the methods above. After one cycle of selection analyze pure recombinant bacteriophage. Hydrolysis of cloned DNA SalI. EcoRI and SalI/EcoRI double digestion and hybridization with the above-described probe showed the presence of identical inserts in all three clones. The insert size of 18 kb (partial Sau3A fragment SalI cut). The transcriptional orientation of the clone defined by the above hybridization of restriction fragments with the above-described probe 1 and Nco I-NsiIII fragment of the probe 1. In the detected area is about 16 kb upstream from the start of transcription. In the downstream direction from the start of transcription was another 1.9 K. p. O. Additional sequencing region -103 +300 confirmed the identity of the clone of the existing analysis of the clone with the following enzymes: NcoI, > PST, kpni restriction sites, BamHI, HindIII, BglII) allowed to obtain the map of restriction flanking the 5'region of the gene the cow Si-casein, shown in Fig.10.

F. Selection of clones containing flanking the 3'region of the bovine aS1-casein

Using previously described methods hybridization using probe 3' aS1-casein selected duplicate nitrocellulose filters with the initial stage of application of the bacteriophage used to highlight the 5'-clones. After two cycles of selection identified 8 positive clones. Phage DNA obtained above method. Subsequent hydrolysis of restrictase SaiI, EcoRI and SalI/EcoRI and hybridization on Southern 3' aS1-probe showed the identity of the inserts in seven of the eight clones. One clone containing the EcoRI insert 18.5 kb, subjected to further analysis using the restriction enzymes BsteII and BamHI. Map of restriction analysis of this clone is shown in Fig.11.

Example 2. Cloning of human lactoferrin

A. Materials

The restriction enzyme, T4 ligase and T7 polynucleotide kinase obtained from the company Boehringer-Mannheim, New England or Biolabs or Bethesda Research Laboratories. Radioisotopes are set by the company Amersham. cDNA library human mammary gland in bacteriophage l-gTll obtained from the company Clontech, PCA selected standard method, plaque hybridization (Maniatis et al. 1982, Molecular Cloning: A Laboratory Manual) using three synthetic oligomers. Two of the oligomer are 30-measures corresponding to the cDNA sequence for Rado et al. supra, the amino acids with the provisions 436 445 and 682 691. The third oligomer is a 21-dimensional probe "best guess" based on the degeneracy of the codon and encodes the amino acid sequence CLF between amino acid residues 18 and 24. Accordingly, the oligomers have the following sequence:

(1) 5'- CTTGCTGTGGCGGTGGTTAGGAGATCAGAC-3' (Seg. ID N: 9)

(2) 5'- CTCCTGGAAGCCTGTGAATTCCTCAGGAAG-3' (Seg. ID N: 10)

(3) 5'- ACCAAGTGCTTCCAGTGGCAG-3' (Seg. ID N: 11).

Radiolabelled probes (Crouse et al. 1983, Methods Enzymol. 101. 78-98) used for selection of aftermarket filters. The filters are washed at a final concentration of 2 X SSC, 37oC.

C. analysis of the nucleotide sequence of

DNA fragments allocate using low-melting agarose (Crouse et al. supra) and subcloning in bacteriophage M 13 mp 18 or M 13 mp 19 (Messing et al. 1983, Methods Enzymol. 101, 20-78). The sequence is determined using enzyme Sequenase (modified T7 DNA polymerase) (Tabor et al. 1987, Proc. Natl. Acad. Sci. USA, 84, 4767-4771). All reactions are carried out in accordance with the manufacturer's instructions (US Biochemicals). The sequence prevedello) and subcloning in HindIII and EcoRI site of plasmid pUC 19 with the formation of pUS 119 Lacto 4,1. This clone contains the complete coding sequence of the Mature form CLF, but does not have full signal sequence.

Example 3. The design CAT vectors of bovine aS1-casein

To determine whether obtained in example 1, fragments of aS1-casein promoter and other components necessary for the expression of heterologous gene construct expression plasmids containing various amounts of flanking 5'- and 3'regions of the gene aS1-casein. In the data vector constructs as a heterologous gene used gene chloramphenicolchloramphenicol (CAT). CAT gene can be used to determine the level of expression of a construct containing a heterologous gene, since it is usually not present in mammalian cells and makes them easily identifiable fermenting activity (Corman et al. 1983, Mol. Cell. Biol. 2, 1044-1051), which can be quantified in cells or in an animal, containing the expressed gene.

A. DNA sequences

From flanking genomic 5'-clone of example 1 PCR amplification is isolated in the form of a Nco I-HindIII fragment (about 830 p. O.). aS1-casein promoter size 681 p. O. plus the first non-coding exon, plus the first terminating the placenta is barb primers consist of:

5'-TCCATGGGGGTCACAAAGAACTGGAC-3' (Seg. ID N: 12) and

5' TGAAGCTTGCTAACAGTATATCATAGG-3' (Seg. ID N: 13),

what made the conclusion based on the sequence published by Yu-Lee et al. 1986, Nuc. Acids Res. 14, 1883 1902.

From cow flanking genomic 3'- clone of example 1 PCR amplification allocated flanking the 3'sequence aS1-casein approximately 1.6 kb (fragment 2, Fig. 12). This area contains the previously described signal splicing within noncoding 3'region of the gene aS1-casein. Fragment 2 subcloned in the SmaI site of the plasmid pUC19.

Sequences of the primers consist of:

5'-GAGGGACTCCACAGTTATGG-3' (Seg ID N: 14)

5'-GCACACAATTATTTGATATG-3' (Seg. ID N: 15),

what made the conclusion based on the sequence published by Stewart et al. 1984, Nucl. Acids Res. 12, 3895 3907.

