MXPA96003890A - Milk of non-human transgenic mammals accounting 2'fucosil-lact - Google Patents

Abstract

The present invention relates to the milk of a transgenic mammal, which is not a human being. Milk is characterized in that it contains heterologous components produced as the secondary gene products of a heterologous gene contained in the genome of the transgenic mammal, which is not a human being. the heterologous gene encodes a heterologous catalytic entity, such as a human enzyme, selected from the group consisting of glycosyltransferases, phosphorylases, hydroxylases, peptidases and sulfotransferases. Especially useful in the practice of this invention are human glycosyltransferases. The desired heterologous components include oligosaccharides, glycoconjugates. Oligosaccharides and glycoconjugates can be isolated from the milk of transgenic mammals and used in the preparation of pharmaceuticals, diagnostic equipment, nutrients, and the like. The whole transgenic milk can also be used to formulate nutritional products that provide special advantages. Transgenic milk can also be used in the production of enteric nutritious products, specializing in

Description

TRANSGENIC PRODUCTION OF OLIGOSACARIDOS AND GLICOCONJUGADOS TECHNICAL FIELD This invention relates to the in vivo production of heterologous glycosyltransferase secondary gene products. These glycosyl transferases are expressed in the breast tissue of animals, not humans, which lead to the production of oligosaccharides as well as various glycoconjugates carried by those oligosaccharides in the milk of the transgenic animal.

BACKGROUND OF THE INVENTION Carbohydrates are an important class of biological compounds. The term "saccharides" encompasses a wide variety of carbohydrate-containing compounds. These include polysaccharides, oligosaccharides, glycoproteins and glycosides with aglycones without carbohydrate. Biological macromolecules composed of protein or lipids containing portions of oligosaccharide are collectively known as glycoconjugates. The carbohydrate portion provides many biological functions. In cells, carbohydrates function as structural components, where they regulate viscosity, storage energy, or are key components of the cell surface. Complex oligosaccharide chains of several glycoconjugates (especially glycoproteins and glycolipids) adjust or modulate a variety of biological procedures. For a general review of the bioactivity of carbohydrates, see: (a) Biology of Carbohydrates, Volume 2, Ginsburg and others, Wiley, N .Y. (1984); and (b) P.W. Macher et al., Anual Review of Biochemestry, Volume 57, p. 785, 1988. Among other things, it is known that: (a) the carbohydrate structures are important to the stability, activity, location and degradation of glycoproteins; (b) certain oligosaccharide structures activate the secretion in plants of antimicrobial substances; (c) glycoconjugates are often found on the surfaces of several cells and are important, among other things, for cellular interactions with the surrounding environment, since they function as receptors or regulators when they bind to cell surfaces of, for example, peptides, hormones, toxins, viruses, bacteria and during cell-cell interaction; (d) the carbohydrate structures are antigenic determinants (eg, antigens of the blood group); (e) carbohydrates function as cell differentiation antigens during normal tissue development; (f) carbohydrates are important in oncogenesis, since specific oligosaccharides have been found to be antigenic determinants associated with cancer; and (g) oligosaccharides are important for sperm / egg interaction and for fertilization. It is known that isolated oligosaccharides inhibit the agglutination of uropathogenic coliform bacteria with erythrocytes. It has been shown that other oligosaccharides possess potent antithrombotic activity by increasing plasminogen activator levels. This same biological activity has been used, covalently linking these oligosaccharides to the surface of medical instruments, to produce surfaces that have anticoagulation effects. These surfaces are useful in the collection, processing, storage and use of blood. Still, it has been found that other oligosaccharides are useful as gram-positive antibiotics and disinfectants. In addition, certain free oligosaccharides have been used in the diagnosis and identification of specific bacteria. It is considered a future market for fine chemicals based on biologically active carbohydrates. Universities and industry, at present, are working intensively on the development of additional uses of biologically active oligosaccharides. These efforts include, but are not limited to: (a) the development of novel diagnostics and reagents for blood group determination; (b) the development of a novel type of therapy as an alternative to antiobiotics, based on the prevention of adhesion of bacteria and viruses to cell surfaces by means of specific oligosaccharides; and (c) the use of olosaccharides to stimulate plant growth and provide protection against certain plant pathogens. A large number of oligosaccharide structures has been identified and characterized: the smallest block or unit of development of an oligosaccharide is a monosaccharide. The main monosaccharides found in mammalian glycoconjugates are: D-glucose (Glc), D-galactose (Gal), D-mannose (Man), L-fucose (Fue), N-acetyl-D-galactose-amine (GalNAc) , N-acetyl-D-glucose-amine (GIcNAc) and N-acetyl-D-neuramic acid (NeuAc). The abbreviations within the parentheses are the normal abbreviated thermology for monosaccharides according to the recommendations of the International Union of Physics, Chemistry and Biology Council; Journal Biological Chemestry, Volume 257, pgs. 3347-3354, (1981). These abbreviations will be used from now on. Despite the relatively small number of fundamental development blocks, the number of possible combinations is very large, because both the anomeric configuration (alpha- or β-glycosidic bond) and the position of the O-glycosidic bond can vary. In this way, a great variety of oligosaccharide structures can exist. It is known that the bioactivity of oligosaccharides is specific in terms of both conformation and composition of sugar. The individual monosaccharides provide an element of bioactivity, but also contribute to the total conformation of the oligosaccharide, thus providing another level of specific character and bioactivity. It is the diversity of the glycoconjugates and the oligosaccharides la- that produces specific biological character of certain structures. However, this diversity also causes a particular problem for the practical utility of these compounds. Glycoconjugates are typically potent immunogens and the specific biocharacter, as noted above, is determined not only by the particular monosaccharide sequence, but also by the nature of the glycosidic linkage. Consequently, it is usually not possible to use oligosaccharide structures found in an animal species in other species. Similar restrictions on use can also be applied on an individual basis. For example, since it is known that certain blood group antigens are formed of specific oligosaccharides, it is necessary to take special care when conjugating a blood group oligosaccharide to a protein and then using that glycoprotein, therapeutically. Careful consideration must be given to the potential aspects of the immunogenic character. Despite these strong difficulties, it is well accepted that there is a need to produce large quantities of oligosaccharides and / or human glucoconjugates carrying those oligosaccharides. Numerous methods have been contemplated as adequate means to achieve this goal. Such methods include the synthesis of oligosaccharides by conventional organic chemistry or the use of in vitro enzymes. Immobilized enzymes are currently preferred for the production, in large scale, in vitro, of oligosaccharides. This is due to a high regio- and stereoselectivity of the enzyme, as well as a high catalytic efficiency under moderate reaction conditions. The literature describes an oligosaccharide synthesis number catalyzed by enzymes. For example, see the scientific review articles by Y. Ichikawa et al., "Enzyme-catalyzed Oligosaccharide Synthesis" in Analytical Biochemestry, Volume 202, p. 215-238, (1992); and K. G. I. Nillson, "Enzymatic Synthesis of Oligosaccharides", Trends in Biotechnology, Volume 6, pgs. 256-264, (1988). Both hydrolases and transferases have been used to facilitate the production of oligosaccharides. Glycosidase enzymes, a subclass of the hydrolases, are especially useful in the synthesis of oligosaccharides, through a reversal procedure of the degradation cycle. Nevertheless, in general, the enzymatic synthesis of oligosaccharide is based on the biosynthetic trajectory. Since the biosynthetic pathway of oligosaccharide synthesis is mainly regulated by the gene encoding the production of each glycosyltransferase, the actual structures of the oligosaccharide are determined by the specificity of the substrate and the acceptor of the individual glycosyltransferases. The oligosaccharides are synthesized by transferring the monosaccharides from sugar nucleotide donors to acceptor molecules. These acceptor molecules can be other free oligosaccharides, monosaccharides or oligosaccharides linked to proteins or lipids.

