RU2390562C2 - Method of creating transgenic mammals which produce exogenous proteins in milk, and transgenic mammals created using said method - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to biotechnology, animal husbandry and medicine. Proposed is a transgenic animal from the family of bulls which produces in its milk a recombinant human growth hormone in amount of at least 92 ng/ml per month on average. Also disclosed is a method of creating such a transgenic animal which involves cloning the genetic make-up which codes the hGH gene and a beta-casein promoter in an expression vector. Further, there is transfection into fetal somatic cells of cattle, usually fibroblasts, and nuclear transfer into enucleated oocytes of cattle, that way forming transgenic embryos. The invention can be used in animal husbandry and medicine.

EFFECT: invention enables to obtain a large amount of the human growth hormone.

43 cl, 5 dwg, 3 tbl, 9 ex

Description

BACKGROUND

Protein factors and hormones used in healthcare are currently obtained in the pharmaceutical industry by extraction or, in recent decades, using recombinant technology. The expression of genetic constructs containing the desired genes was successfully carried out in bacteria, yeast or mammalian cell lines. However, the use of mammalian cell cultures to produce complex proteins, such as proteins that require the right type of glycosylation, involves expensive methods.

Over the past decade, more and more recombinant DNA techniques have been used to produce commercially important biological materials. To this end, DNA sequences have been cloned that encode many medically important human proteins. These include insulin, a plasminogen activator, alpha-1 antitipsin, and coagulation factors VIII and IX. Currently, even with new recombinant DNA-based techniques, these proteins are usually purified from the blood and tissue in an expensive and time-consuming way, which may be associated with the risk of transmission of infectious agents such as agents that cause AIDS and hepatitis.

Although the expression of DNA sequences in bacteria to obtain the desired medically important protein seems attractive, in practice, bacteria are often unsatisfactory as hosts, since foreign proteins are unstable in the bacterial cell and cannot be properly processed.

Recognizing this problem, attempts were made to express cloned genes in mammalian tissue culture and, in some cases, obtained a suitable technique. However, periodic fermentation of animal cells is an expensive and requiring certain technical conditions method.

Therefore, there is a need for a highly productive low-cost method for producing biological substances, such as properly modified eukaryotic polypeptides. The absence of agents that are infectious to humans may be an advantage of this method.

The possibility of obtaining transgenic animals, like cattle, in relation to the desired gene, in order to obtain a large amount of human protein in milk is of great interest to industry. Several research groups report in the literature on their success in obtaining human serum albumin, alpha antitrypsin, and some other examples from transgenic cows or goats.

Many experiments have been conducted previously in mice or rats and always preferred that the transgenic expression be limited to the mammary glands, since beta-casein or lactalbumin promoters that only respond to breast transcription factors in females during lactation were used.

Expression of a heterologous protein exclusively in milk implies the possibility of avoiding undesirable effects on the health of the host animal and providing a simple method of purification.

Currently, attention is devoted to the development of several systems to increase the yield of transfection or selection of cells and the choice of the source of a homologous embryonic somatic cell to increase the viability and immunity of cloned animals.

On the other hand, there is great interest in the transfer of somatic cell nuclei, mainly to make it possible to reproduce elite domestic animals and construct transgenic animals for agricultural and biomedical purposes. Briefly, nuclear transfer (NT) consists in removing the nucleus from the recipient's oocyte, followed by transfer of the donor nucleus to the perivitelline space in close contact with the recipient's cytoplasm and fusion. Development is induced artificially by chemical or physical activation. Obtaining cloned offspring through somatic cell nucleus transfer has been successfully achieved in sheep (Campbell, K. H., et al., Nature 380: 64-66 (1996), 1996; Wells, DN, et al., Biol Reprod 57: 385- 393 (1997); Wilmut, I., et al., Nature 385: 810-813 (1997)); goats (Baguisi, A., et al., Nat. Biotechnol. 17: 456-461 (1999)) and cows (Cibelli, J. B., et al., Science 280: 1256-1258 (1998); Kato, Y., et al., Science 282: 2095-2098 (1998); Wells, DN, et al., Reprod Fertil Dev 10: 369-378 (1998)).

There are several factors that influence NT results, including methods for removing nuclei, fusion, activation, and synchronization of the cell cycle of the recipient donor. High efficiencies in the removal of nuclei from recipient oocytes have been achieved using DNA-specific stains used intravitally to visualize chromatin (Stice, SL, and Keefer, C. L., Biol Reprod 48: 715-719 (1993); Westhusin, ME, et al., J Reprod Fertil 95: 475-480 (1992)). The fusion of a donor cell with a recipient oocyte depends on the accuracy of the alignment of the cells in the pulsed field, the contact of the donor cell with the recipient oocyte and the size of the donor cells (Collas, P., et al., Anal. Biochem. 208: 1-9 (1993)). Activation of the NT-reconstructed embryo has been improved, and blastocyst development rates are equivalent to those for in vitro fertilized oocytes (Liu, L., et al., Mol. Reprod. Dev. 49: 298-307 (1998)).

Successful development of NT embryos was accomplished using mature oocytes (Willadsen, SM, Nature 320: 63-65 (1986)), zygotes (McGrath, J., and Solter, D., Dev. Biol. NY 4: 37-55 ( 1985)) and crushed embryos (Tsunoda, Y., et al., J. Reprod. Fertil. 96: 275-281 (1992)) as recipient cytoplasts; however, it depends on the source of the donor nucleus. Cell cycle compatibility between recipient cytoplasts and donor cells is one of the important factors that influence the development of NT embryos. Appropriate synchronization is necessary to prevent ploidy of the reconstructed embryo.

The mitotic cell cycle has the following successive phases: the interval preceding replication (G ₁ ), the DNA synthesis phase (S), the interval preceding mitosis (G ₂ ), and mitosis (M). During a single cell cycle, all genomic DNA is replicated once before mitosis. An interphase donor nucleus transferred to a mature oocyte lacking a nucleus (metaphase II) undergoes several morphological changes. After fusion, but before the destruction of the membrane of the donor nucleus (NEBD), the chromosomes condense (PCC). These changes are induced by the activity of the maturation / mitosis / meiosis stimulating factor (MPF) and mitogen-activated protein kinase (MAPK) (Collas, P., and Robl, JM, Biol. Reprod. 45: 455-465 (1991)). MPF and MAPK activities are found in all meiotic and mitotic cells and become the highest in metaphase, and in mammalian oocytes, these high levels also induce a delay in metaphase II. A decrease in the activity of MPF and MAPK during fertilization or activation by a calcium ionophore is a signal for completion of meiosis, isolation of the second polar body, decondensation of the sperm nucleus, and pronucleus formation.

The direct effect of NEBD and PCC on donor chromatin depends on the cell cycle of the donor nucleus during transfer. Diploid nuclei G ₀ / G ₁ undergo condensation to form separate chromatids, and tetraploid nuclei G ₂ undergo condensation to form double chromatids. However, the nuclei in the S phase during the transfer have a characteristic “atomized” appearance; PCC causes a lot of DNA damage. Therefore, the correct ploidy can be obtained by transferring nuclei in G ₁ or G ₀ to oocytes in metaphase II during activation or before activation. The second method involves transferring nuclei to a preactivated oocyte in S phase, in this case, a donor cell in phase G ₁ , G ₀ or S can be used. Since MPF and MAPK are low, chromatin decondenses and undergoes DNA replication without PCC and NEBD .

A third synchronization pattern was reported for mice in which live offspring were obtained using a donor cell in G ₂ or metaphase and an oocyte-free nucleus in metaphase 2 (Cheong, H.T. et al., Biol Reprod 48: 958-963 (1993 ); Kwon, OY, and Kono, T., Proc Natl Acad Sсi USA 93: 13010-13013 (1996)). Extrusion of the polar body from an NT-reconstructed embryo was reported, resulting in a single diploid embryo and diploid polar body (Kwon, OY, and Kono, T., Proc. Natl. Acad. Sci. USA 93: 13010-13013 (1996) ) However, there are no reports of the formation of polar bodies after NT into nuclear-free MII oocytes in cattle, sheep, or pigs, which indicates the differences between species in the mechanisms for regulating the formation of an intact division spindle and extrusion of polar bodies.

It has been confirmed that the stages of the cell cycle of the donor cell and the recipient are also important for reprogramming the nuclei of donor cells. An increase in the period between donor nucleus transfer and transcription in the zygote can improve reprogramming of the nucleus. For this reason, several authors activated the oocyte several hours after fusion (Сibelli, J. B., et al., Science 280: 1256-1258 (1998); Wakayama, T., et al., Nature 394: 369-374 ( 1998); Wells, DN et al., Biol Reprod 60: 996-1005 (1999)). Other reports have used sequential nuclear transfer (Stice, S. L., and Keefer, C. L., Biol. Reprod. 48: 715-719 (1993)).

An unexplored way to increase the reprogramming time of the donor nucleus is to transfer the nuclei before metaphase II. After the destruction of the germinal vesicle (GVBD), all nuclear events are regulated by a significant increase in the cytosolic MPF and MAPS in the oocyte, which prevent the restoration of the nuclear envelope and entry into the S phase until fertilization or activation. Therefore, a maturing oocyte can be a universal recipient for a donor cell in metaphase or G ₂ . You can even use cells in G ₁ or G ₀ as donor cells if activation induces an S phase before cell division.