To obtain pMH-I synthesized and introduced into pUC 18 with a unique restriction sites flanking both sides of the plasmid, hybrid splicing signal, containing the 3' splice site of the immunoglobulin gene (Bothwell et al. 1981, Cell 24, 625-637). This plasmid is shown in Fig.15. Sites NcoI and HindIII created such that ligation of fragment 1 of bullish 5' genomic clone leads to functional hybrid splice sequence (Fig.25).

The sequence polyadenylate Acad. Sci, 19, 6777-6781).

Bacterial sequence encoding a CAT, subcloned in pUC19 as > PST -BamHI fragment.

B. Design of pSl 3'5'CAT

Fragment 1aS1-casein promoter subcloned in pMH-1 (Fig.15) between sites NcoI and HindIII education pMHSl 5'flank.

The sequence SV 40 polyadenylation (fragment 3) subcloning in the form of a BamHI-DraI fragment of pUC 19 immediately flanking the aS1 - casein 3'sequence (fragment 2) with the formation of pUC19 3' UTR/SV 40. This allowed to remove continuous EcoRI-SalI fragment (containing flanking the 3'sequence and poly(A) sequence), which was subcloned in pMH-1 with the formation of pMHSl3'UTR (Fig.13), used later for constructing pMHSI 3'UTR hlf containing encoding human lactoferrin sequence.

EcoRI-SalI sequence (fragments 2 and 3) subcloned in the EcoRI-SalI sites pMHS15'flank with the formation of pSl3'5'flank.

> PST -BamHI CAT fragment (fragment 4 in Fig.13) after dephosphorylation BamHI site of the enzyme maple subcloning in pSl 3'5'flank (Fig.13) between sites > PST and SmaI education pSl3'5'CAT.

C. Design pSl5'CAT

The CAT fragment (fragment 4 in Fig.13, PST -BamHI) and SV40 fragment polyadenylation (fragm dotirovanie CAT

Each of the resulting CAT-plasmids transfection in 293 cells S man (Graham F. L. et al. 1977, J. Gen. Virol. 36, 59-72) by the method of coprecipitation with calcium phosphate (Gorman, C. M. et al. 1983, Science 221. 551; Graham F. L. et al. 1973, Virology, 52, 456-467). Cells are harvested after 44 h after transfection and analyzed for CAT activity (Gorman, C. M. et al. 1982, Mol. Cell. Biol. 2, 1011; deCrombrugghe, B. et al. 1973, Nature [London] 241, 237-251 modification Nordeen, S. K. et al. 1987, DNA 6, 173-178). A control plasmid expressing the CAT under the action of an instant early promoter of cytomegalovirus (Boshart, M. et al. 1985, Cell 41, 521), transfection in 293 S human cells for analysis of efficiency of transfection.

In these cells Express pSl3'5'CAT at the level of about 30 to 100 times lower than for the control plasmids, but sufficiently above background. Analysis using primer extension pointed to the initiation of transcription in the expected region.

When transfection pSl5'CAT in 293S cells are also found expression.

Example 4. Bovine aS1-casein/human lactoferrin expression cosmid cGP1HLF

A. Construction of DNA sequences

Bovine aS1-casein flanking the 5'-sequence in 16 kb isolated from a bovine genomic library (phage GP1) in the form of a SalI-BglII fragment. BglII site lies at the junction of the first is tewart et al. 1984, Nucl. Acids Res. 12, 3895) obtained from synthetic DNA, synthesized in the synthesizer Cylone Plus(Millgen/Biosearch I), and it contains the complete signal sequence plus Xhol and Cla I sites, attached to the 5'-end, and Nael to the 3'- end (fragment 8, Fig.16).

Splitting pUC119 Lacto 4.1 enzyme EaeI reveals plasmid exactly the codon for the first amino acid of Mature CLF. Processing enzyme maple used to fill in the excess 5'-end. Further hydrolysis enzymes AccI and EcoRI gives two fragments: (a) EaeI AccI fragment containing the first 243 p. O. Mature CLF (fragment 5, Fig.18), and (B) related AccI EcoRI fragment (fragment 6, Fig. 18) the size of 1815 p. O. containing the rest of the coding sequence, in addition to the five terminal codons.

The obtained synthetic linker containing the last five codons CLF starting at the EcoRI site and leaving on four grounds for a stop codon. To 3'-end of the kpni restriction sites added to the site (fragment 7 in Fig. 18).

From a genomic library cows selected EcoRI 3'-fragment of 8.5 kb (Fig. 11) containing the sequence, starting immediately downstream of the region encoding S1-casein and BstEII site in about 350 p. O. from the 5'-end. This fragment subcloned in pMH-1 EcoRI sites pMH3'UTR (Fig. 16) subcloning CLF 3'-linker (fragment 7, Fig.18) with the formation of pMH3'UTRhLF2linker (Fig. 16).

Then the synthetic signal sequence of bovine aS1-casein (fragment 3) subcloning in XhoI and SmaI sites pMH3'UTRhLF2linker education pSl 3'hLFl/2L (Fig.17).

Two coding CLF fragment (fragments 5 and 6 in Fig.18) subcloning in the NaeI and EcoRI sites pSl3'hLFl/2L (Fig.17) with the formation of pSl3'UTRhLF (Fig. 18).

From pMH 3'E 10 emit large 3'UTR fragment of a S 1-casein (Fig.16) in the form of BstEII-SalI fragment and subcloning in the same sites pSl 3'UTRhLF education phLF3'10 kb (Fig.19).

Kosmidou cGP1HLF receive as a result of ligation of the fragments (Fig.20):

(1) flanking the 5'-sequence of size 16 kb of phage GP 1 (example 1, Fig.10) modify the linker joining two adapters. The SalI site at the 5'-end are ligated with a NotI-SalI linker. The BglII site at the 3'-end are ligated with BglII-XhoI linker;

(2) the region encoding CLF, flanked at the 5'-end of the signal sequence aS1-casein and the 3'-end flanking the 3'sequence aS1-casein size of about 8.5 kb, separated from phLF3'10 kb in the form of a XhoI-SalI fragment. The SalI site at the 5'-end are ligated with SalI-NotI linker;

(3) kosmidou pWE15 (Stratagene, Inc.) translate in a linear form in the presence of NotI.