The enzymatic synthesis of the oligosaccharide has been generally conducted only on a small scale, because enzymes, particularly glycosyltransferases from natural sources, are difficult to isolate. Also, sugar nucleotide donors are very difficult to obtain from natural sources and are very expensive when they are derived from organic chemistry synthesis. However, recently, a recirculation and reuse strategy has been developed to synthesize large amounts of oligosaccharides. The E. U .A. 5, 1 80, 674, incorporated herein by reference, discloses a novel method of affinity chromatography, wherein the reaction products are repeatedly recirculated onto the glycosyltransferases bound to the matrix or to the resin. In addition, recent progress in gene cloning techniques has made available several glycosyltransferases in sufficient quality and quantity to make the enzymatic synthesis of oligosaccharides more practical. The literature is full of descriptions of the recombinant or transgenic expression of a heterologous glycosyltransferase. However, before continuing a discussion of the literature, it is necessary to clarify the meaning of several terms, as used in the present and in the claims. (a) Host, host cell or host animal: these terms are used to refer to the cell or mammal, which is responsible for the biosynthesis of the biological material. (b) Homologous: this term means that the entity, so characterized, is normally present or is produced by the guest. (c) Heterologist: this word means that the entity, so characterized, is not normally present or produced by the guest. In other words, the entity, so characterized, is foreign to the host. (d) Catalytic activity: this term is used to refer to the inherent property of certain biological compounds to facilitate chemical change in other substances. (e) Catalytic entity: this term is used to refer to biological compounds, which inherently possess catalytic activity, which results in the production of new, different or altered compounds. The examples are enzymes and antibodies. An enzyme is a biochemical catalyst of a specific biochemical reaction. An enzyme product is formed as a result of the catalytic activity of the enzyme on a substratum material. (f) Genarna: this word is used to refer to the complete genetic material found in the host. This material is arranged in chromosomes. (g) Gene: this word refers to a functional portion of the genome, which is responsible for the biosynthesis c (e a specific biological entity. (h) Insertion: this word is used to refer to the procedure, whereby a portion of Heterologous DNA or a heterologous gene is introduced into the genome of a host.The DNA, which is inserted, is referred to as an "insert." (I) Transgen: this refers to the heterologous genetic material, which is transferred, by insertion, from the genome of an animal species to the genome of another animal species Simplest, a transgene is a gene, which is heterologous to the host.The transgene encodes a specific biological material. (j) Transgenic mammal or transgenic host: these terms are used to refer to a mammal or cell, to which a transgene has been inserted into its genome As a result of this insertion, the transgenic host produces heterologous biological material, which would not be normally heterologous entities are present or are produced by a transgenic host, as a result of the insertion of genetic material foreign to the host cell genome. (k) Primary gene product: this refers to a biological entity, which is formed directly as a result of the transcription or translation of a homologous or heterologous gene. Examples of these include proteins, antibodies, enzymes and the like. (I) Secondary gene product: this refers to a product, which is formed as a result of the biological activity of a primary gene product. An example thereof is an oligosaccharide, which is formed as a result of the catalytic activity of an enzyme. (m) Biological products: this term is used to refer to products produced or synthesized by a transgenic host as a result of the insertion of a transgene into the genome of the mammal. More specifically, the term means biological products that are secondary gene products. An example thereof, as described below, are human oligosaccharides produced by transgenic cows. Human oligosaccharides are produced as a result of the catalytic activity of human glycosyltransferases. As discovered herein, when the gene encoding human glycosyltransferases is inserted into the murine genome, the resulting transgenic mouse produces heterologous human glycosyltransferase as the primary gene product. Human glycosyltransferase, using heterologous substrate materials, produces oligosaccharides and glycosylated proteins. The oligosaccharide, formed as a result of the activity of the enzyme of the primary gene product, is also appropriately termed as a secondary gene product. Glycoconjugates are another example of the class of compounds referred to herein and in the claims referred to as "biological products". (n) Product: this word is used to refer to the secondary gene products of the present invention and is used as an alternative for "biological product". (o) Humanized milk: this refers to milk obtained from a mammal, which is not a human being, which, through the alteration of the host genome, is made to produce milk, which closely resembles the milk of the human being. An example of humanized milk is cow's milk that contains products found in human milk, but not normally found in cow's milk. Human oligosaccharides are produced in cow's milk as a result of the insertion of the gene encoding human glycosyltransferases into the bovine genome. Humanized milk also contains glycosylated proteins with human oligosaccharides. As noted above, there is a considerable body of literature describing the recombinant or transgenic expression of heterologous glycosyltransferases. However, the literature does not disclose or in any other way suggest the production of secondary gene products in the milk of non-human transgenic mammals, as claimed in the present invention. Examples of the literature are: 1) Patent of E. U.A. No. 5,032,519 by Paulson, which teaches a method for genetically processing, by means of engineering, cells, so as to produce soluble and secretable Golgi processing enzymes, instead of enzymes bound to the naturally occurring membrane. 2) Patent of E. U .A. No. 5, 047, 335 by Paulson, which teaches the alteration by the engineered, genetic process of the genome of Chinese Hamster Ovary Cell (CHO), so that the CHO cells produce a sialitransferase. 3) International Patent Application No. PCT / US91 / 08216, which teaches a transgene capable of producing heterologous recombinant proteins in the milk of transgenic bovine species. This published patent application teaches a method for obtaining only the primary gene product. This published patent application also describes methods for producing and using the altered milk obtained from these transgenic animals. 4) International Patent Application PCT / US91 / 05917 which teaches methods for producing, intracellularly, DNA segments by homologous recombination of overlapping DNA fragments. This published patent application teaches a method for obtaining only the primary gene product. 5) International Patent Application PCT / GB871 / 0458 teaching methods for producing a peptide, said method involves the incorporation of a DNA sequence encoding the peptide, to the gene of a mammal coding for a whey protein, in such a way that the DNA sequence is expressed in the mammary gland of the adult female mammal. This published patent application teaches a method for obtaining only the primary gene product, the peptide, in the milk of the transgenic mammal, and also describes methods for producing and using the altered milk obtained from these transgenic animals. 6) International Patent Application PCT / GB89 / 01343 which teaches methods for producing proteinaceous materials in transgenic animals that have genetic constructs integrated into their genomes. The construct comprises a 5 'side sequence of a mammalian milk protein gene and DNA encoding a heterologous protein other than a milk protein. This published patent application teaches a method for obtaining only one product of the primary gene, the heterologous protein, in the milk of the transgenic mammal. 7) European Patent Application No. 88301 1 12.4 teaches methods for making genes specific to the mammary gland, which results in the efficient synthesis and secretion of biologically important molecules into the milk of these transgenic animals. This published patent application also teaches methods for producing and using the altered milk obtained from these transgenic animals, and a method for obtaining only the primary gene product in the milk of the transgenic mammal. 8) International Patent Application No. PCT / DK93 / 00024 which teaches methods for producing human kapa-casein in the milk of transgenic animals. The genetic construct comprises a 5 'side sequence of a mammalian milk protein gene, such as casein or whey acid protein, and DNA encoding human kapa-casein. The DNA sequence contains at least one intron. This published patent application teaches a method for obtaining only the primary gene product. , the heterologous human kapa-casein, in the milk of the transgenic mammal. 9) International Patent Application No. PCT / US87 / 02069 which teaches a method for producing mammals capable of expressing recombinant proteins in their milk. These publications teach, in one way or another, means to obtain the product of the transgene's primary gene, said gene product being the active protein or enzyme, which is encoded by the transgene. This literature describes transgenic media for obtaining glycosyltransferases in non-human milk. However, none of the aforementioned publications teaches or suggests the use of transgenic animals as means to obtain a desired, secondary gene product, which is the product of the active enzyme. More particularly, if however, none of the aforementioned publications teaches or suggests or in any other way describes the use of transgenic human glycosyltransferases in non-human milk to produce human or glycoconjugate oligosaccharides carried by those oligosaccharides. These oligosaccharides, which are the product of active glycosyltransferases, are hereinafter referred to as the "secondary gene product." In this way, the various oligosaccharides found in human milk are formed as a direct result of genetically regulated expression. In this regard, oligosaccharides can appropriately be considered as "secondary gene products", since they are synthesized as a result of the biochemical activity of the primary gene product, the heterologous glycosyltransferase enzymes. human milk contains a variety of oligosaccharides and proteins.Soluble, free oligosaccharides are not normally produced by animal cells and tissues, with the exception of highly differentiated lactating mammary glands.The oligosaccharides constitute the main portion of the total carbohydrate content of human milk and of bovine. The main carbohydrate component of milk from mammals is disaccharide lactose. Lactose is typically found at a concentration greater than 10 mg / ml and is synthesized by the binding of galactose to glucose. This reaction is catalyzed by the enzyme, β-1, 4-galactosyltransferase. The milk of most mammals, including cows, contains only very small amounts of some additional oligosaccharides. In contrast, human milk contains substantial amounts of a number of additional, soluble oligosaccharides that are greater than lactose. All human oligosaccharides are synthesized by the sequential addition of monosaccharides to lactose. The representative oligosaccharides found in human milk are set forth in Table 1.