When blastomeres in G ₂ or M are used as donor cells, reprogramming of nuclei is possible (Cheong, H. T., et al., Biol Reprod 48: 958-963 (1993); Kwon, OY, and Kono, T., Proc Natl Acad Sci USA 93: 13010-13013 (1996); Liu, L., et al., Mol Reprod Dev 47: 255-264 (1997)). One explanation is that some factors are displaced from chromatin as a result of condensation of chromosomes. Indeed, in the case of nuclear transfer, it was believed that NEBD and PCC are morphological signs of core reprogramming. In addition, during fertilization, sperm chromatin is highly condensed, and its volume is much smaller than the volume of somatic cell nuclei, and the oocyte has the ability to remove sperm nuclear proteins. The oocyte chromosomes are also condensed during the fusion of the spmatozoid-oocyte. Perhaps the conformation of condensed chromatin may have some biological significance. Therefore, when simulating this situation by transferring metaphase nuclei, a nucleated metaphase recipient can improve the NT result. However, several researchers used this approach for domestic animals and used blastomeres as donor cells (Liu, L., et al., Mol. Reprod. Dev. 47: 255-264 (1997)).

One of the objectives of this invention is the characterization and improvement of the existing method for transferring somatic cell nuclei to develop a reliable and economical method for producing genetically identical calves from donor cells of an adult organism.

SUMMARY OF THE INVENTION

The invention relates to a transgenic mammal other than humans, characterized by the production of unexpectedly high levels of recombinant growth hormone in milk. Recombinant growth hormone may be human growth hormone, but without limitation specified. A transgenic mammal other than humans may be, without limitation, an animal belonging to a species of cattle.

The invention further relates to a plasmid that provides expression of a protein of interest in mammalian cells, in which expression is regulated by a beta casein promoter. The protein of interest may be, without limitation, human growth hormone.

The invention also relates to various methods for producing a transgenic non-human mammal that produces recombinant growth hormone in milk. Recombinant growth hormone may be human growth hormone, but without limitation specified. A transgenic mammal other than humans may be, without limitation, an animal belonging to a species of cattle.

The invention also relates to a method for producing a protein of interest, comprising the preparation of a transgenic non-human mammal that produces said protein in milk, the production of said milk from a transgenic non-human mammal, and the purification of said protein of interest from milk. The protein of interest may be human growth hormone, but without limitation. A transgenic mammal other than humans may be, without limitation, an animal belonging to a species of cattle.

The invention also relates to a method for producing and purifying recombinant growth hormone from transgenic animal milk. Recombinant growth hormone may be human growth hormone, but without limitation specified. A transgenic mammal may be, without limitation, an animal belonging to a species of cattle.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. 1A-1B show the daily volume of milk collected from a transgenic cow obtained by fusion of a nucleated oocyte and fibroblast previously transfected with a plasmid containing a gene that encodes human growth hormone (hGH) and a promoter that controls its expression in mammary cells.

In FIG. 2A-2C show the count of bacteria found in milk collected from the same transgenic cow.

In FIG. 3A-3B show the biological activity of hGH found in the milk of the same transgenic cow.

In FIG. 4A shows the daily weight of hGH produced in the milk of the same transgenic cow. The indicated value and daily volume of milk collected from the transgenic cow are shown together graphically in FIG. 4B.

In FIG. 5A shows the concentration of hGH and insulin-like growth factor-1 (IGF-1) in the serum of the same transgenic cow and the daily mass of hGH produced in the milk of the same transgenic cow. The concentration of hGH in the serum of the transgenic cow and the daily mass of hGH produced in the milk of the transgenic cow are shown together graphically in FIG. 5B. In FIG. 5C together graphically depicts temporal concentration profiles of hGH and IGF-1 in serum of a transgenic cow.

In FIG. 6A and 6B show the serum concentration of hGH and insulin-like growth factor-1 (IGF-1) in two transgenic calves obtained by subcloning a cow, which is transgenic for the production of hGH in milk. In FIG. 6C and 6D together graphically depict temporal serum concentration profiles of hGH and IGF-1 for each of these transgenic calves.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a transgenic mammal other than humans, characterized by the production of unexpectedly high levels of recombinant growth hormone in milk. The specified mammal may be, without limitation, an animal belonging to the species of cattle. Other types of transgenic mammals may include, without limitation, the type of pig, the type of sheep, the type of goat or the species of rodent.

Recombinant growth hormone may be, without limitation, human growth hormone. Said molecule, also known as somatotropin, is a protein consisting of 191 amino acids with a molecular weight of approximately 22 kD. It is important for linear growth, and its applications are well known.

The invention also relates to a transgenic mammal, characterized in that the recombinant growth hormone produced in its milk self-stimulates the mammary glands of the animal to produce more milk containing the specified hormone.

The invention also relates to a plasmid containing a gene encoding a protein of interest operably linked to a beta casein promoter and a β-lactamase gene. The indicated protein of interest may be, without limitation, human growth hormone. The indicated plasmid may be the plasmid pRβhGH.

In a further embodiment, the plasmid further comprises a neomycin resistance gene for the selection of geneticin resistant cells. An example of such a plasmid is the plasmid pRNeo.

In a further embodiment, the plasmid contains a gene encoding a green fluorescent protein, such as GFP, which is under the control of the cytomegalovirus (CMV) promoter. An example of such a plasmid is the plasmid pRNeoGreen.

The invention also relates to a plasmid, such as the plasmid described above, which has been linearized by restriction digestion. In particular, the ApaLI restriction enzyme is used and the β-lactamase gene is excised.

The procedure for depositing the above plasmids is carried out under the terms of the Budapest Treaty. Name and address of depository DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany. Corresponding access numbers will be presented over time.

The invention further relates to a plasmid constructed on the basis of a plasmid containing the Neo resistance gene into which a modified shorter region of beta-casein was inserted above the region encoding hGH, such as pVEβcashGH. A linear fragment was obtained from plasmid pVEβcashGH by excision of the beta-lactamase gene.

The invention further relates to a method for transfecting genetic constructs using a combination of cationic lipids for use in liposomes.

Neo-mycin-resistant cell selection methods are also described in appropriate media, which are green fluorescence transgenic cell selection methods. These cells were carefully harvested so as to avoid cell damage.

The invention also relates to a method for transferring nuclei of cells trapped in G ₀ or in other periods of the cell cycle to nucleated oocytes of cows.

The invention relates to a method for transferring a transgenic embryo into a hormone-stimulated uterus of a cow.

A method for determining the health parameters of an animal according to the invention is described. Assays are performed using both serum and animal milk to determine such parameters.

The invention also relates to a method for producing a transgenic non-human mammal, which comprises obtaining a gene that encodes growth hormone, cloning a gene in a plasmid, the gene being operably linked to a promoter that will control gene expression in mammalian cells, producing as a result, an expression plasmid, transfection of somatic cells with an expression plasmid in order to incorporate the plasmid into the cell genome, resulting in transgenic somatic cells, removing e nuclei from a mature oocyte to produce an enucleated oocyte, fusion of a transgenic somatic cell with an enucleated oocyte, resulting in a unicellular embryo, implantation of the embryo in the uterus of a ready-for-fertilization mammal and monitoring pregnancy until the birth of the transgenic mammal.

The invention also relates to a method for producing a transgenic non-human mammal, comprising extracting somatic cells from the body of a female mammal, which is transgenic for the production of recombinant growth hormone in milk, optionally fibroblasts, removing the nucleus from a mature oocyte, to obtain the result of an enucleated oocyte, the fusion of one transgenic somatic cell with an enucleated oocyte, to obtain a unicellular embryo, implantation of the embryo in the uterus ready for fertilization of a mammal and monitoring of pregnancy up to the birth of a transgenic mammal.

The invention also relates to a method for producing a transgenic non-human mammal, comprising superovulation of a female non-human mammal, which is transgenic by production of recombinant growth hormone in milk, artificial insemination of a mammal with sperm obtained from a male non-transgenic mammal, excellent from a person to receive embryos, collection of embryos, implantation of embryos in the uterus of a ready-to-fertilize mammal, and pregnancy monitoring It is up to birth of the transgenic mammal.

The invention also relates to a method for producing a transgenic non-human mammal, comprising superovulation of a female non-human mammal, which is transgenic from the production of recombinant growth hormone in milk, artificial insemination of a mammal with sperm obtained from a male mammal other than a person who is transgenic for the production of the indicated recombinant growth hormone to obtain embryos, collection of embryos, implantation of embryos in the uterus ovogo to fertilize mammalian and monitoring of pregnancy until the birth of the transgenic mammal.

The invention further relates to a method for producing a transgenic non-human mammal, comprising superovulating a female non-transgenic non-human mammal, artificially inseminating a mammal with sperm obtained from a male non-human mammal, which is transgenic from the production of recombinant growth hormone to obtain embryos, collection of embryos, implantation of embryos in the uterus of a ready-to-fertilize mammal and monitoring pregnancy up to Denia transgenic mammal.

Recombinant growth hormone may be, without limitation, human growth hormone. A transgenic mammal other than humans may be, without limitation, an animal belonging to a species of cattle.

The invention further relates to a method for producing a protein, comprising producing a transgenic non-human mammal that produces unexpectedly high levels of protein of interest in milk, obtaining milk from a transgenic non-human mammal, and purifying the protein of interest from milk .

The invention also relates to a method for producing a protein of interest in a transgenic non-human mammal obtained by a method comprising obtaining a gene that encodes the protein of interest, cloning the gene in a plasmid, the gene being operably linked to a promoter that will control expression gene in mammalian cells, resulting in an expression plasmid, transfection of somatic cells, optionally fibroblasts, with a plasmid, in order to enable plasmid the midway into the genome of these somatic cells, receiving transgenic somatic cells, removing the nucleus from a mature oocyte, obtaining an enucleated oocyte, merging one transgenic somatic cell with an enucleated oocyte, obtaining a unicellular embryo, implanting the embryo into the uterus of a mammal ready for fertilization and monitoring pregnancy up the birth of a transgenic mammal.