Primeape, Inc.) obtaining cGP1HLF.

Example 5. Expression plasmids bovine aS1-casein/CLF

A. Design pSl3'5'hLF

HindIII-SalI fragment from pl3'UTRhLF subcloning in the same sites pMHSl5'flank with the formation of pSl3'5'hLF (Fig.20). The obtained plasmid consists of a sequence of the promoter of bovine aS1-casein size 681 p. O. hybrid intron aS1-casein/IgG signal sequence aS1-casein, coding CLF flanking the 3'sequence aS1-casein size of about 1.6 kb and sequence region late polyadenylation SV 40.

Century pSl5'hLF

Plasmid pSl3'5'hLF (Fig. 20) is treated with enzymes and kpni restriction sites BamHI on the border with flanking 3'-sequence aS1-casein size of 1.6 kb. The larger vector fragment is purified dephosphorylated on Lenovo and after Smolevichi get pSl5'hLF.

C. Radioimmunoassay analysis CLF

Enriched immunoglobulin fraction ascitic fluid of monoclonal antibodies to human lactoferrin, not entering into cross-react with bovine or murine protein, obtained by precipitation with 50% ammonium sulfate and connection with CNBr-activated separate 4 B (20 mg protein per 1 g sepharose). Beads sepharose suspended (2 mg/ml) in phosphate BU.) NaN3, pH 7,4). Suspension sepharose (0.3 ml) is incubated for 5 h at room temperature by rotating the type head over the head" with the sample (typically 50 μl) in polystyrene test tubes (2 ml Beads sepharose then washed with brine (5 times with 1.5 ml) and incubated for 16 h at room temperature with 50 μl (1 kBq) 125I-labeled, purified by affinity chromatography polyclonal rabbit antibodies to human lactoferrin with 0.5 ml PBS, 0.1% (wt./about.) Tween-20. After that separate again washed with brine (4 times with 1.5 ml), and determine the associated radioactivity. The results are expressed as percentage of binding of the added labeled antibodies. The content of lactoferrin in the samples expressed in nanomolar using as a standard the cleaned milk lactoferrin man (serial dilution in PBS, 10 ml add, 0.1% (wt./about.) Tween-20.

Repeated tests of standard in a variety of cases revealed a high reproducibility of this RIA, intra - and manalytics the coefficients of variation were in the range of 5 to 10% of Human lactoferrin in the amount of only 0.1 nanogram is easily detected in this RIA.

D. Expression in 293S cells

293S cells transfection vishey is I the efficiency of transfection). After 44 h after transfection of the cells, remove the medium and analyzed by CLP the above method, RNA is selected by the method Stryker et al. 1989, EMBO J. 8, 2669. The results can be summarized as the following.

1. The efficiency of transfection is the same for two CLF plasmids.

2. CLF is expressed in the cells and secreted into the environment. In both cases, the content is about 0.4 mg/ml of medium using 3x106cells.

3. Protein manifests itself as CLF in the milk sample human reaction dose when determining the number associated with125I-antilactoferrin antibodies.

4. Based on Western blotting protein has approximately the same size (80 KD), and that in the sample of milk man.

5. based on Northern blotting CLF RNA produced in the cells that have the correct dimensions and its content is the same for both plasmids.

The data obtained show that these two plasmids are able to Express CLF. All applicable standards identical protein CLF present in the milk of human rights. Action heterologous signal sequence is shown in the fact that it promotiom secretion of the protein from the cells into the environment. A casein régua.

Example 6. In vitro maturation, fertilization and cultivation of bovine oocytes

Immature oocytes is available in large quantities (400-600 per day) aspiration of follicles transfer from ovaries obtained from a slaughterhouse. Immature oocytes grown in vitro until they can fertilize. After maturation, the oocytes are fertilized with sperm, also enjoy or become capable in vitro. The pronucleus of fertilized oocyte is then injected into the transgene encoding the expression and secretion of human lactoferrin. The zygote, resulting from such in vitro fertilization, then grow to the stage of morula or blastocyst (5-6 days) in the environment, prepared or "conditioned" by the tissues of the oviduct. Then blastocyst transfer nonsurgical by the recipient of cattle to continue the pregnancy or analyze the above method for integration of the transgene.

In vitro maturation (IVM). The ovaries receive immediately after slaughter from a local slaughterhouse and from them derive the oocytes. Or oocytes receive from live cattle surgical, endoscopic or by transvaginal ultrasound. In all cases, the oocytes are sucked off from the ovarian follicle (diameter and calf and incubated for 24 h at 39oC (Sirard et al. 1988, Biol. Reprod. 39, 546-552).

In vitro fertilization (IVF). Mature oocytes are fertilized with fresh or thawed sperm. Sperm are prepared for fertilization, getting in early populations of sperm enriched in mobility resulting from the division by the method of "floating" (Parrish et al. 1986, Theriogenology, 25. 591-600). Moving sperm is then introduced into the environment fertilization, consisting of a modified solution Tyrode (Parrish et al. 1986, supra) with the addition of heparin for induction of maturation of sperm (Parrish et al. 1988, Biol. Reprod. 38. 1171-1180). Gaining capacity is the final moment of the process of sperm maturation, essential for fertilization. Sperm and oocytes together stand 18 hours an Important feature of this method IVF is that (in the case of thawed sperm) sustainable, reproducible results are achieved after you have determined the optimal conditions for fertilization particular seminal fluid (Parrish et al. 1986, supra).

In vitro cultivation (IVC). Conventional systems of cultivation intended for the development of a mouse, rabbit or human eggs, do not support the development of bovine embryos after stage 8-16 cells. This difficulty is overcome pre-conditioning cloth a stage in 8-16 cell to blastocyst stage in vitro (Eyestone and First, 1989, J. Reprod. Pert. 85, 715-720).