TABLE 1 OLIGOSACCHARIDES PRESENT IN HUMAN MILK Structure Common name Concentration (mg / l) 1. Gal-ß-1,4-Glc Lactose 50,000 2. Fuc-a-1,2-Gal-ß-1,4-Glc 2-fucosil-lactose 200 3. Gal-ß-1,3-GlcNAc-ß-1,3-Gal-ß-1,4-Glc Lacto-N-tetraose 400 4. Gal-ß-1,4-GlcNAc-ß-1,3-Gal-ß-1,4-Glc Lacto-N-neotetraose 60 . Fuc-a-1,2-Gal - / - 1,3.GlcNAc-ß- 1,3-Gal-ß-1,4-Glc Lacto-N-fucopentaose I 200 6. Gal-β-1,3 [Fuc-a-1,4] GlcNAc-β-1,3-Gal-β-1,4-Glc Lacto-N-fucopentase II 20 7. Gal-β-1,4 [Fuc-a-1,3] GlcNAc-β-1,3-Gal-β-1,4-Glc Lacto-N-fucopentase III 50 8. Fuc-a-1,2- Gal-ß-1,3 [Fuc-a-1,4] - GlcNAc-ß-1,3-Gal-ß-1,4-Glc Lacto-N-difucopentaose I 25 9. NeuAc-a-2,6-Gal-a-1,4-Glc 6-siali-lactose 25 . NeuAc-a-2,3-Gal-ß-1,4-Glc 3-sialyl lactose 10 11. NeuAc-a-2,3-Gal-ß-1,3-R Sialyltetrasaccharide at 10 12. Gal-ß-1,3 [NeuAc-a-2,6] GlcNAc- ß-1,3-R Sialyltetrasaccharide b 35 13. NeuAc-a-2,6-Gal-ß-1,4-GlcNAc- ß-1,3-R Sialyltetrasaccharide c 50 14. NeuAc-a-2,3-Gal-ß-1,3 [NeuAc-a-2,6] -GlcNAc-β-1,3-Gal-β-1,4-Glc Disialyltetrasaccharide 60 . NeuAc-a-2,3-Gal-ß-1,3 [Fuc-a-1,4] -GlcNAc-β-1,3-Gal-β-1,4-Glc Sialyl Lacto-N-fucopentaose 50 -a-: denotes a glycosidic alpha-binding R: Gal-ß-1,4-Glc Oligosaccharides in human milk are present as a result of the activity of certain specific glycosyltransferases, found in the breast tissue of human beings. For example, fucose residues linked to alpha 1, 2, in structures 2,5 and 8 are produced by a single human fucosyltransferase and characterize a phenotype known in the field of immunohaematology as "secretors". These individuals are characterized in this way because they synthesize substances from the blood group of humans in their salivary secretions and other mucosal secretions, wherein the oligosaccharides are covalently bound to various proteins. The fucose residues bound to alpha 1, 4 in structures 6, 8 and 15 are formed as a result of the enzymatic action of a different fucosyltransferase. These oligosaccharides represent a phenotype present in individuals characterized because they have a "Lewis positive" blood type. Such individuals use this fucosyltransferase to synthesize an oligosaccharide structure corresponding to an antigen of the blood group of humans. This oligosaccharide is also found in saliva, and in other mucosal secretions, and is covalently bound to lipids found on the red blood cell membrane of individuals with "Lewis positive". The structure 5 is related to the H antigen of the blood group ABO; structure 6 is the blood group antigen of "Lewis a"; structure 8 is the blood group antigen of "Lewis b".

At least fifteen human milk proteins have been identified. Some of these proteins are generally recognized to be glycosylated, that is, they are covalently linked to certain specific oligosaccharides. The particular oligosaccharides, which are covalently bound to the protein, are the same as, or similar to, the oligosaccharides described above, and their formation is the result of the normal, genetically regulated expression of certain specific glycosyltransferase genes. The presence of a heterologous glycosyltransferase could also affect the post-translational modification of proteins. Proteins glycosylated by a heterologous glycosyltransferase are also appropriately known as "secondary gene products". Both homologous and heterologous proteins could be modified by the glycosyltransferase in a form different from that resulting from the activity of homologous glycosyltransferases. It has been known that these oligosaccharides and glycosylated proteins promote the development of desirable bacteria in the intestinal tract of the human being. It is also believed that oligosaccharides, in human milk, inhibit the binding of harmful microorganisms to the mouth and throat. These human oligosaccharides and specifically glycosylated proteins, are absent or present in markedly different amounts, in bovine milk. In addition, as noted above, bovine milk contains predominantly lactose. Human milk contains not only lactose, but also numerous other oligosaccharides. Also, the amino acid composition of human milk proteins is significantly different from the amino acid composition of the corresponding cow's milk proteins. As a consequence, babies who are fed infant formula, which includes cow's milk, may be more susceptible to intestinal disorders such as diarrhea, or their relationships and plasma amino acid levels in blood may differ from milk-fed infants. maternal For the same reasons, immunocompromised and critically ill patients of advanced age also have an urgent need for the availability of a nutritional product, which closely resembles the composition of human milk biochemically. The complicated chemistry of proteins and oligosaccharides in human milk has made its synthesis on a large scale extremely difficult. Before they can be incorporated into the commercial nutritive product, a practical method must be devised to obtain large quantities of proteins and oligosaccharides from human glycosylated milk. A potential solution to this problem is the use of transgenic animals, more particularly transgenic cows that express genes or enzymes that encode cDNA (s), which catalyze the formation of oligosaccharides and / or glycosylated proteins with the same human oligosaccharides. Domestic animals that have transgenic milk, such as rabbits, pigs, sheep, goats and cows, are proposed here as means to produce milk containing human oligosaccharides and glycosylated proteins with human oligosaccharides. More particularly, transgenic cows are highly suitable for the production of oligosaccharides and recombinant proteins, since a single cow can produce more than 10,000 liters of milk containing as much as 300 kilograms of protein (mainly casein) per year at a minimal cost. In this way, transgenic cows appear to be the least expensive production route than other recombinant protein production methods, since there is no need to invest in fermentation facilities. Also, the mammary glands of the cow have a more effective cost than the cultured cells, probably continuous production, and since the milk is collected several times a day, the time between the actual synthesis and the harvest can be as short as few hours. The genetic stability of the cow is greater than the production systems based on microbes or cells. Also, cows are relatively easy to reproduce using artificial insemination, embryo transfer techniques and embryo cloning. In addition, the downstream processing of cow's milk containing human transgenic proteins may require little or no purification. Below are publications that teach such methods. Yes*? However, none of these publications teaches, describes or otherwise suggests the production of secondary gene products in the milk of non-human transgenic mammals, as claimed in the present invention.

"Molecular Farming: Transgenic Animáis as Bioreactors" by J. Van Brunt, Biotechnology, Volume 6, pgs. 1 149-1 154, 1988, describes the alteration of the genome of several higher, domestic, milk-bearing animals, which produce transgenic animals capable of producing several heterologous entities. This publication suggests methods to obtain only the primary gene product.