The invention also relates to a method for producing a protein of interest in a transgenic mammal other than a human obtained by a method comprising extracting somatic cells from the body of a female mammal, which is transgenic for the production of said protein of interest in milk, not necessarily fibroblasts, removal of the nucleus from a mature oocyte, to obtain an enucleated oocyte, fusion of an ode to a transgenic somatic cell with an enucleated oocyte, to obtain one full-time embryo, implantation of the embryo into the uterus of a mammal ready for fertilization and monitoring of pregnancy up to the birth of a transgenic mammal.

The invention also relates to a method for producing a protein of interest in a transgenic non-human mammal obtained by a method comprising superovulation of a female non-human mammal that is transgenic for the production of said protein of interest in milk, artificial insemination of a mammal with sperm obtained from a male non-transgenic non-human mammal to receive embryos, collection of embryos, implantation of embryos in the uterus odotvoreniyu mammal and monitoring of pregnancy until the birth of the transgenic mammal.

The invention also relates to a method for producing a protein of interest in a transgenic non-human mammal obtained by a method comprising superovulation of a female non-human mammal that is transgenic for the production of said protein of interest in milk, artificial insemination of a mammal with sperm obtained from a male mammal other than a human that is transgenic in the production of said protein of interest to obtain embryos, collection of embryos, implantation of embryos in the uterus of a mammal ready for fertilization and monitoring of pregnancy up to the birth of a transgenic mammal.

The invention also relates to a method for producing a protein of interest in a transgenic non-human mammal obtained by a method comprising superovulation of a female non-transgenic non-human mammal, artificial insemination of a mammal with sperm obtained from a male mammal other than a human, which is transgenic according to production of said protein of interest to obtain embryos, collection of embryos, implantation of embryos in the uterus ready for fertilization NIJ mammal and monitoring of pregnancy until the birth of the transgenic mammal.

Transgenic mammals characterized by the production of unexpectedly high levels of protein of interest in their milk can be, without limitation, animals of the species of cattle. Other types of transgenic mammals may include, without limitation, the type of pig, the type of sheep, the type of goat or the species of rodent. The protein of interest, without limitation, may be human growth hormone.

The invention also relates to a transgenic mammal other than humans, a species of cattle that produces recombinant human growth hormone in milk, the genome of which contains an integrated plasmid, wherein the plasmid contains the human growth hormone gene and beta casein promoter , which controls the expression of said gene in mammalian mammary gland cells.

The invention further relates to a transgenic mammal that produces unexpectedly high levels of hGH, but there is no physical growth expected at such a high level of hGH production. Since transgenic cattle are affected by the presence of human growth hormone, it can be assumed that animals could grow at a rate faster than the growth rate of non-transgenic animals and suffer from conditions such as diabetes mellitus, hypertension, an increased risk of cardiovascular disease and enlargement of the organs of the body, including the liver, spleen, kidneys and heart. Such high levels of hGH should theoretically render the animal non-viable. However, this is not the case. A mammal, such as a cow, with dangerously high levels of foreign blood hormone, but which has excellent health and excellent recombinant protein performance, makes an unexpected inventive contribution.

The recombinant human growth hormone according to the invention is produced at unexpectedly high levels. The level of human growth hormone produced is approximately more than 1.0 g / l of milk. The hGH level may be more than about 2.0 g / l of milk. The level of hGH produced may also exceed approximately 3.0 g / l of milk. In another embodiment, the produced level of hGH may be approximately more than 4.0 g / l of milk. In yet another embodiment, the level of hGH produced may be greater than about 5.0 g / l of milk. In a further embodiment, the level of hGH produced may be about more than 6.0 g / l of milk. In a further embodiment, the level of hGH produced is from about 1.0 g / l of milk to 7.0 g / l of milk. In a further embodiment, the level of hGH produced is from about 2.0 g / L milk to 6.0 g / L milk. In another embodiment, the level of hGH produced is from about 2.0 to 5.0 g / l of milk.

In addition, the invention relates to a method for purifying recombinant growth hormone from milk of a transgenic mammal, as well as to analyzing said hormone. Purification methods may include chromatography and concentration steps. Various types of chromatography can be used, and these include ion exchange chromatography, reverse phase chromatography, molecular size exclusion chromatography, or affinity chromatography. Ion exchange chromatography may be anion exchange chromatography. Affinity chromatography may be immunoaffinity chromatography. In addition, multiple chromatography steps can be performed.

The invention also relates to a method for purification of recombinant growth hormone from transgenic non-human milk, which produces recombinant growth hormone, including clarification of transgenic non-human mammal milk, to obtain clarified milk and chromatography of clarified milk, c obtaining purified recombinant growth hormone as a result.

The invention also relates to a method for purification of recombinant growth hormone from transgenic non-human milk, which produces recombinant growth hormone, including clarification of transgenic non-human milk, to obtain clarified milk, chromatography of clarified milk by anion exchange chromatography using an enlarged layer, to obtain the material subjected to anion exchange chromatography, the implementation of reversed phase chromatography of the material subjected to anion exchange chromatography to obtain material subjected to reverse phase chromatography, performing anion exchange chromatography of the material subjected to reverse phase chromatography to obtain material subjected to anion exchange chromatography, performing molecular size exclusion chromatography of the material subjected to anion exchange chromatography, to obtain molecular size exclusion chromatography, concentration of ma Methods and material subjected to molecular exclusion chromatography, to obtain a concentrated material and implementation of molecular size exclusion chromatography of the concentrated material to obtain the purified recombinant growth hormone.

The invention also relates to a method for purifying recombinant growth hormone from transgenic mammal milk other than humans, which produces recombinant growth hormone, which includes clarifying milk obtained from a transgenic mammal to obtain clarified milk, immunoaffinity chromatography of clarified milk to obtain material subjected to immunoaffinity chromatography, reverse phase chromatography of a material subjected to immunoaffinity chromatography ii, obtaining a material subjected to reverse phase chromatography, performing anion exchange chromatography of a material subjected to reverse phase chromatography, obtaining a material subjected to anion exchange chromatography, performing molecular size exclusion chromatography of a material subjected to anion exchange chromatography, to obtain a material subjected to molecular size exclusion chromatography, the concentration of the material subjected to molecular exclusion chromatography phase, obtaining concentrated material and performing molecular size exclusion chromatography of the concentrated material to obtain purified recombinant growth hormone.

The recombinant growth hormone in the above purification methods, without limitation, may be human growth hormone. A transgenic mammal, without limitation, may be a cattle mammal.

The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of a variety of conditions and parameters commonly encountered in the enzymatic production of chemicals and in protein purification methods that are obvious to those skilled in the art are the essence of the invention and are within the scope of the invention.

EXAMPLE 1

The construction of expressing plasmids

The authors created a construct that carried most of the promoter of the bovine beta casein gene, including a short fragment of the 5'-non-coding region of the beta casein gene fused to the coding sequence of the human growth hormone gene. The beta casein region used in various designs was reduced from 3.8 kb. up to about 1.3 kb The hGH gene spans from about 2 to 2.2 kb, depending on whether the internal polyA signal is turned on.

The expression cassette was placed in a polylinker of a conventional pUC or pBS cloning vector.

The specified promoter provides tissue-specific and developmentally regulated gene expression under its control, similar to beta-casein, and in this case heterologous hGH.

The most typical plasmid is pRβhGH, which carries a full-sized bovine beta casein promoter fused to the human growth hormone gene coding sequence.

The other constructs described are mainly derived from the original plasmid, which is indicated so as to improve the selection of transfected cells or to increase the efficiency of DNA integration into the bovine cell genome.

In the initial period, cotransfection was carried out with a plasmid containing the gene for resistance to geneticin, facilitating selection, but then other constructs carrying the NPT gene for resistance to neomycin in the same vector containing the hGH expression cassette were used. An example of such a plasmid is the plasmid pRNeo.

Another plasmid was obtained for the constitutive expression of a green fluorescent protein, which contains the CMV promoter, a plant-derived enhancer (alpha-alpha), and a green fluorescent protein gene from A. victoria jellyfish. An example of such a plasmid is the plasmid pRNeoGreen.

In addition, another plasmid was created. The plasmid was constructed on the basis of a plasmid containing the Neo resistance gene, into which a region of a modified shorter beta-casein promoter was inserted above the region encoding hGH. This plasmid is a plasmid pVEβcashGH.

Other constructs were created in which the β-lactamase region was excised by ApaLI restriction, and the linear fragment containing the complete expression cassette was purified after agarose gel electrophoresis and extracted from the gel.

Constructs were analyzed using restriction enzymes and DNA sequencing, and their ability to induce hGH expression was pre-tested in mammary cell lines using recognition with a fluorescent antibody.

The preparation of plasmid pVEβcashGH will be described in detail as an example in this part of the genetic constructs.

Obtaining pVEβcashGH

The aim of this design is to obtain minimal elongation of the beta-casein promoter region for controlling the specifically regulated transcription of the hGH gene, fused directly further along with its polyA signal in the host organism.

The use of pVEX as a starting vector makes it possible to use the neomycin resistance gene regulated by the tk promoter already present in this plasmid. The early SV40 promoter, containing 554 bp, was removed by restriction with StuI and NdeI enzymes, and the last site was filled using Klenov enzyme and self-ligation of the resulting vector.

A short beta-casein promoter was obtained after PCR amplification of a 1.3 kbp fragment. from the promoter region of the original gene of 3.8 kb, using the following oligonucleotides as primers:

PB1 5'TCTACTCGAGGATCATCTATCTGTCCCAAAG (SEQ ID NO: 1)

and

PB2 5'CTAGGATCCAATGATCTGATTTTTGTGG (SEQ ID NO: 2).

This fragment contains 1230 bp canonical promoter plus 49 bp the first non-coding exon of the beta casein gene.