Proven immunity bovine embryos grown in vitro. This partly stems from the existence of a "block" to divide in vitro on stage in 8-16 cells. This block can be bypassed by growing embryos in the oviduct rabbits (for an overview, see Boland, 1984, Theriogenology, 21, 126-137) or sheep (Willadeen, 1982, in "the Mammalian Egg Transfer, E. Adams, ed. pp.185-210; Eyestone et al. 1987, Theriogenology, 28, 1-7). However, such an alternative in vivo in less than ideal in that (1) requires the maintenance of a large number of animals-recipients, (2) requires surgical intervention to gain access to the fallopian tubes to transfer and secondary surgical intervention (or Backspace) to extract embryos, (3) it is rarely possible to extract all transferred embryos and (4) access to the embryos in the growing area for observation or treatment completely eliminated. No culture systems in vitro prevented the development of various methods (such as gene transfer pronucleus injection), preventing the accumulation of the necessary information about the history and ontogeny the development of the calf and complicating the process of growing embryos to the stage, sufficient for non-surgical embryo transfer and is compatible with cryopreservation methods (for example, to the stage of late is in 8-16 cell while Camous et al. 1984, J. Reprod. Fert. 72, 479-485 showed the ability of the division to 216 cells in joint cultivation of embryos with trophoblastic cloth.

The technique of joint cultivation was expanded by using tissue oviduct on the basis of the ability of Homo - or heterogeinity to support development from zygote to blastocyst. So, cow embryos grown in conjunction with the tissue of the oviduct or in the environment, air-conditioned oviduct tissue, developed in vitro from zygote to blastocyst (Eyestone and First, 1989, J. Reprod. Fert. 85, 715-720; Eyestone, W. H. 1989, "Factors affecting the development of early bovine embryos in vivo and in vitro Ph. D. Thesis, University of Wisconsin). Blastocysts were created in the system after sirovasthi and artificial insemination or in vitro in the maturation and fertilization of immature oocytes. Created in this way blastocysts resulted in pregnancy and the birth of calves after transfer of animals-recipients. Results see table. 4.

Thus, when the daily initial fee of 500 oocytes can be expected approximately 55 successful pregnancies.

Preparation of oviduct tissue, culturing and conditioning environment

1. To get the cow oviduct after slaughter or salpingectomy.

2. Collect 10 ml of the modified solution tradeshops (Parrish et al, 1988, Biol. Reprod. 38, 1171-1180).

4. Re-suspending the pellet tissue in M199 + 10% fetal calf serum in a ratio of 1 volume of fabric: 50 volume environment.

5. Suspension fabric can be used for co-cultivation of the embryo.

6. Or the environment may be conditioned for 48 hours After centrifugation of the suspension supernatant can be used as a medium for embryo culture. If desired, the conditioned medium can be stored at -70oC. When growing embryo conditioned medium should be used without dilution (Eyestone, 1989, ibid).

Example 7. Microinjection CLF-transgene in cow pronucleus

The DNA fragment containing the link expression CLF, separated from the vector using the appropriate restriction enzymes and separated on agarose gel. Fragment purified by elektrobudowa, extraction with phenol and chloroform and ethanol precipitation (Maniatis et al.). The fragment is dissolved and cialiswhat in 10 mm Tris-HCl, 0.1 mm edtk (pH of 7.2) at a concentration of 1 to 2 µg/ml Needles for microinjection fill cialisovernight the DNA solution.

Before in vitro fertilization extra cells are separated from the eggs or intensive mixing 2 min pronucleus in principle is injected as the pronucleus of mouse (Hogan B. et al. 1986, in "Manipulating the mouse embryo". Cold Spring Harbor Laboratory) with the additional step of centrifugation for the purpose of visual inspection pronucleuses. Injecting conduct after 18-24 h after fertilization. The time varies depending on the source of the seed of the bull. Various batches of seed do the core visible through different periods of time.

Bovine oocytes matured and fertilized in vitro, centrifuged in the Eppendorf tube in 1 ml tyrodes-Gennaro solution (Parrish, 1987) at 14500 g for 8 min (Wall et al. 1985, Biol. Reprod. 32, 645-651). The embryos are transferred into the drop tirades-Gennaro solution on a microscope plate, covered with paraffin oil. With the help of hydraulic system oocytes fixed in the holder of the eggs so that both pronucleuses become visible (use interferention-contrast and phase-contrast optics). If necessary, the oocytes rotate in the holder eggs for inspection pronucleuses. Then through the transparent zone, the cytoplasm into the pronucleus needle. Small volume 1-3 PL injected (contains 20-100 copies of DNA into the pronucleus using either a continuous flow or pulsatile flow (using the switch) of the DNA solution from the needle. Or embryos studiosorum. Then injected embryos are transferred in a drop of medium for culturing according to the method of example 6 to develop to the stage of morula or blastocyst.

Example 8. Early detection of transgenesis CLF - transgene

After microinjection construct in the oocytes grow them. A suitable site each embryo split and subjected to lysis (D. King et al. 1988, Molecular Reproduction and Development, i, 57-62), proteolysis (Higuchi, R. 1989, "Amplifications (A forum for PCR Users", 2, 1-3) and DPNI hydrolysis. PCR is done according to previously described methodology (Ninomiy T. et al. 1979, Molecular Reprod, and Devel. 1, 242-248) series of two primers: one in aS1, and the other in CLF cDNA sequence. For example, in PCR, where the leading primer (30 ñ timer) aS1-sequence is the sequence:

ATG AAA CTT ATC CTC ACC TGT CTT GTG (Seg. ID N: 16),

reverse primer (30 ñ timer) in CLP - sequence is the sequence GGG TTT TCG AGG GTG CCC CCG AGG ATG GAT (Seg. ID N: 17); (971-1000 in Fig. 1-7), the fragment will be created in 990 p. O. Fragment contains inactivated DpNI site due to loss adenosylmethionine for 934 p. O. from the beginning of the leading primer.