International Patent Application No. PCT / US 91/08216 describes a transgene capable of producing heterologous recombinant proteins in the milk of transgenic bovine species. This published patent application teaches a method for obtaining only the primary gene product. This application also describes methods for producing and using the altered milk obtained from these transgenic animals. International Patent Application No. PCT / GB 87/00458 describes methods for producing a peptide, said method involves the incorporation of a DNA sequence encoding the peptide, to the gene of a mammal coding for a whey protein , in such a way that the DNA sequence is expressed in the mammary gland of the adult female mammal. This published patent application teaches a method for obtaining only the primary gene product, the peptide, in the milk of the transgenic mammal. This application also describes methods for producing and using the altered milk obtained from these transgenic animals. International Patent Application No. PCT / GB 89/01343 teaches methods for producing proteinaceous materials in transgenic animals that have genetic constructs integrated into their genomes. The construct comprises a 5 'side sequence of a mammalian milk protein gene and DNA encoding a heterologous protein other than a milk protein. This published patent application teaches a method for obtaining only one product of the primary gene, the heterologous protein, in the milk of the transgenic mammal. European Patent Application No. 88301 1 12.4 describes methods for making genes specific to the mammary glands, which results in the efficient synthesis and secretion of biologically important molecules into the milk of these transgenic animals. This published application also describes methods for producing and using the altered milk obtained from these transgenic animals, and a method for obtaining only the primary gene product in the milk of the transgenic mammal. International Patent Application No. PCT / US 87/02069 teaches a method for producing mammals capable of expressing recombinant proteins in the milk of lactating animals. This patent application does not disclose or in any other way suggest the production of secondary gene products in the milk of non-human transgenic mammals, as claimed in the present invention. Since transgenic animals can be used for the production of large quantities of human proteins, they have not been used for the production of secondary gene products, such as human oligosaccharides or proteins and glycosylated lipids with certain specific oligosaccharides, or proteins and milk lipids human glycosylated with certain specific oligosaccharides. None of the aforementioned publications describes or suggests a method for producing human oligosaccharides and glycoconjugates in milk from non-human mammals. The aforementioned publications neither describe nor suggest a method for obtaining glycoconjugates in milk from non-human mammals, where the glycosylation is with the desired oligosaccharides. To obtain this result it is required that the genome of mammals, who carry milk, who are not humans, be altered in order to ensure that the mammary tissue selectively expresses a desired human glycosyltransferase, which could then glycosylate certain proteins with the desired oligosaccharide . This approach requires that the DNA encoding the human glycosyltransferase, desired, be incorporated into said genone. The literature also does not describe or suggest a method for obtaining human glycosylated proteins in milk from mammals, which are not human beings, where the glycosylation is with the desired oligosaccharides. The literature also does not disclose or suggest a method for obtaining human milk glycosylated proteins in milk from non-human mammals, where the glycosylation is with the desired portions of oligosaccharide. To achieve this result it would be required that the genome of mammals carrying milk, which are not human beings, be altered to ensure that their breast tissue selectively expresses both the human glycosyltransferase and the human proteins, which are then appropriately glycosylated with the desired oligosaccharides by active human glycosyltransferase. This approach requires not only that the DNA encoding the desired glycosyltransferase be inserted into said genome, but also that the DNA encoding the desired human proteins also be incorporated into said genome. Accordingly, it is an aspect of the present invention to provide methods for successfully detecting the transgenesis of fertilized oocytes before implantation, so that the transplanted oocytes contain the genetic constructs required to achieve the desired glycosylation and production of oligosaccharides. It is also an aspect of the present invention to provide non-human, transgenic milk-bearing mammalian species which are capable of producing human glycosyltransferases that are secreted extracellularly by the mammary tissue of said mammalian species. Furthermore, it is also an aspect of the present invention to provide a non-human species of mammals carrying milk, transgenic, which are capable of producing human glycosyltransferases that are secreted extracellularly by the mammary tissue into the milk produced by said species of mammals.

Furthermore, it is an aspect of the present invention to provide a species of mammals that carry milk, transgenic, that are not human beings, which are capable of producing proteins and glycosylated, human oligosaccharides that are secreted extracellularly by the mammary tissue into the milk produced. for these mammalian species. The present invention also relates to non-human, transgenic mammalian milk-bearing species which are capable of producing glycosylated human milk proteins and lipids in the milk of said transgenic animals. It is also an aspect of the present invention to provide transgenic milk-bearing mammalian species, which are not human beings, which are capable of producing human oligosaccharides in the milk of said transgenic animals. The present invention also relates to food formulations containing glycosylated human proteins, lipids and oligosaccharides of said transgenic milk. The present invention also relates to pharmaceutical, medical diagnostic and agricultural formulations containing glycosylated proteins, lipids and oligosaccharides obtained from the milk of transgenic animals. It is also an aspect of the present invention to provide transgenic bovine species that are capable of producing glycosylated proteins, such as proteins and lipids of glycosylated human milk in their mammary glands. It is a further aspect of the present invention to provide transgenic bovine species that are capable of producing human oligosaccharides in the milk of said transgenic cows. The present invention also relates to food formulations containing glycosylated proteins, lipids and oligosaccharides of said transgenic bovine milk. The present invention also relates to pharmaceutical, medical diagnostic and agricultural formulations containing glycosylated proteins, lipids and oligosaccharides obtained from the milk of transgenic cows.