The hGH gene fragment was obtained by PCR on the original bovine hGH genomic clone using the following oligonucleotides as primers:

PB4 5'CTAGGATCCATGGCTACAGGTAAGCGCC (SEQ ID NO: 3)

and

GHTE 5'ATGCTGTGTCTGGACGTCCT (SEQ ID NO: 4)

The ends of the beta-casein promoter fragment were blunted by Klenov enzyme, and the fragment was inserted into the BamHI site to be filled in pVEX. After selecting recombinant clones, the authors chose a specific direction suitable for using the unique HindIII site located below in pVEX to embed the hGH coding region.

To this end, the ends of the hGH fragment were also blunted and also filled the HindIII site of the plasmid. Clones were selected that contain the beta-casein promoter and hGH, correctly fused to express hGH only under the control of this promoter.

The size of this plasmid is approximately 8.5 TPN

Somatic cell transfection

The plasmids pRβhGH (together with another plasmid with the gene for resistance to geneticin), pRNeo, pRNeoGreen or pVEβcashGH were then used to transfect the primary culture of somatic cells using a method based on calcium phosphate or liposomes. Typically, fetal calf fibroblasts were used for transfection.

Transfected cells were selected by adding geneticin to the culture. After a period of 2 to 8 weeks, cells that were resistant to geneticin were suitable for use as donor cells to obtain transgenic clones. Selected transfected cells were analyzed by PCR for expression cassette contents to ensure appropriate nuclear transfer to create transgenic embryos.

EXAMPLE 2

Removal of nuclei from oocytes and transfer of metaphase nuclei to mature enucleated oocytes

In vitro collection and maturation of cow oocytes

Cow oocytes were aspirated from the ovaries obtained from the slaughterhouse, and they matured in TCM-199 + 5% FCS at 39 ° C for 24 hours. The ripening medium was equilibrated with CO ₂ for at least 2 hours before use. Mature oocytes were stripped by shaking for 2 minutes in warm TL-HEPES with 1 mg / ml bovine testicular hyaluronidase.

Nuclear transfer using cumulus cells

Kernel Removal

Nuclei were mechanically removed from oocytes using Narishige hydraulic micromanipulators and Nikon Diaphot microscopy. Nuclei were removed using cone-shaped and pointed pipettes at 20 μm. Oocytes were pre-stained with 5 μg / ml bisbenzimide dye (Hoechst 33342, Sigma Chemical Co., St. Louis, MO, USA) for 20 minutes. The content of nuclei from metaphases was removed by visualization of stained chromosomes in ultraviolet light. Metaphase chromosomes were evaluated after aspiration inside the pipette. The transgenic somatic cell was transferred to the perivitelline space and placed close to the enucleated oocyte.

Merger

The transgenic somatic cell and enucleated oocyte were manually aligned in the fusion chamber so that the fused membranes were parallel to the electrodes. This was accomplished using a glass pipette to manipulate the embryos.

Electrofusion

The fusion was carried out using a single electric pulse of 180 volts / cm for 15 μs (BTX Electro 200 cell manipulator BTX Inc., San Diego, CA, USA), and controlled by observing in the BTX graphic pulse optimizer analyzer. The chamber for pulsed action on embryos consisted of two electrodes of 0.5 mm steel wire mounted at a distance of 0.5 mm from the microscope slide. Putative zygotes were monitored for fusion, lysis, and fragmentation.

Development Assessment

Zygotes were evaluated 48 hours after fertilization in relation to crushing and after 7-9 days in relation to the development of morula or blastocysts.

EXAMPLE 3

Cell and embryo culture

Various donor cells, systems for culturing and processing recipient oocytes were tested in an experiment, the purpose of which was to simplify the methods and increase the survival rate of embryos in the cattle cloning program. Three reconstructed embryo culture systems were used in the case of using adult fibroblasts as donor cells: TCM-199 + 5% FCS, Menezo + 5% FCS (in both cases using VERO cells as a co-culture) and SOF without co-culture, but at a lower concentration of O ₂ . SOF medium was also used to cultivate reconstructed embryos when the donor cell was a genetically and non-genetically modified fetal fibroblast. Finally, when genetically modified fetal fibroblasts were used as donor cells, the recipient oocytes were pretreated with roskovitin (R) to stop meiosis and optimize the usability of the recipient. Oocytes were aspirated from ovaries obtained from a slaughterhouse, and they matured in TCM-199 + 5% FCS at 39 ° C for 24 hours. In the case of the R-treated group, before maturation, the oocytes were incubated with 25 μM R in TCM-199 + 5% FCS for 24 hours at 39 ° C. Mature oocytes were stripped by shaking for 3 minutes in TL HEPES with 1 mg / ml hyaluronidase from bovine testes. Metaphases were determined and nuclei were removed from oocytes upon imaging using Hoechst 33342 (5 μg / ml) in UV light (<6 seconds). Adult body fibroblasts from an Angus bull and fetal fibroblasts from a 45-day-old female Jersey female were used as donor cells. Transfection with constructs containing the resistance gene was carried out using liposomes. After selection with geneticin for 10-15 days, the donor cells in the G ₀ / G ₁ stage were fused with enucleated oocytes using an electric pulse. After 3 hours, activation was induced by incubation in TL-HEPES with 5 μM ionomycin for 4 minutes and 2 mm 6-DMAP for 3 hours. The oocytes were then washed with TL-HEPES and cultured either in TCM-199 + 5% FCS + 10 g / l albumin, or in Menezo + 2% FCS, together with VERO cells, or in SOF medium and atmosphere 5% CO ₂ + 5% O ₂ + 90% N ₂ . Typically, two blastocysts were transferred non-surgically to the recipient cow and pregnancy was determined at 30-35 days by ultrasonography. Crushing (48 hours), development to blastocysts (7-9 days) were recorded and analyzed using the Chi-square test. Crushing and development rates of pre-blastocysts were higher when embryos were cultured in SOF. However, no differences were found in the proportion of pregnancy cases associated with different cultivation conditions or the source of donor cells. Stopping meiotic maturation for 24 hours did not jeopardize the ability of recipient oocytes to develop. Therefore, treatment with roskovitin can be used to increase the suitability of oocytes for NT methods. See table 1 below.

The percentages in columns with different superscripts were different (P <0.05).

The implanted cows were provided with the opportunity for a normal pregnancy to progress through to natural birth. Under appropriate conditions, it is possible to use the surgical method (cesarean section) for delivery. Newborns are fed colostrum rich in Ig for the first 48 hours and then use synthetic, later natural feeds (all feeds do not contain animal compounds).

EXAMPLE 4

Testing transgenic calves and produced

recombinant protein

In this example, the authors provide a complete description of the tests performed on a particular transgenic calf, which was obtained as a result of the method described in examples 1-3, and testing the recombinant protein produced by it. However, it should be understood that the same set of analyzes is carried out in the case of animals that were born due to the use of other methods for producing transgenic calves, such as the methods that will be described in examples 5 and 6 below.

Using PCR reactions performed on DNA purified from calves leukocytes, using DNA from non-transgenic Jersey calves as a negative control, it was confirmed that the cow beta-casein promoter and the gene encoding hGH are included in the transgenic calf cell genome. They can be identified together in the form of a unique DNA fragment that differs from the homologous beta-casein gene of the animal.

Using a Pharmacia automatic sequencer, it was confirmed that the inserted gene sequence corresponds to 100% the gene encoding hGH. It contains introns, a secretion signal, and a terminator. The cow beta-casein promoter, which regulates the expression of the same hGH gene in the calf, was also sequenced. All of these elements exactly coincided with the theoretically expected sequence from the genetic construct used to transform the cells from which the clones were created.

Knowing the accuracy of the genetic phase of the experiment, the authors went on to prove that the produced recombinant protein is a putative protein and coincides in its physical and chemical characteristics with natural hGH.

For this purpose, the authors needed to get milk from the heifer, since the beta-casein promoter provides expression of the recombinant protein only in the mammary glands when the animal is at the age of milk production.

Then, the transgenic heifer was induced to produce milk when it reached ten months of age. By this time, the heifer weighed 240 kg (approximately 530 pounds).

The first phase of this treatment was the combined subcutaneous administration of estrogens (estradiol benzoate, hysteren, Instituto Rosenbusch) and progestogens (medroxyprogesterone acetate, Pronal, Aton), which included 5 consecutive uses of each drug at a dose of 0.1 mg / kg and 0 , 25 mg / kg, respectively, every 48 hours (i.e. on days 1, 3, 5, 7, and 9, assuming that treatment begins on day 1).

The second phase included subcutaneous administration of dexamethasone (decadron, Sidus) and oxytocin (orastin, Hoechst Marion Rousseb); however, the total amount of the first agent, amounting to 20 mg, was injected over a period including 18-20 days (one third of the indicated total weight every day), and 3 applications of 50 MEU of the last agent in 21-23 days.

The above information is summarized in table 2:

As expected, the cow began to give colostrum a day after the end of the treatment, and then the gradually produced liquid turned into milk in quality. The collected fluid was appropriately stored and carefully analyzed.

Several tests were carried out, the results of which are shown in FIG. 1-4, using colostrum and milk (for simplicity, colostrum and milk will hereinafter be referred to as "milk", unless it is necessary to distinguish between them). First, the volume of milk collected was measured. The initial milk yield (the first five days of lactation) was 1650 ml / day. During the first month of production, two manual milking per day were carried out, one in the morning and one in the evening (the contribution of each udder to the final volume could be noted); whereas from the second month, as milk production began to increase, three milking per day (morning, noon and evening) were performed. Daily volumes grew more or less continuously until about 10,000 ml were reached three months after the first milking. Detailed information on this topic (FIG. 1A) can be seen on the curve of daily milk volumes indicated by dates (FIG. 1B).