Example 9. Production CLF with milk of cows of different breeds

Cow morula developed from microinjection of oocytes cleaved by the method of the STU. The other half is subjected to DNA analysis by the method of example 8. After the result of the analysis is known, the morula kept in culture for development into blastocysts or used as a source for nuclear transfer in nuklearna zygote. Transfer of blastocysts in synchronized cows carried out according to the method Betteridge (Betteridge K. J. 1977, in "Embryo transfer in farm animals: a review of techniques and applications).

CLF found in the milk of lactating transgenic offspring with the use of RIA in example 5.

Example 10. Plasmids expressing the bovine aS1-casein/CSA

To construct the expression vector for CSA used three overlapping phage clone containing the complete gene CSA. Clones identified as lHAL-HA1, lHAL-3W and lHAL-H14. The clones described in Urano et al. 1986, J. Biol. Chem. 261, 3244 3251; Urano et al. 1984, Gene, 32, 255 - 261. The sequence of the gene plus the surrounding area published in Minghetti et al. 1986, J. Biol. Chem. 261. 6747 6757. The only phage containing the complete gene CSA, construct it as follows.

Clone HA-1 treated with BstEII and AhaII. A fragment of approximately 1400 p. O. extending from the provisions of 1784 (in the first exon, immediately downstream of the ATG) to 3181, allocate and attach a synthetic linker catalinotto, environmental ATG, and several common restriction sites. This fragment is called fragment #1.

Process clone 3W in the presence AhaII and SacI and produce a fragment of about 13,1 kb extending from the provisions 3181 up 16322, and a synthetic linker attached to the SacI site to facilitate cloning in phage EMBL3. This fragment is called fragment #1.

These two fragments are ligated and clone in the phage EMBL3. After identifying the correct phage fragment extending in an upward direction immediately after BstEII site (which introduced unique restriction sites) to SacI site, separated from the SacI SalI fragment (extending from position 16322 to - 21200), isolated from clone H-14. These two fragments then are ligated and clone in EMBL4.

After processing ClaI (in the upward direction immediately from the newly introduced BstEII site) and BamHI (in the downward direction immediately from the SalI site in ragovoy DNA) this new clone gives the fragment containing the complete gene CSA with flanking 3'-sequence of approximately 2.5 kb.

To construct the expression vector for CSA kosmidou cGP1HLF partially hydrolyzing in the presence of ClaI and BamHI. This removes the signal sequence, the coding sequence of CSA, 3'-UTR and Fri with the above fragment CSA and the resulting kosmidou called cGP1HSA.

Educated in the expression vector contains: (1) the sequence of the promoter of size 16 kb originating from gene aS1-casein, (2) the first exon and intron of this gene present in GP 1, (3) signal sequence CSA gene, complete genomic gene encoding CSA, including 2.5 kb downstream of this gene, and (4) 8 kb from flanking the 3'sequence derived from a gene aS1-casein.

The transgene used to generate transgenic cows of different breeds, producing CSA in milk, by means similar to those used for producing CLF in the milk of cows of different breeds.

Example 11. Cleanup CSA from the milk of cows of different breeds

The secretion of heterologous proteins from milk is easier, because after precipitation of casein these proteins are mostly found in the whey fraction, less contaminated compared with getting protein in microbial cells and other cellular systems.

To clean CSA from cow's milk are recommended chromatographic methods. This approach achieved better yields and higher purity of albumin compared with ethanol fractionation (Curling, 1980, in: "Methods of Plasma Protein Fractionation", Curling, ed. Academic Press, London, UK; C, what its important role in maintaining massoudieh osmotic pressure is also better preserved during chromatographic purification (Unevenness, 1982, Joint Meeting of the ISH-ISBT, Budapest).

To highlight CSA produced in the milk of transgenic cows, apply the following stage.

1. The precipitation of the Caseins (about 80% of milk protein) and basically just milk fat at pH 4.5 and/or the addition of chymosin. Serum fraction contains albumin.

2. Affinity chromatography of albumin on Sebatron blue 3 GA Sepharose CL-6 B (Harvey, 1980, in: "Methods of Plasma Protein Fractionation" op. cit. ). This stage is to remove non-albumin proteins and reduce processed approximately 30 times. Albumin elute from the matrix 0.15 M NaCl and 20 mm of sodium salicylate at a pH of 7.5.

3. Buffer exchange on Sephadex G-25 desalting in of 0.025 M sodium acetate, establishing pH 5.2, followed by filtration.

4. Anion-exchange chromatography on DEAE-Sepharose CL-6B. Desorption of albumin at pH 4,5.

5. Cation-exchange chromatography on CM-Sepharose CL-6B. Elution of albumin On,11 M sodium acetate at pH 5.5 and the concentration of albumin, 6% (wt./about.) solution by ultrafiltration.

6. Gel filtration on Sephacryl S what Yu (Monomeric albumin) are concentrated by ultrafiltration and injected into the compositions.

It should be noted that the stage 3 6 is essentially identical to the method presented Curling et al (Curling, 1980, op. cit. Curling et al. 1982, op. cit. Berglof et al. 1982, op. cit.) to clean CSA of plasma.