DESCRIPTION OF THE INVENTION The present invention uses transgenes encoding a heterologous catalytic entity to produce secondary gene products in the milk of transgenic mammals, which are not human beings. More particularly, the present invention uses transgenes encoding heterologous glycosyltransferases to produce heterologous oligosaccharides and glycosylated glycoconjugates in the milk of transgenic mammals, which are not human beings. Milk from a transgenic mammal, which is not a human being, is described, characterized in that it comprises heterologous components produced as the secondary gene products of at least one heterologous gene contained in the genome of said transgenic mammal, which is not a human being . Also described is a product produced in the milk of transgenic mammals, which are not human beings, wherein the product results from the action of a catalytic entity selected from the group consisting of heterologous enzymes and heterologous antibodies, and wherein said mammal Transgenic ífero, that is not a human being, contains in its genome, at least a heterologous gene that codifies for said catalytic entity. Examples of the aforementioned product are oligosaccharides and glycoconjugates. The production of transgenic milk containing human oligosaccharides and / or glycosylated proteins with certain olosaccharides is desirable since it provides a milk matrix, where little or no additional purification is needed for human consumption, and wherein said transgenic milk It looks biochemically to human milk. Humanized milk is described, wherein said milk is produced by a transgenic mammal, which is not a human being, wherein the genome of the transgenic mammal, which is not a human being, contains at least one heterologous gene coding for a human catalytic entity. The catalytic entity produces oligosaccharides and glycoconjugates that are present in the milk of a transgenic mammal, which is not a human being. Also disclosed is a method for obtaining a human milk, said method comprising the steps of: (a) inserting into the genome of a mammal, which is not a human being, a heterologous gene that encodes the production of a human catalytic entity , wherein said catalytic entity produces a secondary gene product in the milk of said mammal that is not a human being; and (b) milking said mammal, which is not a human being. Also disclosed is a method for obtaining a humanized milk biological product, said method comprising the steps of: (a) inserting into the genome of a mammal, which is not a human being, a heterologous gene that encodes the production of a heterologous catalytic entity , wherein said catalytic entity produces a secondary gene product in the milk of said mammal, which is not a human being; and (b) milking said mammal, which is not a human being; and (c) isolating the biological product from said milk. Also described is a transgenic mammal, which is not a human being, characterized in that the genome of said mammal contains at least one heterologous gene which codes for the production of the heterologous catalytic entity selected from the group consisting of enzymes and antibodies, and in wherein said catalytic entity produces a second heterologous product in the milk of said mammal. Also described is a transgenic cow, characterized in that the genome of said cow contains at least one heterologous gene which codes for the production of a heterologous glycosyltransferase selected from the group consisting of fucosyltransferase, galactosyltransferase, glucosyltransferase, xylosyltransferase, acetylases, glucuronyltransferases, glucuronylepimerases, sialyltransferases, mannosyltransferases, sulfotransferases, β-acetylgalactosaminyltransferases and N-acetyl-glucosaminyltransferases, and wherein the milk of said cow contains heterologous and glycoconjugate oligosaccharides produced by said glycosyltransferase. Representatives of mammals, which are not human beings, useful in the present invention are mice, rats, rabbits, pigs, goats, sheep, horses and cows. Representative of the heterologous genes useful in the present invention are the genes encoding human enzymes and human antibodies. (Human enzymes and human antibodies, in the description and in the claims, are also referred to as a catalytic entity). Examples of human enzymes useful in the present invention are enzymes selected from the group consisting of glycosyltransferases, phosphorylases, hydroxylases, peptidases and sulfotransferases. Especially useful in the practice of the present invention are the glycosyltransferases. Illustrative of the glycosyltransferases, especially useful in the practice of the present invention, are enzymes selected from the group consisting of fucosyltransferase, galactosyltransferase, glucosyltransferase, xylosyltransferase, acetylases, glucuronyltransferases, glucuronylepimerases, sialyltransferases, mannosyltransferases, sulfotransferases, β-acetylgalactose-minyltransferases and N-acetylglucosami nyltransferases. Examples of the desired heterologous gene gene products of the present invention are oligosaccharides and glycoconjugates. (The heterologous secondary gene products in the description and in the claims are also referred to as a "biological product" or simply as a "product"). Representative of the heterologous oligosaccharides produced as secondary gene products are lactose, 2-fucosyl lactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fuctopentaose I, lacto-N-fuctopentase II, lacto-N- fuctopentase III, lacto-N-difucopentaose I, sialyl lactose, 3-sialyl lactose, sialyltetrasaccharide a, sialyltetrasaccharide b, sialyltetrasaccharide c, disialyltetrasaccharide and sialyl-lacto-N-fuctopentaose. Illustrative of the heterologous glycoconjugates produced as secondary gene products described herein, are glycosylated homologous proteins, glycosylated heterologous proteins and glycosylated lipids. Representative of the glycosylated heterologous proteins, desirable, according to the practice of the present invention, are proteins selected from the group of proteins consisting of human serum proteins and human milk proteins. Examples of human milk proteins are proteins selected from secretory immunoglobulins, lysozyme, lactoferrin, kapa-casein, alpha-lactalbumin, beta-lactalbumin, lactoperoxidase and lipase stimulated with bi-ial salt. An enteric nutrient product containing humanized milk useful in the nutritional maintenance of an animal is also described. Also disclosed is a pharmaceutical product which contains the product of the present invention, which is useful in the treatment of an animal. In addition, a medical diagnosis containing the product of the invention useful in the diagnosis of an animal is described. Also described are agricultural products that contain the product of the invention, useful for the maintenance of grains. Also disclosed is a method for producing a transgenic mammalian species, which is not a human being, capable of producing heterologous secondary gene products in the milk of said species, said method comprising the steps of: (a) preparing a transgene, said transgene consisting of at least one expression-regulating DNA sequence that functions in the mammary secretory cells of said transgenic species, a secretory functional A DNase in the mammary secretory cells of said transgenic species and a recombinant DNA sequence. encoding a recombinant heterologous catalytic entity, said secretory DNA sequence being operably linked to said recombinant A DN sequence to form a secretory-recombinant DNA sequence, and said at least one expression regulation sequence being operably linked to said recombinant-secretory DNA sequence, wherein said transgene is cap az of directing the expression of said secretory-recombinant A DN sequence in mammary secretory cells of said transgenic species containing said transgene, to produce a recombinant heterologous catalytic entity, which when expressed by said secretory mammary cells, catalyzes the production of Secondary gene products in the milk of said transgenic species; (b) introducing said transgene into the embryonic target cell; transplanting the transgenic embryonic target cell formed by the same or the embryo formed from the same to a vessel of female origin; and (c) identifying at least one female offspring, which is capable of producing said secondary gene products in the milk of said offspring. A useful method for producing transgenic, non-human, large animals, such as pigs, goats, sheep, horses and cows capable of producing heterologous secondary gene products in their milk is also disclosed. The method described comprises the steps of: (a) preparing a transgene capable of conferring said genotype when it is incorporated into the cells of said transgenic mammal, which is not a human being; (b) methylating said transgene; (c) introducing said methylated transgene into fertilized oocytes of said mammal, which is not a human being, to allow the integration of said transgene into the A genomic DN of said fertilized oocytes; (d) cultivating the individual oocytes formed therein to pre-plant embryos thus replicating the genome of each of said fertilized oocytes; (e) removing at least one cell from each of the embryos of said preimplantation and lysing at least one cell to release DNA contained therein; (f) contacting said released DNA with a restriction endonuclease capable of cleaving said methylated transgene, but unable to cleave the non-methylated form of said transgene formed after integration and replication of said genomic DNA; and (g) detecting which of the cells of the preimplantation embryos contains a transgene, which is resistant to cleavage by said restriction endonuclease as an indication that preimplantation embryos have integrated said transgene. It is also described, according to the above method, the removal of the first hemi-embryos, which are used and analyzed according to steps (d) to (f), said method also comprises: (g) cloning by at least one of said second hem embryos; and (h) forming a multiplicity of transgenic embryos. It also describes the transplantation of more than one of the transgenic embryos to recipient female descendants to produce a population containing at least two transgenic mammals, which are not human beings, that have the same genotype and transplant the rest of the embryos of preimplantation containing a genetically integrated transgene to a recipient female offspring and identifying at least one offspring having said desired genotype, said genotype being capable of producing a heterologous secondary gene product in the milk of said species, said secondary gene products heterologous are selected from the group consisting of oligosaccharides and glycoconjugates. The DNA sequence forming the transgene, useful in the present invention, comprises at least three functional parts: a) A portion encoding the human glycosyltransferase. This portion of transgene, hereinafter referred to as the "recombinant portion" or "recombinant sequence"; b) A signal portion; and c) A portion of expression regulation. The recombinant portion of the transgene comprises a DNA sequence encoding the desired glycosyltransferase enzyme. The signal portion may be naturally present or genetically processed, by means of engineering in the sequence of A D N. This signal encodes a secretory sequence, which ensures that the glycosyltransferase is transported to the Golgi apparatus of the cell. In the present invention, the signal DNA sequence is functional in mammary secretory cells. These sequences are operably linked to form a recombinant signal-expression DNA sequence. The expression sequence ensures that the transgene is expressed only in certain types of tissue. In the present invention, the expression is regulated to the secretory mammary tissue. At least one expression regulation sequence, functional in the mammary secretory cells of the transgenic species, is operably linked to the recombinant signal DNA sequences. The transgene thus constructed is capable of directing the expression of the recombinant signal DNA sequence in mammary secretory cells containing the transgene. Said expression results in the production of the glycosyltransferase, which is secreted from the secretory mammary cells into the milk of the transgenic species. In addition to the functional parts described above, the transgene may also comprise additional elements. For example, the recombinant portion can code for more than one protein. In this way, in addition to encoding the glycosyltransferase, it can also code for one or more human proteins. Also, multiple transgenes encoding other glycosyltransferases and other heterologous proteins can be transfected simultaneously. All additional transgenes can also be operably linked to the secretory and expression regulation sequences of the glycosyltransferase transgene. The expression of multiple transgenes results, not only in the production of the glycosyltransferase, but also in the other proteins, which are secreted from the mammary secretory cells into the milk of the transgenic species. In the presence of suitable substrate materials, the glycosyltransferase will convert the individual monosaccharide units to the desired oligosaccharides. The desired oligosaccharides will be present in the milk of the transgenic species. The same glycosyltransferase enzyme will also covalently link monosaccharides to proteins via available glycosylation sites. These glycosylated proteins with desired oligosaccharides will also be present in the milk of the transgenic species. The advantages of the present invention will be better understood by reference to the following detailed description, taken together with the appended figures.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1, nucleotide and amino acid sequence of human alpha-1,2-fucosyltransferase; Figure 2 is an illustration of the protocol for achieving the amplification and expression of fucosyltransferase cDNA; Figure 3 is an illustration of the construction of plasmid pWAP-polyA using the regulatory sequence (promoter) of whey acid protein (WAP); Figure 4 is an illustration of the pWAP-fucosyltransferase plasmid for microinjection in mouse embryos; Figure 5 is a Western blot photograph illustrating the presence of human alpha-1,2-fucosyltransferase in the milk of transgenic mice; Figures 6A to 6F show high-pressure liquid chromatography profiles of milk samples obtained from normal or non-transgenic (Frames A and B) and transgenic mice expressing human alpha-1,2-fucosyltransferase (Frames C, D, E and F); Figure 7 is a photograph of a fluorophore assisted by a carbohydrate gel electrophoresis of oligosaccharide material deposited after separation by high pressure liquid chromatography; Figure 8 is a photograph of a fluorophore assisted by a carbohydrate electrophoresis gel after digestion of the oligosaccharide samples with a specific fucosidase for fucose-alpha-1, 2 ligatures; Figure 9 is a photograph of a fluorophore assisted by a carbohydrate electrophoresis gel showing the monosaccharide composition of the oligosaccharide samples isolated from the milk after exhaustive digestion with a mixture of fucosidase and β-galactosidase. The monosaccharide units were labeled with the fluorochrome of 8-a-minonaphthalen-2,3,6-trisulfonic acid (ANTS) to facilitate detection; Figure 10 is a Western blot photograph of milk protein isolated from normal (non-transgenic) and transgenic mice expressing human alpha-1,2-fucosyl transferase. The glycosylation of the proteins marked with ink was detected by means of immunofluorescence using a specific lectin for the ligation of alpha-1,2-fucose. The figure demonstrates the presence of glycosylated milk proteins with the H antigen product of the transgenic enzyme. Figures 1 to 10 are supplied in accordance with 37 C. F. R. 1 .81.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the expression in vivo in mammary tissue of non-human mammals of heterologous, catalytically active glycosyltransferases, which control the production of secondary gene products resulting from the specific activity of the glycosyltransferase enzyme. These glycosyltransferase enzymes control the synthesis of free oligosaccharides or the covalent attachment of oligosaccharides to proteins or lipids. This expression is achieved in a cell using genetic engineering to instruct the cell to produce specific, heterologous glycosyltransferases (primary gene product), and then employ the specific catalytic activity associated with each glycosyltransferase to produce a specific product, the gene product. secondary. In the case of glycosyltransferases, the secondary gene product includes not only the synthesized oligosaccharides but also the glycosylated proteins and lipids. Oligosaccharides and glycosylated proteins / lipids are secreted and found in free form in the milk of transgenic mammalian species. As used herein and in the claims, the term "glycosylation" represents the post-translational modification of a protein or lipid by an enzymatic procedure facilitated by the expressed glycosyltransferase, which results in the covalent attachment of the protein or lipid of one or more monosaccharide units. This glycosylation is achieved by instructing the cell to produce both the glycosyltransferases as well as the protein or lipid of interest. The protein or lipid of interest can be an entity whether homologous or heterologous. As used herein and in the claims, the term "homologous" refers to a composition or molecular form normally produced by the host cell or animal. As used herein and in the claims, the term "heterologous" refers to a composition or molecular form that is not normally produced by the host cell or animal. Genetic engineering techniques are used to incorporate a foreign genetic material, which is a genetic material derived from other species, into the genome of the host animal. As used herein and in the claims, the terms "transgenic cell" or "transgenic animal" refers to a cell line or a host animal that contains such transformed genomes. As used herein and in the claims, "transgenic products" refers to products derived from said transgenic entities; For example, milk derived from a transgenic cow is called transgenic milk. The present invention is based, in part, on the production of a transgenic mammal, which is not a human being, wherein the cells comprising the mammary gland contain a transgene, which expresses a desired glycosyltransferase. (The transgenic mammary cell genome can also be transfected with a gene, which encodes a human protein). The resulting glycosyltransferase, when expressed in the transgenic host mammary cell, is useful to produce soluble, free oligosaccharides in the milk produced by said transgenic animal. The expressed glycosyltransferase is also useful in the glycosylation of milk proteins homologous or heterologous human proteins, when the transgenic mammary cell also expresses said proteins. The same concept can be applied to the modification of lipids. The present invention has wide application in the synthesis of oligosaccharides by various glycosyltransferases such as fucosyltransferase, galactosyltransferase, glucosyltransferase, sialyltransferases, mannosyltranferases, xylosyltransferase, sulfotransferases, glucuronyltransferases, β-acetylg to lactose minyltransferases and N-acetyl-glucosaminyltransferases. The products of other kinds of enzymes of the Golgi apparatus, such as acetylases, glucuronylepimerases, glycosidases, acetyltransferases, mannosidases and phosphotransferases can also be synthesized by the method described.