In parallel with milk volume measurements, microbiological analyzes of milk were carried out, the results of which (FIG. 2A) are visible on the corresponding CFU graphs (colony forming units) depending on the date and in FIG. 2B-2C.

Biological activity (hGH) was also evaluated in an in vitro biological activity assay in NB2 cell culture (FIGS. 3A-3B). It was confirmed that the biological activity of the recombinant hGH produced in heifer milk, within the limits of the method error, has normal values for natural human hGH. In addition, the resulting milk was examined using Western blotting, which revealed the main band corresponding to intact hGH. Additional minor bands corresponding to split variants and aggregates were also identified, as expected in such production systems.

Based on information related to biological activity and daily volumes, daily production of hGH can be calculated using equation 1, in which mhGH means the mass of hGH obtained per day in milligrams; BA - biological activity in international units; SA is the specific activity of hGH (3 IU / mg) and V is the daily volume of milk collected. The data are shown in FIG. 4A. A graph of daily mass hGH is shown in FIG. To establish a visual correlation between the daily mass of hGH and the daily volume of milk, the last indicator was also plotted on the graph in FIG.

(Equation 1)

As can be seen on the basis of this information, the daily mass of hGH in milk and the daily volume of milk tend to increase as the cow develops (although the former is more so), which tends to increase productivity in relation to recombinant hormone (gram hGH per liter of milk). It can be noted that this productivity varies from 2 g of hGH per liter (first milking), when the product is colostrum, to an average of 5 g of hGH per liter of milk, starting from the second month of lactation. Thus, an unexpectedly high yield of human growth hormone was obtained in the form of productivity, which increases more or less continuously as the fluid is qualitatively converted from colostrum to milk.

Although at present the mass of hGH produced per day is really outstanding, it should be noted that since the cow has not yet fully grown, the trend towards an increase in the mass of recombinant protein produced per day should continue in the future until reaching a maximum.

In parallel with the tests carried out on milk, a different set of assays for cow serum was performed, the results of which are shown in FIGS. 5A-5C. First, measurements were made of the concentration of hGH in the serum of the cow. The results (FIG. 5A) are plotted along with the daily mass of hGH in milk in order to be able to compare both values (FIG. 5B).

Reference serum GH limits (in humans) are 0.06-5 ng / ml. Assuming that the cattle have a hypothetically similar range, it can be noted that, with the exception of the beginning, the entire hGH curve in the serum of a transgenic cow, depending on the date, lies well above the indicated limit. Despite this fact, it must be taken into account that although hGH and the corresponding bovine hormone (bGH) are very similar in terms of their amino acid sequence and 3D structure, the first, although fully functional, is not fully active in cattle.

Therefore, in order to evaluate the potential health risk for the cow of these high serum hGH levels, serum concentrations of IGF-1 (insulin-like growth factor 1, also known as somatomedin C) were measured (Fig. 5A). Growth hormone functions primarily through IGF-1, which is produced in the liver. Since IGF-1 mediates many of the effects of growth hormone in vivo cell division and its metabolic effects, the evaluation of IGF-1 is a valuable diagnostic tool for indirectly evaluating suspected growth hormone-related disorders. Thus, IGF-1 is a reliable indicator of bioavailable growth hormone. The results of these analyzes are shown in FIG. 5C together with the results corresponding to hGH in order to be able to simultaneously visualize both values.

In addition, serum IGF-1 and hGH were measured in the group of non-transgenic cows in order to have a control group and thus obtain a comparison with data corresponding to the transgenic cow. The average measurement values of both the protein in the case of a nontransgenic group and the protein of the transgenic cow are shown in table 3 below.

Table 3 Averages [IGF-1] in serum [hGH] in serum Non transgenic 123.40 0.28 Transgenic 484.02 656.98

It should be noted that the average serum concentration of hGH in the nontransgenic group lies within the expected range, while the average value for the transgenic cow greatly exceeds the upper limit of this range. In addition, it is indisputable that the average level of IGF-1 in the serum of a transgenic animal is certainly higher than the level corresponding to non-transgenic cows, which is the main difference.

Therefore, although a high concentration of hGH in the serum of a transgenic cow (which is known to cause disorders in a person with serious consequences, such as diabetes mellitus, hypertension, an increased risk of cardiovascular disease and enlargement of organs of the body, including the liver, spleen, kidneys and heart) should theoretically to make an animal non-viable, this, obviously, cannot constitute an obstacle to its health and well-being. A cow with dangerously high levels of a foreign hormone in its blood, but with excellent health and outstanding recombinant protein productivity, represents an unexpected and inventive contribution.

Another innovative aspect of the present invention is that recombinant hGH, which penetrates the blood circulation, stimulates the mammary gland to produce more milk. The specified action is achieved indirectly, i.e. through the action of IGF-1. This molecule increases blood flow through the mammary gland, providing it with important precursors for the synthesis of milk fat, protein and lactose. Thus, IGF-1 acts by directing nutrients through the bloodstream to cells in the udder, where they contribute to milk production. Therefore, a self-stimulating animal is obtained, since the recombinant hGH produced in the milk of a transgenic cow stimulates a prolonged increase in the volume of milk given to the animal by stimulating its mammary gland, with corresponding release of more hGH in the milk of the animal.

EXAMPLE 5

Obtaining transgenic calves by subcloning

Five tissue samples were taken from the ear of the transgenic calf using a needle with a diameter of 1.5 mm, the end of which was previously beveled for this purpose. Samples were sent chilled to the laboratory in a PBS-based medium containing antibiotics and antifungal agents.

Then, tissue samples were incubated for 72 hours in MEM medium with 10% fetal bovine serum antibiotics at 39 ° C and in an atmosphere of 5% CO ₂ . At the end, tissue samples were removed and fibroblasts at the periphery of the plate were allowed to grow until the layer merged. After reaching the indicated fibroblasts were incubated for at least 5 days without changing the culture medium in order to achieve their synchronization at the stage G ₀ , which was evaluated using visualization under a microscope.

After trypsin treatment, individual fibroblasts were fused with enucleated bovine oocytes according to Example 2, and the embryos thus obtained were cultured in SOF medium and 5% CO ₂ + 5% O ₂ + 90% N ₂ atmosphere up to the blastocyst stage. After this, as a rule, two fibroblasts were transferred non-surgically to the recipient cow, and pregnancy was determined on day 30-35 by ultrasonography.

The implanted cows were provided with a normal course of pregnancy up to natural birth. Under appropriate conditions, it is possible to use the surgical method (cesarean section) for delivery. Newborns are fed colostrum rich in Ig for the first 48 hours and then use synthetic, later natural feeds (all feeds do not contain animal compounds).

In FIG. 6A and 6B show measurements of the serum concentration of hGH and IGF-1 performed in parallel for two transgenic animals obtained by subcloning a transgenic cow, and the results of these analyzes are shown in FIG. 6C and 6D, respectively, in order to be able to simultaneously visualize both values for each animal. Since the calves are still young, the values of both hGH and IGF-1 are still in the corresponding range of the standard, but it is assumed that they will be increased in the same way as in the transgenic cow, from which these two clones were obtained.

EXAMPLE 6

Obtaining transgenic calves by artificial

transgenic cow fertilization

In this example, an alternative method for producing transgenic cattle will be described. This method includes superovulation of a transgenic cow using hormonal treatment; artificial insemination of said cow; re-collection of the embryos thus obtained; implantation of said embryos with surrogate cows; and the development of pregnancy up to the birth of animals. A description of this method is presented below.

Superovulation

On the morning of 1 day (i.e., the day the method began) 150 μg of prostaglandins (D (+) - chlorprostenol, arsaprost, Arsa) were administered intramuscularly to the transgenic cow. The animal was subjected to a diet, giving 4 kg of feed per day containing about 15% protein (before 1 day, the animal was given 2 kg of feed with the same protein content). On the morning of day 8, the progesterone device for CIDR (controlled internal drug release) was placed intravaginally. In addition, 50 mg of progesterone and estradiol were administered intramuscularly. The amount of feed fed to the animal increased to 6 kg / day with the same protein content. Then, two intramuscular injections of FSH and LH (PLUSET®, Calier) were introduced on days 12, 13 and 14 (one in the morning and the other in the evening). The next day (day 15), the treatment was continued by intramuscular injection of two PLUSET® injections (one in the morning and the other in the evening) and two injections of prostaglandins of 150 μg each (the same administration schedule). On the morning of day 16, CIDR was removed. The total amount of PLUSET® administered during the superovulation phase was 350 IU.

Fertilization

This phase included three consecutive injections (the first and second - in the morning and evening on day 17, respectively, and the last - in the morning on day 18) of sperm from a donor-bull of the Jersey breed, which was received in advance and stored frozen to maintain viability sperm cells.

Embryo collection and implantation in surrogate cows

Embryos were collected by washing both horns of the cow uterus using 1 L of DMPBS (Nutricell®) in the morning on day 26. Immediately after this, two embryos were transferred non-surgically to the recipient cow, and pregnancy was determined on day 30-35 by ultrasonography.

The implanted cows were provided with the opportunity for a normal pregnancy to progress through to natural birth. Under appropriate conditions, it is possible to use the surgical method (cesarean section) for delivery. Newborns are fed colostrum rich in Ig for the first 48 hours and then use synthetic, later natural feeds (all feeds do not contain animal compounds).

Since the formation of biological offspring obeys Mendel’s law, half of the animals born as a result of using the method described above must be transgenic, and half of them must be males and the other half females. Therefore, there is a high probability of obtaining a transgenic male (founder animal), the sperm of which can be used to found the main bank of the Jersey transgenic sperm used to fertilize superovulated transgenic / non-transgenic cows in order to breed the transgenic herd.