Example 12. Transgenic mouse having a transgene, human serum albumin (CSA), created by homologous recombination

For introducing CSA gene in transgenic mice using three overlapping genomic clones of CSA: (lHAL-HA1, lHAL-H14 and lHAL-3W, shown in Fig. 22 in accordance with the message Urano et al. 1984, Gene, 32, 255-261 and Urano et al. 1986, J. Biol. Chem. 261, 3244-3251. Briefly, genomic library design by the partial hydrolysis of the EcoRI restriction enzyme fibroblasts human DNA. For clones lHAL-H14 and lHAL-3W this library is selected using the32P-labeled genomic clones of human albumin by hybridization about the day when 65oC in 1M NaCl, 50 mm Tris-HCl (pH 8.0), 10 mm etc, 0,1% VAT, 100 kg/ml hydrodynamically fragmented DNA salmon sperm and 10x denhardt's solution after prehybridization in 3SSC and 10x denhardt's solution. After hybridization the filters are washed at 65oC 0.2 SSC and 0.1% VAT. The selection of clone lHAL-HA1 performed identically, except that for screening libraries of human fibroblasts used Bgl II-EcoRI fragment from the 5'-end lHAL-3W which together constitute the entire gene CSA and flanking region. Consisting mainly of 5'-sequence of fragment I is EcoRI-EcoRI fragment isolated from lHAL-HA 1; middle fragment II is AcyI (=AhaII)-SacI fragment from lHAL-3W and consisting mainly of 3'-sequence of fragment III is XhoI SalI fragment from lHAL-H14 (Fig.16-21). Slices treated with DNA polymerase maple and dNTP to fill the excess sticky ends. In some experiments dephosphorylation leaf fragments are then treated with bacterial alkaline phosphatase to remove 5'phosphate groups from each fragment. Overlapping DNA fragments then concentrate together and injected into the male pronucleus of fertilized mouse eggs according to published methods (Hogan et al. 1986, in "Manipulation of The Mouse Embryo: A Laboratory Manual", Cold Spring Harbor Laboratory). While the number of injectable molecules was varied in the range from 25 to 100, the ratio of the individual fragments was approximately 1:1:1. The embryos are implanted in the uterus of female mice according to the method of Hogan et al. supra.

For the analysis of correct homologous recombination of three overlapping fragments and integration emerged transgene into the genome of mouse genomic DNA newborn mice subjected to the following specific hydrolysis with subsequent hybridization on Sautoy recombination;

Nco I: cut the overlapping area and gives the strip 8 and 9.3 kb in the case of proper recombination;

Nco I + Hind III cuts in several positions outside the area of overlap indicates the presence of intact fragments;

Hinc II: cut the overlapping area, gives several bands, indicating the correctness of rearrangements in these parts.

In the initial experience with born 28 transgenic animals for 22 shows the correct recombination of all three fragments. 20 of these 22 animals taken a blood on the analysis of the presence of the protein CSA (radioimmunoassay analysis). 15 of these 20 animals showed expression at the level of 0.5 to 5 μg/ml of a single animal, which is not detected recombination and non-transgenic, did not show any expression. Using blokirovaniya RNA was detected only in two cases (two mice with the highest protein level) for the band. Currently implemented blokirovanie RNA, allowing to determine the presence of mRNA (e.g., poly (A) + RNA). The application of the reverse transcriptase for cDNA synthesis and subsequent PCR detected a correlation between the presence of RNA and protein. However, in these experiments could not determine the size(s) RNA.

Pranie two transgenes CLF, one of which contains a 5'-sequence regulating the expression of aS1-casein approximately 16 kb (pGP1hLF (16 kb), also known as p16,8HLF 4), and the second contains the 5'-sequence regulating the expression of aS1-casein approximately 7.9 kb (pGP1hLF (8 kb), also known as p 8.8 HLF 4). A General strategy for the design reflected in Fig.23.

From phage clone GP1 allocate EcoRI-BglII fragment of 1.8 kb (fragment C in Fig.23). This fragment extends from the provisions of -100 in the website of the start of transcription in the second exon of the gene aS1-casein. The BglII site is in the articulation of the first introna and the second exon of the gene aS1-casein. Containing a BglII site to the 3'end are ligated with a synthetic BglII-ClaI linker and subcloning in the plasmid pUC19. The obtained plasmid is designated as pEBS.

Fragment B (Fig. 23) was isolated as EcoRI fragment and clone into the EcoRI site of pEBS. Fragment B contains sequences from position -7500 to position -100 in the website of the start of transcription in the gene aS1-casein. Formed in the resulting plasmid is designated as the pEB 3 S and it contains a combination of fragments B and C, EcoRI-Cla I fragment 8.9 kb, extending from position -7500 to position +1400 customers transcription starts. EcoRI-ClaI fragment 8.9 kb of 3 pEB S, obtained by complete hydrolysis in the presence of ClaI and partially the e, 1986, 46, 269 276 contains the NotI restriction site) with the formation of pNE3BS.

From a phage GP1 allocate ClaI-EcoRI fragment of 8.5 kb (fragment A in Fig. 23) extending from the provisions of 1600 to position -7500 site of the start of transcription. Then the fragment subcloning in pUC19 and get pSE. Using synthetic nucleotide enter a unique NotI site in the ClaI site destruction in the past. The obtained plasmid is designated as pNE.

From pNE allocate an insert in the form of a NotI-EcoRI fragment and together with the EcoRI-ClaI insert from pNE3BS are ligated into the cloning vector pKUN 2. The obtained plasmid pGP1 (D2ex) contains aS1-casein promoter in 16 kb plus 5' end of the gene attached to the BglII site at the border of the second exon.

The final plasmid (p 16,8HLF4) containing the transgene, is obtained using NotI-ClaI fragment from clone pGP I (D2ex) and Xho-Not I fragment from clone pHLF 3' 10 kb. The structure of the transgene same as in the previously described transgene.

As a small modification of this plasmid carried out the destruction of the plasmid SalI site in the processing SalI and implementation of a linker containing a NotI site. Then the SalI site is introduced in a downward direction immediately after the sequence CLF processing Cloned customers in this position and adding the ü CLF is surrounded by two unique restriction sites (ClaI and SaII) and can be replaced by any recombinant DNA sequence, with ClaI site at the 5'end and a SalI site at the 3'-end.