BEST MODE FOR CARRYING OUT THE INVENTION The following describes the incorporation of DNA encoding the production of a fucosyltransferase, particularly human alpha-1,2-fucosyltransferase (hereinafter also referred to as Fuc-T), to the genome of cells that form non-human mammary glands. An example of a Fuc-T product is 2'-fucosyl lactose. This is one of the oligosaccharides in human milk and has the chemical formula, fucose-alpha-1,2-Gal-β-1, 4-Glc. Other Fuc-T products will include glycoproteins containing terminal β-galactose residues, which can be fucosylated by means of Fuc-T. The resulting carbohydrate structures, fucose-alpha-1, 2-galactose-β-R, wherein R is selected from the group consisting of β-1,3-GlcNAc, β-1, 4-GlcNAc, and the like, are known in the field of blood group serology as the "H antigen". It will be recognized by one skilled in the art that other glycosyltransferases and Golgi processing enzymes may also be used in accordance with the present invention. In the non-limiting examples, described below, transgenic mice were employed. Mouse genomes do not contain or express the DNA encoding Fuc-T. Therefore, if the transgenic mice produce either Fuc-T, 2'-fucosyl-lactose, or the H antigen then the successful incorporation of the gene encoding Fuc-T into the murine genome should occur. It is well known in the art that it is possible to insert the DNA encoding glycosyltransferases into the genome of transgenic host cells. Some of the cell lines that can be used for the transgenic expression of glycosyltransferases are Chinese Hamster Ovary (CHO) cells, mouse L cells, mouse A9 cells, baby hamster kidney cells, C-127 cells, PC8 cells, insect cells, yeast cell lines and other eukaryotic cells. In the preferred embodiment of the present invention, the host cells are mammary cells, said cells comprising tissue from the mammary glands of transgenic mammals, which are not human beings. Preferred embodiments of the present invention utilize mice, rats, rabbits, pigs, sheep, goats, horses, or transgenic cows. Particularly preferred embodiments use transgenic sheep, goats or cows. A particularly preferred embodiment of the present invention is the use of bovine mammary tissue in transgenic lactating cows. The precise procedure used to introduce the altered genetic material to the host cell is not critical. Any of the well known procedures for introducing foreign nucleotide sequences can be used. These include the use of plasmid vectors, viral vectors and any other known methods for producing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material, to the host cell. It is only necessary that the particular genetic engineering method, used, be able to successfully introduce at least one transgene to the host cell, which is capable after expressing the desired glycosyltransferase. A preferred technique in the practice of the present invention is the transfection of an embryonic target cell, transplantation of the embryonic, transgenic target cell formed therefrom into a recipient substitute offspring, and identifying at least one female offspring that is capable of of producing the free human oligosaccharide (s) or human recombinant protein, glycosylated, in its milk. A highly preferred embodiment of the present invention comprises the steps of transfecting an embryonic target cell of a bovine species, transplanting the embryonic, transgenic target cell formed therefrom, to a recipient bovine offspring and identifying at least one bovine offspring. female that is capable of producing the free human oligosaccharide (s) or recombinant protein, homologous or heterologous, glycosylated, in its milk. The following examples demonstrate the disruption of the genome of mammalian host cells, which is not a human being, by inserting therein heterologous DNs encoding specific glycosyltransferases. Then, the transgenic host expresses specific, catalytically active glycosyltransferases, which facilitate the production of a secondary gene product, more specifically a specific oligosaccharide. The glycosylation of milk proteins is also demonstrated. If also, in addition to the DNA encoding the oligosaccharides, heterologous DNA encoding human milk proteins is inserted into the host genome, then the human milk proteins will also be expressed by said host. Since the same host will also express the glycosyltransferase, the glycosylation of human milk proteins will occur with certain specific oligosaccharides. The human milk proteins of interest include secretory immunoglobulins, lysozyme, lactoferrin, kapa-casein, lactoperoxidase, alpha-lactalbumin, β-lactalbumin and lipase stimulated with bile salts. This aspect of the oligosaccharide synthesis and protein / lipid glycosylation has several advantages over the other methods currently available. The aspect lies in the novel combination of: (a) the use of transgenic mammary cells for the synthesis of sugar nucleotides from natural sources of carbon, such as glucose; (b) the expression of recombinant, heterologous glycosyltransferase genes in transgenic mammalian cells; (c) the production of heterologous oligosaccharides of desired structure by the natural lactating mammary glands of transgenic animals, said production being the result of the enzymatic activity of the expressed heterologous glycosyltransferase enzymes; and (d) The use of the heterologous glycosyltransferase enzyme to glycosylate proteins or homologous or heterologous lipids. The experiments described below illustrate the following points: a) A human alpha-1, 2-fucosyltransferase gene was isolated and cloned from a cell line of human epidermal carcinoma. This enzyme is responsible for the synthesis of the oligosaccharide 2'-fucosyl lactose and the glycosylation of proteins with blood group-specific antigen H; b) The functional nature of the gene was demonstrated by its ability to express catalytically active alpha-1, 2-fucosyltransferase in cultured, non-human cell lines. The presence of 1,2-fucosyltransferase was demonstrated by enzymatic activity analysis specific for this enzyme. The presence of catalytically active alpha-1, 2- fucosyltransferase was also demonstrated using the immunofluorescence technique to demonstrate the presence of the H antigen on the surface of the cells expressing the enzyme; c) The utility of this gene in the formation of transgenic animals, which are not human beings, capable of expressing the product of the alpha-1, 2-fucosyltransferase gene, was demonstrated by the successful development of transgenic mice carrying the gene of human alpha-1, 2-fucosyltransferase, which is capable of expressing catalytically active alpha-1,2-fucosyltransferase; d) The expression in the breast tissue of a transgenic animal, which is not a human, of human alpha-1, 2-fucosyltransferase, catalytically active. The presence of the enzyme was established through direct analysis of enzymatic activity and immunofluorescence using antibodies that exhibit a specific binding character for the enzyme; e) The formation of secondary gene products resulting from the catalytic activity of human alpha-1, 2-fucosyltransferase expressed in non-human milk. Such products include the release of the human oligosaccharide, 2'-fucosyl lactose, to milk and the glycosylation of milk proteins with the H antigen product of the enzyme. The presence of secondary gene products was established through biochemical analysis of the immunofluorescence compounds using lectins that exhibit a specific binding character for the H antigen. The following examples are provided as representatives of the scope of the invention and should not be considered as limiting the invention claimed herein. Examples 1 and 2 use tissue culture systems. These in vitro experiments were taken to prove that said expression of enzymatically active glycosyltransferases was possible. The Examples 1 and 2 are not critical to the capability of the present invention and are intended only for the purpose of ensuring an understanding and appreciation of the invention. Examples 3, 4, 5 and 6 prove that the in vivo production of heterologous secondary gene products in the milk of transgenic mammals, which are not human beings, is possible. Examples 3-6 are provided for the purpose of enabling the teachings, scope and claims of the invention. In view of the foregoing, the applicants believe that a deposit of biological material under 37 C. F. R. § 1 .802 is not required.