EXAMPLE 7

Purification of Recombinant hGH from Milk

In the event that the verified approximate molecular weight, Western blotting results, and the biological activity of the recombinant hGH produced in the calf’s milk turn out to be appropriate, they will undergo a complete purification process, since in the production of a biopharmaceutical product it is imperative that the protein of interest is purified to homogeneity so that avoid the presence of possible contaminants in the specified product. The specified method includes the stage of obtaining skim milk by centrifugation and dilution of the supernatant obtained in order to achieve better solubility of recombinant hGH, which, under appropriate conditions, may be retained in casein micelles (clarification); passing the resulting solution through an enlarged layer anion exchange chromatography column (an alternative to this step is to use an immunoaffinity column, see Example 8 below); the resulting solution is subjected to reverse phase HPLC (C4); then fractions enriched in recombinant hGH are subjected to anion exchange chromatography.

The purified material is desalted, concentrated and subjected to molecular size exclusion chromatography. In this case, molecular weight separation is carried out in order to obtain pure recombinant hGH.

The method of purification of human growth hormone (hGH) from milk includes the following stages in order: (a) clarification, (b) anion exchange chromatography using an expanded layer, (c) reverse phase chromatography, (d) anion exchange chromatography, (e) molecular size exclusion chromatography (desalination), (f) concentration and (g) molecular size exclusion chromatography.

Lightening

Fresh milk was mixed with a sufficient amount of Tween 80 to obtain a 0.5% solution. After tween 80 was added, 2M Tris-HCl was added until a pH of 7.3 ± 0.1 was obtained. The product was then homogenized for 30 minutes and then centrifuged at 14,000 g to separate the fat layer. Then, the resulting solution was diluted with 0.5% Tween 80 until a conductivity of less than or equal to 1500 μS / cm was obtained. The pH was adjusted to 7.3 ± 0.1 using 2M Tris-HCl, and then the product was filtered through a 0.8 μm diameter pore membrane and stored accordingly.

Anion exchange chromatography

using extended layer

The substance obtained in the previous step is chromatographed using an anion exchange matrix according to the following parameters:

1. Equipment:

A. Column:

1) diameter: 5 cm;

2) layer height: 30 cm (compact layer);

3) matrix:

a) Streamline Q XL (Amersham);

b) volume: 600 ml.

2. Solutions and buffers:

A. 0.5 N NaOH;

B. 20% ethanol;

C. Buffer A: 20 mm Tris-HCl, pH 7.3;

D. Buffer B: 500 mm Tris-HCl, pH 7.3;

E. Buffer C: 20 mm Tris-HCl, 150 mm NaCl, pH 7.3;

F. Buffer D: 20 mm Tris-HCl, 500 mm NaCl, pH 7.3;

3. Material subjected to chromatography:

A. clarified milk;

B. Sample parameters:

1) volume: 25 ± 5 l;

2) conductivity: <1500 μS / cm;

3) pH: 7.3 ± 0.1.

To balance the column, 1.5 column volumes (“vol. Col.”) (900 ml) of purified water were passed through it at a flow rate of 115 ± 5 cm / hour (downward flow). Then, the following solutions or buffers were successively passed through it in the amounts indicated in detail below with a flow rate of 230 ± 30 cm / h (upward flow): 3.0 vol. (1800 ml) 0.5 N NaOH; 3.0 vol. (1800 ml) purified water; 1.0 vol. (600 ml) of buffer B and finally 3.0 vol. (1800 ml) of buffer A.

After equilibration of the column, a chromatography material was applied. The specified application was carried out at 12 ± 3 ° C and a flow rate of 230 ± 30 cm / hour. Then elution was carried out with a flow rate of 115 ± 15 cm / h and at the same temperature. Firstly, a sufficient amount of buffer A (upstream) was passed through the column, and secondly, the solutions and buffers passed in detail below, in the following order (downward flow): 1.5 vol. (900 ml) buffer A; 2.0 vol. (1200 ml) of buffer C and finally 2.0 vol. (1200 ml) of buffer D.

After this stage, the following solutions and buffers were successively passed through the column in amounts indicated in detail below to purify it: 1.5 vol. (900 ml) purified water; 1.5 vol. (900 ml) 0.5N NaOH; 2.0 vol. (1200 ml) purified water; 1.0 vol. (600 ml) buffer B; 1.5 vol. (900 ml) of purified water and finally 1.5 vol. (900 ml) 20% ethanol.

Selected fractions containing hGH were analyzed for total proteins (Bradford method) and for the protein of interest (via RIA) and stored at 2-8 ° C.

Reverse phase chromatography

The substance obtained as a result of the previous stage was subjected to chromatography according to the following parameters:

1. Equipment:

A. Column:

1) diameter: 4 cm;

2) layer height: 48 cm;

3) matrix:

a) BakerBond-butyl broad-porous (C4), with a pore size of 15 μm, prepared for an LC column (Baker);

b) volume: 600 ml.

2. Solutions and buffers:

A. Mobile phase 1 (MP1): 30 mm NaHCO ₃ , pH 7.2: purified water: acetonitrile (35:55:10);

B. Mobile phase 2 (MP2): 30 mm NaHCO ₃ , pH 7.2: purified water: acetonitrile (20:10:70);

C. 50% methanol.

3. Substance subjected to chromatography:

A. The combined selected fractions obtained as a result of the previous stage;

B. Sample parameters:

1) volume: 30 ± 15 l;

2) pH: 7.3 ± 0.3.

In order to balance the column, the following solutions or buffers were passed through it in the amounts detailed below, with a flow rate of less than or equal to 478 cm / h: 0.3 vol. (180 ml) 50% methanol; then a gradient of 50% methanol-MP2 was applied, starting from a ratio of 100: 0 of said solutions to achieve a ratio of 0: 100 of said solutions in a total volume of 1.0 vol. (600 ml); after the end of the gradient, 1.0 vol. (600 ml) MP2; then the gradient MP2-MP1 was applied, starting with a ratio of 100: 0 of these solutions until a ratio of 0: 100 of these solutions in a total volume of 1.0 vol. (600 ml), and finally 2.0 vol. Col. Was passed through the column. (1200 ml) MP1.

After equilibrating the column, the material to be chromatographed was filtered through a membrane with a pore diameter of 0.45 μm and then immediately applied. The specified application was carried out at 20 ± 5 ° C and a flow rate of less than or equal to 238 cm / hour. Then, elution was carried out at a rate of 478 ± 78 cm / h at the same temperature and then the solutions and buffers, which are described in detail below, were passed in the following order: 1.0 vol. (600 ml) MP1; gradient MP1-MP2, starting from the ratio of 65:35 of these solutions until the ratio of 45:55 of these solutions in a total volume of 18.0 vol. (9000 ml); 1.0 vol. (600 ml) MP1-MP2 in the ratio of 45:55, and finally 2.0 vol. (1200 ml) MP2.

After the end of this stage, a gradient of MP2-50% methanol was used to clean the column, starting from a ratio of 100: 0 of said solutions until a ratio of 0: 100 of said solutions in a total volume of 1.0 vol. (600 ml) and finally 2.0 vol. Col. Was passed through the column. (1200 ml) 50% methanol.

The fractions obtained by this chromatography were analyzed in homogeneous 20% SDN-PAGE and in relation to oxidized hGH, and, depending on the results, selection was performed. Then, the selected hGH-containing fractions were analyzed for total proteins (Bradford method) and stored at 2-8 ° C.

Anion exchange chromatography

The material obtained from the previous step was chromatographed using an anion exchange matrix as follows:

1. Equipment:

A. Column:

1) diameter: 5 cm;

2) layer height: 25 cm;

3) matrix:

a) Source 30Q (Pharmacia);

b) volume: 500 ml.

2. Solutions and buffers:

A. 20% ethanol;

B. Solution K: 0.5N NaOH, 3M NaCl;

C. Solution L: 50 mm Tris, pH 7.50;

D. Solution M: 0.1 N HCl, 3M NaCl;

E. Mobile phase 3 (MP3): L solution: acetonitrile (70:30);

F. Mobile phase 4 (MP4): 50 mm Tris, 0.1 M NaCl, pH 7.50: acetonitrile (70:30).

3. Substance subjected to chromatography:

A. Selected fractions obtained from the previous step;

B. Sample parameters:

1) volume: 4.5 ± 1 l;

2) pH: 7.2 ± 0.2.

In order to balance and disinfect the column, the following solutions or buffers were successively passed through it in the amounts detailed below, with a flow rate of less than or equal to 183 ± 20 cm / h: 1.0 vol. (500 ml) purified water; 1.0 vol. (500 ml) of solution K; 1.0 vol. (500 ml) of solution L and, finally, 1.0 vol. (500 ml) MP3.

After equilibration of the column, a chromatographic material was applied. The specified application was carried out at 20 ± 5 ° C and with a flow rate of less than or equal to 183 cm / hour. Then, elution was carried out with a flow rate of 183 ± 20 cm / h at the same temperature, and then the solutions and buffers, which are described in detail below, were passed in the following order: 1.0 vol. (500 ml) MP3; gradient MP3-MP4, starting from the ratio of 15:85 of these solutions until the ratio of 25:75 of these solutions in a total volume of 5.0 vol. (2500 ml), and finally 2.0 vol.col. was passed through the column. (1000 ml) MP4.