Designed and another transgene identical to above, except that only has about 8 kb of 5'sequence regulating the expression of aS1-casein. The transgene construct a selection of pNE3BS NotI-ClaI fragment and merge directly with Xho-NotI fragment from clone pHLF3'10 kb. The obtained plasmid is designated as pGP1hLF (7 kb) (also known as p 8.8 HLF4).

Plasmid 16,8hLF4 modify the introduction of its hybrid splice signal (aS1-casein-IgG), described in examples 3 and 5. The resulting plasmid is designated as 16,8hLF3 and identical 16,8hLF4, except for the presence of hybrid intron instead of "natural" casein intron in 5'-UTR.

CLF signal sequence can also be used in all cDNA constructs disclosed herein, instead of casein signal sequence. This can be done as follows. Get synthetic oligomer containing full CLF signal sequence (Fig. 22-23) plus the ClaI restriction site in the 5'-end and the EagI restriction site at the 3'end. These restriction enzymes cut sites also border with casein signal sequence in the other plasmids (e.g., p16,8hLF 4). Poluchennuyu plasmid hydrolyzing in the presence of ClaI and EagI and used as a vector for accommodation ClaI-EagI fragment, containing a sequence CLF. Of the positive clones produce cDNA in the form of a ClaI-SalI fragment and inserted into gidralizovanny ClaI and SalI plasmid p16,8hLF4 with the creation of p16,8hLF5. Similarly, this Cla-Sal fragment containing CLF-cDNA plus signal sequence CLF, can be implemented in any CLF cDNA vector.

Example 14. Production of recombinant human lactoferrin in the milk of transgenic mouse

Transgenic mouse create use of several described in the examples of transgenes. Used transgenes are shown in table. 5. In each case, the 5'- and 3'-sequences regulating the expression came from gene bovine aS1-casein, RNA splice signal in the untranslated 5'-region was either gene aS1-casein, or hybrid casein-IgG intron.

Recombinant DNA in each case came from cDNA clones.

Example 15. Construction of transgenic cassettes for genomic recombinant DNA

All of these plasmids contain the field originating from retranscribing areas a S1-casein cows (including intron). If the expression is subject to genomic gene that already contains untranslated regions and introns, allowing for high expression, in this case vasani with untranslated regions subject to gene expression. Such an expression cassette is p16kb, CS, and its design as follows. As a matrix in PCR experiments used a plasmid pSl3'5'hLF. This plasmid contains 680 p. O. sequence of the promoter of the gene aS1-casein, as well as its first exon. The rest of this plasmid for this experience is not of interest. Upstream primer is located immediately upstream from the insertion in plasmid portion (immediately upstream from the NotI restriction site). His sequence: 5' CGA CGT TGT AAA ACG ACGG-3'.

Downstream primer is located in exon 1. Its sequence corresponds exactly to the first 19 p. O. exon, and also includes dehybridization region 17 p. O. containing ClaI and SalI site. Primer has the following sequence:

5'-ATTGTCGACTTATCGATGGGTTGATCAAGGTGA-3'.

Amplificatory fragment of hydrolyzing in the presence of NotI and SalI and are ligated with pKUN2 (see example 13). In the resulting plasmid (p-680CS) in the connected proximal promoter fragment from -680 to +19 plus two restriction site immediately downstream from these 19 p. O.

This plasmid hydrolyzing in the presence of NotI (immediately upstream from -680) and NsiI (-280) and used as etc) is 80), selected from p16,8hLF4 (example 13). In this plasmid (p-16kb, CS), thus, connected fragment of the promoter from -16000 to +19. A plasmid can be used to insert genomic genes, which carry their own UTR's and poly (A) signal. After the introduction of a genomic gene in the form of a ClaI-SalI fragment flanking the 3' sequence aS1-casein can be inserted in the form of a SalI fragment.

Example 16. The design of the transgene for producing protein C

Genomic sequence of the protein C published in the work of Foster et al. 1985, Proc. Natl. Acad. Sci. USA 82, 4673-4677. However, this sequence does not include the first exon identified using the cDNA sequence published by Beckman et al. 1985, Nucl. Acids Res. 13, 5233-5247. The first exon of the protein C is located in the position from -1499 to -1448 in sequence by Foster. The transgene for expression and secretion of protein C in the milk of cows of different breeds is shown in Fig. 24. This transgene construct as follows.

Genomic library of human rights in EMBL-3 (Multicopy) probe sequence specific for protein C. purified ragovoy DNA containing the complete gene for protein C, is subjected to the selection. The phage isolated from E. coli strain with Dam-phenotype, such as strain GM113. The result is a clone of the I.

Allocate ClaI-NheI fragment, extending from +1333 to 11483, and denote it as fragment 1.

Plasmid pGEM7 (Stratogene, Inc.) hydrolyzing in the presence of SphI and SmaI. The area between them to replace the corresponding region of the plasmid pKUN (Gene, 1986, 46, 269-276). The obtained plasmid is designated as pGEM7A. It is characterized by the following card restriction in the relevant field:

< / BR>
Synthesize two primers. Primer GP 125 has the following sequence:

< / BR>
Primer GP126 has the following sequence:

< / BR>
Primer GP 125 overlaps with exon O (position 654 675 gene protein C) and enters the ClaI site in the untranslated 5'-region. Exon O this exon, not identified by Foster et al. Primer GP126 covers the area from 1344 until 1315 protein c gene. This area contains a ClaI site.

The region between positions 654 and 1344 amplified using as a matrix of human DNA, or ragovoy DNA. The product of amplification hydrolyzing in the presence of ClaI and clone in the vector pGEN7a education pPCCC. This vector multiply in dam-negative strain, such as GM113, and partially hydrolyzing in the presence of ClaI (interest only plasmids, treated with ClaI in position 1340) and completely in the presence of the XbaI. In the floor. the C this plasmid is isolated in the form of a ClaI-SalI fragment of the transgene protein C, which are ligated with p16 kb, CS (see example 15) with the formation of the transgene, is able to Express the protein C with cow's milk, and denote this plasmid as p16 kb, CS, PC.