EXAMPLE 1 Isolation of the Human Alpha-1, 2-fucosyltransferase Gene from a Human Epidermal Carcinoma Cell Line. The cDNA encoding alpha-1, 2-fucosyltransferase was isolated from a collection of epidermal carcinoma cell line cDNA (A 431), whereas alpha-1,2-fucosyltransferase was previously cloned from this source (VP Rajan et al., J Biological Chemistry, Volume 264, pp. 1 1 158-1 1167, 1989). This description is incorporated herein by reference. After amplification mediated by the polymerase chain reaction (PCR) of the protein encoding the sequence, the cDNA was cloned into a bacterial vector to determine the cDNA sequence of the amplified gene. The DNA sequence was determined from each of the six independently isolated clones of alpha-1,2-fucosyltransferase. This nucleotide sequence and the corresponding amino acid sequence are shown in Fig. 1. To determine the aforementioned cDNA, two initiators of alpha-1,2-fucosyltransferase, each containing 31 nucleotides (31 -dores), were engineered, based on the alpha-1, 2- cDNA sequence. published fucosyltransferase. The initiator contained BigNH2 initiating the methionine residue in position 27, where the transcription of Fuc-T (beginning of an open reading frame) began. The second BigCOOH initiator, contained a stop codon at position 10. The primers are indicated in Figure 2. The PCR reaction included approximately 1 μM of each primer, 1 μg of the template with PCR buffer and Taq polymerase. The PCR reaction was carried out in a thermal cycler (Perkin and Elmer, Model 840) using a temperature cycle of 94 ° C for one minute, 60 ° C for three minutes, 72 ° C for three minutes for 30 cycles followed for an extension of 5 minutes at 72 ° C. The electrophoresis was applied to the PCR reaction product in a low melting point agarose of 0.8% w / v. A 1.1 kilobase fragment was detected. This fragment was cut and subcloned into the PCR II cloning vector. One of the transformants, hereinafter referred to as the selector, was selected and characterized by both restriction and nucleotide sequence analysis. The DNA sequence was performed using an Applied Biosystems automatic DNA sequencer, Model 373A. The restriction pattern of the insert indicated similarity with the coding region of alpha-1,2-fucosyltransferase. The nucleotide sequence of this candidate clone was identical to the published sequence, except at position 340. Site-directed mutagenesis, in vitro, was employed to correct this single defective base, thus forming the wild-type sequence that was used in the transfection experiments described below.

EXAMPLE 2 Host Cell Expression of Human Glycosyltransferases. This example describes the transfection of mouse L cells and cultured Chinese Hamster Ovary (CHO) cells with a gene capable of expressing human specific glycosyltransferase, alpha-1, 2-fucosyltransferase or Fuc-T. These cell lines were selected for transfection, since their natural genomes do not carry the DNA that encodes Fuc-T. The transfection of the cell lines is shown to produce either Fuc-T or its enzymatic products (2'-fucosyl-lactose or H antigen bound to glycoproteins), then successful transfection was demonstrated. This was demonstrated by the immunofluorescence technique, using specific antibodies and / or a specific lectin that binds selectively to the antigen H. The Fuc-T gene used for transfection was obtained as described in Example 1. Next the transfection and the materials used in it are described.

Phenyl-β-D-galactoside was obtained from Sigma Chemical Co. The nucleotide sugar GDP-L- (U-? 4C) fucose, with a specific activity of 278 mCi / mmole, was purchased from Amersham Corporation. The human epidermal carcinoma cDNA collection, A431, was obtained as a donation from Dr. Nevis Frigiep, University of Miami, Oxford, Ohio. The PCR I I vector was purchased from Invitrogen Corporation. The expression vector pQE 1 1 was purchased from Qiagen Inc. Plasmid pSV2-neo was obtained from Pharmacia Fine Chemicals Corporation. Plasmid pMet-FucT-bGH was obtained from Drs. Xhou Chen and Bruce Kelder at the University of Ohio, Athens, Ohio. This construct contains the cDNA encoding Fuc-T. The primers were synthesized by Operon Technology or Fischer Scientific Corporation. The mouse monoclonal antibody for the H antigen was purchased from Dako Corporation. The goat anti-mouse antibodies labeled with fluorescein isothiocyanate were purchased from Sigma Chemical Company. Rabbit polyclonal antibodies to alpha-1,2-fucosyltransferase emerged as a means to detect the expression of this enzyme to elicit sufficient enzyme to act as the antigen in the induction of the antibody, the selector insert was subcloned into a vector of inducible expression in the frame with a 6XHis tag (pQE1 1). A protein labeled with 6XHis was easily purified with a nickel affinity chromatography column. To avoid any possible toxicity of the cell, the hydrophobic region of alpha-1,2-fucosyltransferase was eliminated. To achieve this, two initiating knots were built. The first, a Bam H I-N H2 hybridized to the model at position 60; the second initiator, Sal I COO encompasses the stop codon. The BamHI and Sal I sites were processed, by engineering, on the upstream and downstream initiators. The PCR product was subcloned in frame to a BamHI / Sal I site of the expression vector pQE1 1 allowing fusion with the 6XHis tag. Three milligrams of the fusion protein (alpha-1, 2-fucosyltransferase-6XHis) were purified using a Ni-agarose affinity column. This material was used to raise rabbit polyclonal antibodies that exhibit specific character against Fuc-T.

Cell Line v Culture: Mouse L cells were obtained from CHO cells of the American Tissue Culture Collection (ATTCC) in Washington, D.C. Cells were grown in a minimal, essential medium of alpha (alpha-MEM, G IBCO, Grand Island, New York), supplemented with 10% fetal bovine serum (GIBCO), 80 μ / ml penicillin (Sigma), 80 μg / ml streptomycin / Sigma) and L-glutamine (Sigma), hereinafter referred to as alpha-MEM / 10% FCS. Transfected L cells were grown on alpha-MEM containing G418 (GIBCO) in 400 μg / ml. Transfected CHO cells were grown on alpha-MEM containing G418 (GI BCO) in 1000 μg / ml.

Momentary Transfection: L cells were grown on 8-chamber chamber slides (Lab-Tek) at a 75% confluence level. A transfection cocktail was added to each chamber, pMet-Fuc-bGH DNA (2 μg), lipofection (2 μl) and 200 μl of Opti-MEM medium (GIBCO). After 6 hours of incubation at 37 ° C, 200 μl of alpha-MEM / 10% FCS was added, and after 48 hours of further incubation at 37 ° C, the slides were processed for indirect immunofluorescence, as described below . The ability of the cloned cDNA fragment to encode functional alpha-1, 2-fucosyltransferase was tested, demonstrating the presence of the catalytic product of this enzyme, i.e., the H antigen, on the cell surface of mouse L cells, cultured . (L cells do not normally have the H antigen on their membranes). The wild-type insert of the selector, observed in Example 1, was subcloned into the pMet-bGH plasmid at an EcoR L site. In this alpha-1,2-fucosyltransferase expression construct, the activity is under the control of the metallothionein promoter. This promoter is zinc inducible. The mouse L cells were transfected momentarily with the pMet-Fuc-bG H construct, and the presence of the structure of the H antigen on the cell surface was confirmed using the immunofluorescence technique with anti-antigen mouse monoclonal antibodies. H, as described below. Secondary antibodies labeled with fluorescein were goat anti-mouse antibodies. In addition, the presence of the H antigen was confirmed using the fluorescein-labeled lectin, Ulex europaeus aglutinnin 1, which specifically binds to the structures of fucose-alpha-1,2-galactose.