After the end of this stage, the following solutions or buffers were successively passed through the column in amounts indicated in detail below to purify it: gradient MP4-purified water, starting from a ratio of 100: 0 of said solutions until reaching a ratio of 0: 100 of said solutions in a total volume of 0 , 5 about.col. (250 ml); 0.5 vol. (250 ml) purified water; gradient purified water-solution K, starting from a ratio of 100: 0 of said solutions until a ratio of 0: 100 of said solutions in a total volume of 0.5 vol. (250 ml); 1.0 vol. (500 ml) of solution K; gradient solution K-purified water, starting from a ratio of 100: 0 of said solutions until a ratio of 0: 100 of said solutions is reached in a total volume of 0.5 vol. (250 ml); 0.5 vol. (250 ml) purified water; gradient purified water-solution M, starting from a ratio of 100: 0 of said solutions up to reaching a ratio of 0: 100 of said solutions in a total volume of 0.5 vol. (250 ml); 1.0 vol. (500 ml) of solution M; gradient solution M-purified water, starting from a ratio of 100: 0 of said solutions until a ratio of 0: 100 of said solutions is reached in a total volume of 0.5 vol. (250 ml); 1.0 vol. (500 ml) purified water; gradient purified water-solution L, starting from a ratio of 100: 0 of said solutions up to reaching a ratio of 0: 100 of said solutions in a total volume of 0.5 vol. (250 ml); 0.5 vol. (250 ml); 0.5 vol. (250 ml) of solution L; gradient solution L-purified water, starting from a ratio of 100: 0 of said solutions until a ratio of 0: 100 of said solutions is reached in a total volume of 0.5 vol. (250 ml), and finally 1.5 vol. (750 ml) of purified water.

Selected fractions containing hGH were analyzed for total proteins (Bradford method) and stored at 2-8 ° C.

Molecular Size Exclusion Chromatography

The material obtained from the previous step was chromatographed using a molecular size exclusion chromatography matrix, as follows:

1. Equipment:

A. Column:

1) diameter: 5 cm;

2) layer height: 25 cm;

3) matrix:

a) Cellufine GH25 (Millipore);

b) volume: 500 ml.

2. Solutions and buffers:

A. 0.5 N NaOH;

B. 20% ethanol;

C. Buffer C: 150 mm NaH ₂ PO ₄ , pH 7.2;

D. Buffer G: 320 mm glycine, 10 mm NaH ₂ PO ₄ , 0.1% tween 80, pH 6.9.

3. Substance subjected to chromatography:

A. Selected fractions obtained from the previous step;

B. Sample parameters:

1) volume: 0.5 ± 0.2 l;

2) pH: 7.5 ± 0.5.

In order to balance and disinfect the column, the following solutions or buffers were successively passed through it in the amounts indicated in detail below with a flow rate of less than or equal to 180 cm / h: 1.0 vol. (500 ml) purified water; 1.0 vol. (500 ml) 0.5N NaOH; 0.5 vol. (250 ml) purified water; 0.5 vol. (250 ml) buffer C and finally 2.0 vol. (1000 ml) of buffer G.

After equilibration of the column, a chromatographic material was applied. The specified application was carried out at 20 ± 5 ° C and a flow rate of 183 ± 20 cm / h. Then elution was carried out at the same flow rate and 1.0 vol. Col. Was passed through the column at the same temperature. (500 ml) of buffer G as many times as needed to complete the cycles.

After this stage, the following solutions or buffers were successively passed through the column in amounts indicated in detail below to purify it: 0.5 vol. (250 ml) purified water; 1.0 vol. (500 ml) 0.5N NaOH; 0.5 vol. (250 ml) purified water; 0.5 vol. (250 ml) buffer C; 0.5 vol. (250 ml) of purified water and finally 1.5 vol. (750 ml) 20% ethanol.

Selected fractions containing hGH were analyzed for total proteins and stored at 2-8 ° C.

Concentration

The fractions obtained from the previous example were concentrated according to the conditions described below:

1. Equipment:

A. Peristaltic pump: Watson Marlow - Catalog No. 302S;

B. Handsets: Watson Marlow - Catalog No. 902.0080.016;

C. Hub: Prep Scale Millipore - CDU Catalog Number F006LC.

2. Solutions and buffers:

A. 0.28% sodium dodecyl sulfate (SDS);

B. 0.06% triton;

C. 0.125n NaOH;

D. Buffer G: 320 mm glycine, 10 mm NaH ₂ PO ₄ , 0.1% tween 80, pH 6.9.

3. The processed substance:

A. Selected fractions obtained from the previous example;

B. Sample parameters:

1) volume: 1.0 ± 0.5 l;

2) conductivity: 1200 ± 100 μS / cm;

3) pH: 6.9 ± 0.1.

The equipment was first cleaned, disinfected, and equilibrated, and solutions and buffers were passed through the equipment in the following sequence: 2 L 0.125 N NaOH; 10 l of purified water and, finally, 2 l of buffer G. After that, the equipment was ready for use to concentrate the selected fractions in accordance with the usual method. Concentration was carried out until a protein concentration of 15 mg / ml was reached (evaluated by the Bradford method).

The selected fractions were filtered through a membrane with a pore diameter of 0.22 μm, analyzed for total proteins (Bradford method) and stored at 4 ° C.

The conductivity and pH of the selected fractions were 1100–1300 μS / cm and 6.9 ± 0.1, respectively.

Molecular Size Exclusion Chromatography

The material obtained from the previous step was chromatographed using a molecular size exclusion chromatography matrix, as follows:

1. Equipment:

A. Column:

1) diameter: 5 cm;

2) layer height: 92 cm;

3) matrix:

a) high-resolution Sephacryl S-200 (Amersham Pharmacia);

b) volume: 1800 ml.

2. Solutions and buffers:

A. 0.5 N NaOH;

B. 20% ethanol;

C. Buffer H: 320 mm glycine, 2.2 mm NaH ₂ PO ₄ , 1.8 mm Na ₂ HPO ₄ , pH 7.30.

3. Substance subjected to chromatography:

A. Fractions selected from the previous step are concentrated;

B. Sample parameters:

1) volume: 40 ± 20 ml;

2) conductivity: 1200 ± 100 μS / cm;

3) pH: 7.3 ± 0.1.

In order to balance and disinfect the column, the following solutions or buffers were successively passed through it in the amounts indicated in detail below, with a flow rate of less than 46 cm / h: 1.0 vol. (1800 ml) purified water; 1.0 vol. (1800 ml) 0.5N NaOH and finally 2.0 vol. (3600 ml) H. buffer

After equilibration of the column, a chromatographic material was applied. The specified application was carried out at 20 ± 5 ° C and a flow rate of 46 ± 15 cm / h. Then, elution was carried out at the same flow rate and temperature, and 1.0 vol. (1800 ml) of buffer H as many times as needed to complete the cycles.

After the end of this stage, the following solutions or buffers were passed through the column in amounts indicated in detail below to purify it: 1.0 vol. (1800 ml) of purified water and 1.5 vol. (2700 ml) 20% ethanol.

Fractions containing purified hGH were aseptically filtered through a 0.22 μm membrane into sterile, pyrogen-free plastic vials, analyzed for total proteins and stored at -20 ° C.

EXAMPLE 8

An alternative way to purify hGH from milk

Instead of the previously described purification method, an alternative scheme can be used to purify the recombinant hGH in milk. The main difference between the method described in example 7 and the alternative method presented in this example is that the second stage of the first method involves anion exchange chromatography using an expanded layer, while the corresponding stage of the latter involves immunoaffinity chromatography. The clarification stages in both methods are also slightly different. Since the remaining parts in both purification schemes are identical, only the first two steps of an alternative method will be described below.

Lightening

Fresh milk was mixed with a sufficient amount of Tween 80 to obtain a 0.5% solution. After the addition of Tween 80, 1M Tris was added to obtain a pH of 7.3 ± 0.3. The product was then homogenized for 30 minutes and then centrifuged at 14,000 g to separate the fat layer. Then, the resulting solution was diluted 20 times with S buffer (50 mm Tris-HCl, 500 mm NaCl, 0.5% Tween 80, pH 7.3) and then filtered through a membrane with a pore diameter of 0.45 μm and stored accordingly .

Immunoaffinity Chromatography

The material obtained from the previous step was chromatographed using an immunoaffinity interaction matrix (Affigel 10 ester agarose, manufactured by BioRad with covalently linked anti-GH monoclonal antibodies produced by Bio Sidus) according to the following parameters:

1. Equipment:

A. Column:

1) diameter: 30 cm;

2) layer height: 15 cm;

3) matrix:

a) Affigel 10, ester agarose (BioRad) with covalently linked monoclonal antibodies against GH (Bio Sidus);

b) volume: 10 l;

2. Solutions and buffers:

A. Buffer A: 50 mm Tris-HCl, 500 mm NaCl, pH 7.2;

B. Buffer B: 100 mm citric acid, pH 3.0;

C. Buffer C: 150 mm NaH ₂ PO ₄ , pH 7.2;

D. Buffer D: 50 mm Tris-HCl, 500 mm NaCl, 500 mm guanidine-HCl, pH 7.2;

E. Buffer E: 50 mm Tris-HCl, 500 mm NaCl, pH 7.2, 0.2% sodium azide, 0.1 g / l gentamicin.

3. Material subjected to chromatography:

A. clarified milk.

B. Sample parameters:

1) volume: 30-50 l;

2) conductivity: 45 ± 15 mS / cm;

3) pH: 7.3 ± 0.3.

In order to balance and disinfect the column, if it has not been used for the last seven days, the following solutions or buffers were passed through it in the amounts indicated in detail below, with a flow rate of less than 51 cm / h: 1.0 column volume (“vol. Col.”) (10 L) buffer A; 2.0 vol. (20 L) buffer D; 2.0 vol. (20 L) buffer A; 1.0 vol. (10 L) buffer B; 2.0 vol. (20 L) buffer C and finally 1.0 vol. (10 L) buffer A.

On the other hand, if the column was used for the last seven days, then the following solutions or buffers were sequentially passed through it in the amounts indicated in detail below, with a flow rate of less than 51 cm / h: 1.0 vol. (10 L) buffer C and finally 2.0 vol. (20 L) buffer A.