Contained in the plasmid p16 Kb, CS, PC transgene treated with NotI and used to generate transgenic cows of different breeds of the above methods. Such transgenic animals capable of producing protein C in the milk.

1. The transgene to obtain the recombinant polypeptide in the milk of transgenic cows, containing at least a fragment of the 5'-flanking sequence of the gene the cow S1-casein, containing the promoter and enhancer, with restrictee card, a DNA sequence encoding a polypeptide that provides secretion in breast cells, a DNA sequence encoding a recombinant polypeptide, a fragment of the 3'-flanking sequence of the gene the cow S1-casein, which includes the site of the terminal polyadenylation, with restrictee card, and a sequence that provides secretion, operatively linked to a sequence that encodes a recombinant polypeptide with the formation of secretory-recombinant posledovatelnostyu, when this transgene provides the expression of secretory-recombinant DNA sequence in the secretory cells of the mammary gland of cows born and heifers containing the same transgene during lactation, the production of recombinant forms of the polypeptide, which when secretion of the secreted breast cells producing a recombinant polypeptide in the milk of transgenic cows and born heifers.

2. The transgene under item 1, characterized in that it further comprises recombinant insertion sequence.

3. The transgene under item 2, characterized in that the recombinant insertion sequence is a hybrid insertion sequence.

4. The transgene under item 3, characterized in that the hybrid insertion sequence contains possible signal of RNA splicing.

5. The transgene under item 1, wherein the recombinant polypeptide is a homologous polypeptide cows.

6. The transgene under item 5, wherein the homologous polypeptide selected from the group consisting of casein, lactoferrin, lysozyme, hydrolases cholesterol and serum albumin.

7. The transgene under item 1, wherein T7, characterized in that the heterologous polypeptide is chosen from the group consisting of human milk proteins, human serum proteins and industrial enzymes.

9. The transgene under item 8, wherein the heterologous polypeptide is a protein of human milk.

10. The transgene under item 9, wherein the human milk protein selected from the group consisting of secretory immunoglobulins, lysozyme, lactoferrin, lactoglobulin, -milk albumin and salt stimulated lipase bile.

11. The transgene under item 10, wherein the milk protein is lactoferrin or lysozyme.

12. The transgene under item 8, wherein the heterologous peptide is a human serum protein.

13. The transgene under item 12, characterized in that the protein human serum selected from the group consisting of albumin, immunoglobulin, Factor VIII, Factor IX and Protein C.

14. The transgene under item 13, wherein the whey protein is an albumin.

15. The transgene under item 8, wherein the heterologous polypeptide is an industrial enzyme selected from the group consisting of about the activities of DNA selected from the group consisting of DNA sequences encoding secretory sequence of human lactoferrin, human serum albumin, human lysozyme and secretory signal sequences cow S1-casein, S2-casein, -casein, K-casein, a-lactalbumin, -lactoglobulin and serum albumin.

17. The transgene under item 16, characterized in that the sequence of secretory DNA is a DNA sequence that encodes a secretory signal sequence of bovine S1-casein.

18. The transgene under item 1, characterized in that the 5'-flanking sequence has a size about 30 KB and 3'-flanking sequence has a size of about 15 KB.

19. The transgene under item 1, characterized in that the 5'-flanking sequence has a size of 1 KB and 3'-flanking sequence has a size of 2 KB.

20. A method of obtaining a transgenic cow, containing the recombinant polypeptide produced in her milk, including the introduction of the transgene under item 1 in embryonic the target cell cows, transplantation of transgenic embryonic target cells or derived from it the cow embryo-recipient and the identification of at least od is. 0, characterized in that it additionally includes the stage of fertilization, embryonic target cells in vitro prior to introduction of the transgene.

22. A method of obtaining a transgenic cow, containing the recombinant polypeptide produced in her milk, including methylation of the transgene under item 1, introduction methylated transgene into fertilized oocytes of this animal for integration of the transgene in the genomic DNA of fertilized oocytes, culturing the thus obtained individual oocytes to preimplantation embryos replication in the genome of each fertilized oocyte, removing at least one cell from each preimplantation embryo and lysis of at least one cell with the release contained in DNA, contacting this selected DNA with a restriction enzyme that can cleave methylated transgene, but not able to cleave demetilirovanny the form of the transgene, formed after integration into genomic DNA and its replicia, with subsequent identification of the preimplantation embryo, the cells of which contain the transgene.

23. Milk from transgenic cows containing recombinant polypeptide produced is the, that recombinant polypeptide is homologous to bovine polypeptide.

25. Milk under item 23, wherein the recombinant polypeptide is a heterologous polypeptide.

26. Milk on p. 25, wherein the heterologous polypeptide is chosen from the group comprising human milk proteins, human serum proteins and industrial enzymes.

27. Milk under item 26, wherein the heterologous polypeptide is a human milk protein.

28. Milk on p. 27, wherein the milk protein is chosen from the group comprising secretory immunoglobulins, lysozyme, lactoferrin, lactoglobulin, -lacto albumin and salt stimulated lipase bile.

29. Milk on p. 28, wherein the milk protein is a lactoferrin or lysozyme.

30. Milk on p. 25, wherein the heterologous polypeptide is human serum protein.

31. Milk under item 30, wherein the human serum protein selected from the group including albumin, immunoglobulin, Factor VIII, Factor IX and Protein C.

32. Milk on p. 31, characterized in that Thu is containing a series of recombinant polypeptide, produced transgenic cow received pursuant to sub. 20 22.

34. Food composition under item 33, wherein the recombinant polypeptide is at least partially purified from transgenic milk.

35. Food mixture according to p. 33, characterized in that it is composed of nutrients, suitable for baby food.

 

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