After equilibration of the column, the material was subjected to chromatography. The specified application was carried out at 5 ± 3 ° C and a flow rate of less than 51 cm / h. Then, elution was carried out at a flow rate of 42 ± 9 cm / h and at the same temperature, and then the solutions and buffers, which were described in detail below, were passed in the following order: 2.0 vol. (20 L) buffer A and 1.5 vol. (15 L) buffer B.

After this stage, the following solutions or buffers were passed through the column in amounts indicated in detail below to clean the column: 2.0 vol. (20 L) buffer D; 2.0 vol. (20 L) buffer A and finally 2.0 vol. (20 L) buffer E.

Selected fractions containing hGH were analyzed for total proteins (Bradford method) and for the protein of interest (using RIA) and stored at 4 ° C.

EXAMPLE 9

Quality control of purified recombinant hGH

Two batches of purified recombinant hGH were subjected to a series of analyzes to confirm that the product was no different from natural hGH. Such methods included SDS / PAGE analyzes, Western blot, in vitro biological activity (nb2 cells) and in vivo (hypophysectomized rats), peptide mapping, determination of the complete amino acid sequence, isoelectric focusing (IEF) and reverse phase and exclusion HPLC, but not limited to. The results obtained in all of these analyzes were exactly the same for both recombinant and natural hGH, which proves that purified recombinant hGH exactly matches natural hGH, thus being suitable for the production of a biopharmaceutical product.

Based on the full description of the invention, it will be understood by those skilled in the art that the same can be accomplished in a wide and equivalent range of conditions, composition, and other parameters without going beyond the scope of the invention or any embodiment thereof. All patents and publications cited in this description are fully incorporated into this description by reference in full.

Claims (43)

1. A transgenic non-human bull mammal that produces recombinant human growth hormone in its milk, with at least 92 ng / ml growth hormone being produced on average per month in the serum of this mammal. 2. The transgenic non-human mammal according to claim 1, wherein said human growth hormone is produced at a level approximately greater than 1.0 g hGH / L of milk. 3. The transgenic non-human mammal according to claim 2, wherein said human growth hormone is produced at a level approximately greater than 2.0 g hGH / L of milk. 4. The transgenic non-human mammal according to claim 3, wherein said human growth hormone is produced at a level approximately greater than 3.0 g hGH / L of milk. 5. The transgenic non-human mammal according to claim 4, wherein said human growth hormone is produced at a level approximately greater than 4.0 g hGH / L of milk. 6. The transgenic non-human mammal according to claim 5, wherein said human growth hormone is produced at a level approximately greater than 5.0 g hGH / L of milk. 7. The transgenic non-human mammal according to claim 6, wherein said human growth hormone is produced at a level approximately greater than 6.0 g hGH / L of milk. 8. The transgenic non-human mammal according to claim 1, wherein the production of said recombinant human growth hormone by said mammal stimulates said mammal to produce more milk containing said growth hormone. 9. The transgenic non-human mammal according to claim 1, the genome of which contains an integrated plasmid, wherein said plasmid contains a growth hormone gene operably linked to a promoter that controls the expression of said gene in mammary cells of said mammal. 10. The transgenic non-human mammal of claim 9, wherein the promoter is a beta casein promoter. 11. The transgenic non-human mammal of claim 10, where the plasmid is a plasmid pRbhGH (DSM 16763). 12. The transgenic non-human mammal of claim 11, wherein said plasmid further comprises a neomycin resistance gene. 13. The transgenic non-human bull mammal of claim 12, wherein said plasmid, which further comprises a neomycin resistance gene, is plasmid pRNeo (DSM 16764). 14. The transgenic non-human mammal of claim 9, wherein the production of said recombinant growth hormone by said mammal stimulates said mammal to produce a large amount of milk containing said growth hormone. 15. A transgenic non-human mammal related to the species of cattle in which recombinant human growth hormone is produced in milk and whose genome contains an integrated plasmid, said plasmid containing a human growth hormone gene and a beta casein promoter that controls expression the specified gene in the cells of the mammary gland of the specified mammal, while in the serum of the specified mammal is produced on average per month at least 92 ng / ml growth hormone. 16. The transgenic non-human mammal of claim 15, wherein said plasmid is plasmid pRbhGH (DSM 16763). 17. The transgenic non-human mammal of claim 16, wherein said plasmid further comprises a neomycin resistance gene. 18. The transgenic non-human mammal of claim 17, wherein said plasmid, which further comprises a neomycin resistance gene, is a pRNeo plasmid (DSM 16764). 19. The transgenic non-human mammal according to claim 15, wherein said integrated plasmid is found in somatic cells and germ cells of said mammal. 20. The transgenic non-human mammal of claim 15, wherein the production of a recombinant human growth hormone by said mammal stimulates said mammal to produce more milk containing said hormone. 21. A method of obtaining a transgenic non-human mammal from a genus of bulls, comprising: a) cloning a gene that encodes human growth hormone into a plasmid, whereby said gene is operably linked to a promoter that will direct expression of said gene in mammary cells, leading to plasmid expression;
b) transfection of bovine somatic cells with said expressing plasmid so that said plasmid is included in the genome of said cells, resulting in transgenic somatic cells;
c) enucleation of a mature bovine oocyte, resulting in an enucleated oocyte;
d) the fusion of one of these transgenic somatic cells with the specified enucleated oocyte, which leads to the production of a single cell embryo;
e) implantation of the indicated embryo into the uterus of the recipient mammal, which leads to the onset of pregnancy in the mammal; and
f) monitoring pregnancy until the birth of a transgenic mammal,
however, in the serum of the indicated transgenic mammal, at least 92 ng / ml of human growth hormone is produced on average per month. 22. The method of claim 21, wherein said promoter is a beta-casein promoter and where said growth hormone is human growth hormone. 23. The method of claim 22, wherein the expression plasmid is plasmid pRbhGH (DSM 16763). 24. The method of claim 23, wherein said expression plasmid further comprises a neomycin resistance gene. 25. The method of claim 24, wherein said expression plasmid, which further comprises a neomycin resistance gene, is plasmid pRNeo (DSM 16764). 26. The method according to item 21, where the specified transgenic mammal is a mammal from the genus of bulls, in which milk produces recombinant human growth hormone, and the genome of which contains an integrated plasmid, wherein the plasmid contains the human growth hormone gene and the beta casein promoter, which controls the expression of the specified gene in the mammary gland cells of the specified mammal. 27. The method of claim 26, wherein said plasmid is plasmid pRbhGH (DSM 16763). 28. The method of claim 27, wherein said plasmid further comprises a neomycin resistance gene. 29. The method of claim 28, wherein said plasmid, which further comprises a neomycin resistance gene, is plasmid pRNeo. 30. The method according to item 21, where the specified somatic cells are fibroblasts. 31. The method according to item 21, where these transgenic somatic cells are obtained by isolating from the body of a female transgenic for the production of the indicated recombinant growth hormone in her milk. 32. The method according to p, where these transgenic somatic cells are fibroblasts. 33. A method of obtaining a transgenic non-human mammal from a genus of bulls, including
a) superovulation of a female non-human mammal that is transgenic in the production of recombinant human growth hormone in her milk;
b) artificial insemination of said mammal with sperm obtained from a male non-transgenic non-human mammal to produce embryos;
c) collecting said embryos;
d) implantation of said embryos into the uterus of a mammal ready for fertilization;
e) monitoring pregnancy until the birth of a transgenic mammal,
however, in the serum of the indicated mammal, at least 92 ng / ml of human growth hormone is produced on average per month. 34. A method of obtaining a transgenic non-human mammal from a genus of bulls, including
a) superovulation of a female non-human mammal that is transgenic in the production of recombinant human growth hormone in her milk;
b) artificial insemination of said mammal with sperm obtained from a male mammal other than a human, which is transgenic from the production of said recombinant growth hormone, to produce embryos;
c) collecting said embryos;
d) implantation of said embryos into the uterus of a mammal ready for fertilization;
e) monitoring pregnancy until the birth of a transgenic mammal,
however, in the serum of the indicated mammal, at least 92 ng / ml of human growth hormone is produced on average per month. 35. A method of obtaining a transgenic non-human mammal from a genus of bulls, including
a) superovulation of a female non-transgenic non-human mammal;
b) artificial insemination of said mammal with sperm obtained from a male mammal other than a human, which is transgenic from the production of said recombinant growth hormone, to produce embryos;
c) collecting said embryos;
d) implantation of said embryos into the uterus of a mammal ready for fertilization;
e) monitoring pregnancy until the birth of a transgenic mammal,
however, in the serum of the indicated mammal, at least 92 ng / ml of human growth hormone is produced on average per month. 36. The method according to any one of paragraphs.21, 31, 33, 34 or 35, where the specified mammal produces human growth hormone at a level exceeding approximately 1.0 g hGH / l of milk. 37. The method according to clause 36, where the specified mammal produces human growth hormone at a level exceeding approximately 2.0 g hGH / l of milk. 38. The method according to clause 37, where the specified mammal produces human growth hormone at a level exceeding approximately 3.0 g hGH / l of milk. 39. The method according to § 38, where the specified mammal produces human growth hormone at a level exceeding 4.0 g hGH / l of milk. 40. The method according to § 39, where the specified mammal produces human growth hormone at a level exceeding approximately 5.0 g hGH / l of milk. 41. The method according to clause 40, where the specified mammal produces human growth hormone at a level exceeding approximately 6.0 g hGH / l of milk. 42. The method according to any one of claims 21, 31, 33, 34 or 35, wherein the production of said recombinant growth hormone by said mammal stimulates said mammal to produce more milk containing said growth hormone. 43. The method according to any one of paragraphs.21, 31, 33, 34 or 35, where the gene encoding the indicated recombinant growth hormone operably linked to a promoter that controls the expression of the specified gene in breast cells is located in somatic cells and germ cells of the specified a mammal.

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