Indirect immunofluorescence. Successful transfection was demonstrated by the presence, on the surface of the cell, of the H antigen. Indirect immunofluorescence analysis was performed using 8 cavity tissue culture chamber slides. Cells were placed in each chamber at an appropriate density, incubated overnight at 37 ° C, and then analyzed for H antigen. The camera holders were washed with phosphate buffered saline (PBS), fixed with 100μl of a 2% solution of formalin in Hanks balanced salt solution (H BSS), and permeabilized with saponin ( 2mg / ml; Sigma) in 1% FCS, and incubated with a dilution of 1 in 1000 of the anti-H antibody for 60 minutes in a humid chamber at room temperature. The slides were then washed three times with PBS and incubated for an additional 60 minutes with a 1: 1000 dilution of goat anti-mouse antibody labeled with FITC, at room temperature in a humid chamber. The wetting prevented the sample from drying out. The efficiency of transfection of L cells, or the percentage of transformed L cells, which express the H antigen, based on immunofluorescence, was approximately 30%. The aforementioned results clearly demonstrate the successful transfection of mammalian cell lines, which are not human beings, with the DNA encoding Fuc-T. Cultured, transfected cell lines not only produce the primary gene product, Fuc-T, but also modify the glycoproteins. As a result of Fuc-T activity, the modified proteins carry the H antigen. These results prove that the cloned cDNA fragment encoding Fuc-T is capable of expressing enzymatically active Fuc-T. Therefore, this cDNA was used for the production of transgenic animals, as described below.

EXAMPLE 3 Transgenic mammal, which is not a human being, which possesses the gene that codes for a specific human glycosyltransferase. This experiment proves that transgenic mammals, which are not human beings, are capable of producing a heterologous, catalytically active glycosyltransferase. More specifically, it is proved that the transgenic animals produce human alpha-1,2-fucosyltransferase. Transgenic mice were produced by microinjection of human Fuc-T cDNA into the embryo genome of mice. The fertilized mouse eggs were isolated in a single cell stage and the male pronuclei were injected with the transgenic construct containing the human alpha-1, 2-fucosyltransferase gene, as shown in Example 1. These embryos were then implanted into pseudo-pregnant mice, which were previously paired with sterile males. The offspring of transgenic founder mice were identified, after approximately 25 days after birth, using PCR amplification to analyze the chromosomal DNA obtained from a tail fragment with probes specific for the inserted human gene. To achieve this desired transformation, normal techniques were used in practice. Such details have been described, fully, in the following references, which are incorporated herein by reference and which were also discussed hereinabove: (a) International Patent Application No. PCT / US90 / 06874; (b) International Patent Application No. PCT / DK93 / 00024; (c) International Patent Application No. PCT / GB87 / 00458; and (d) International Patent Application No. PCT / GB89 / 01343. One aspect of the present invention relates to the expression of a human, catalytically active glycosyltransferase in the milk of a mammal, which is not a human, and to the use of said glycosyltransferase to effect the formation of the desired secondary gene product. To obtain the specific expression of the mammary gland of the human gene during the lactation of transgenic mice, the regulatory sequence (promoter) of the serum acid protein (WAP) of a mouse was used to generate a transgenic construct for the expression of human alpha-1,2-fucosyltransferase. The murine WAP promoter was received as a donation from Dr. L. Henninghauser of the National Institutes of Health, Bethesda, Maryland. This material was used to construct the plasmid pWAP-polyA, shown in Figure 3. This plasmid contains the polyadenylation signal sequence of bovine growth hormone (polyA) at the 3 'end of the fusion gene which results in the effective expression, processing and stability of the RNA messenger. The human alpha-1, 2-fucosyltransferase (Fuc-T) gene was inserted into this plasmid to result in the formation of plasmid pWAP-polyA-Fuc-T, illustrated in Figure 4. This plasmid was used for microinjection of mouse embryos, described above. Using microinjections of DNA at concentrations of 2 and 4 μg / ml, a total of 85 pups were obtained from 16 injections. Only two injections did not result in pregnancy. The size of the bait, from a single injection was normal, averaging 3 to 10 pups per litter. Biopsies of the tail of the 85 young of the mice were performed. By analyzing the tail biopsy, it was determined that nine of the founder population, hereinafter referred to as F0, possessed the gene encoding human alpha-1, 2-fucosyltransferase. This corresponds to a transgenic mouse production efficiency of approximately 1%. This falls within the scale of production efficiency expected, between 5 and 25%. The F0 progeny comprised eight males and one female. Then, six of the founders were spawned with normal mice, resulting in a total of one progeny of 98. Thirty-seven of the offspring (hereinafter referred to as F 1) had the gene encoding alpha-1,2. -human p-yltransferase, as determined from tail biopsies and PCR analysis. This corresponds to an efficiency of F- of approximately 36%. The generation of F1 is composed of nineteen males and nineteen females. Table 1 summarizes the results obtained.

TABLE 2 PRODUCTION EFFICIENCY OF THE F1 GENERATION OF SIX FOUNDING MICE Founder # No. Progeny Total No. of Transgenic Progeny Efficiency of Female Male Transaenesis (%) 6 16 4 2 37.5 28 18 2 2 22.2 29 1 8 4 6 55.6 34 1 3 3 6 69.2 54 1 5 2 1 20.0 72 1 8 4 2 33.3 Fifteen of the females of F 1 (second generation) reached maturity and were pregnant with normal mice. The pregnant F 1 females were allowed to give birth. The milk of four of these F1 mothers was harvested, ten days after birth. The collection of milk was performed using one of the two techniques that are normal in practice: a) Breast suction using a vacuum line connected to a trap flask and a suction cup; or b) Anaesthetize and sacrifice the animal after cutting the teats to release the fluid contents of the mammary gland. The milk samples were kept frozen on dry ice until they were subjected to analytical procedures, as described below. The collected milk samples were prepared to initially separate the oligosaccharides from the milk proteins and lipids. This was achieved using the methods described by A. Kobata (Methods in Enzymology, Chapter 24, Volume 28, pp. 262-271, 1982) and A. Kobata et al. (Methods in Enzymology, Chapter 21, pp. 21 1 -226 , 1978). The milk samples were treated as follows. Samples, typically 90-100 μl, obtained from control (non-transgenic) and transgenic animals, were centrifuged at a relative centrifugal force (RCF) of 10,000 for 20 minutes in conical, conical centrifuge tubes. The centrifugation resulted in the separation of the milk into two layers: an upper layer of cream consisting mostly of lipids, and a lower layer. The lower layer, containing soluble material, was removed and transferred to a new centrifuge tube. Two equivalent volumes of ice-cooled ethanol were added, swirled and centrifuged at 10,000 RCF. The soluble supernatants of ethanol were recovered and concentrated by evaporation of the alcohol using a Speed-Vac concentrator. The insoluble protein pellets of ethanol were kept frozen at -70 ° C until another analysis was performed. After concentration, the extracts containing the oligosaccharide were resuspended in water to the exact volume of the original milk sample. These resuspended samples were kept at 4 ° C in a refrigerated autosampler until re-used. When appropriate, these samples were subjected to compositional analysis, as described in Examples 4 to 7. One aspect of the present invention is the transgenic expression of heterologous glycosyltransferases in the mammary gland of milk-bearing mammals, which are not human . The expression of heterologous glycosyltransferases can be demonstrated in two ways: a) Directly, determining the presence of the same enzyme (primary gene product); and b) Indirectly, determining the presence of the enzyme product (secondary gene product: oligosaccharide or glycosylated protein) in the milk of the transgenic animal. As noted above, the murine genome does not encode the specific alpha-1, 2-fucosyltransferase responsible for the synthesis of the H antigen. In this way, if Fuc-T or Fuc-T products are present in the milk of transgenic mice, then successful transgenesis has occurred, providing unique means to synthesize, and thus to obtain secondary gene products. An important aspect of the present invention is the production of heterologous secondary gene products in the milk of non-human animals. As noted above, the secondary gene products can comprise not only the immediate product of the enzyme, the oligosaccharide, but also the protein or glycosylated lipids, homologous or heterologous, which are glycosylated through the covalent attachment of said oligosaccharide to the protein or lipid. The milk harvested from Example 3 was analyzed for the presence of human alpha-1, 2-fucosyltransferase and also for the presence of secondary gene products, specifically 2'-fucosyl lactose and proteins glycosylated with the H antigen. Examples 4, 5, 6 and 7 prove the production of human Fuc-T and Fuc-T products in the milk of non-human animals.

EXAMPLE 4 Analysis to Test the Production of a Specific Glycosyltransferase in the Milk of Transgenic Mammals, which are not Human Beings. This example demonstrates the feasibility of obtaining human alpha-1, 2-fucosyltransferase in the milk of transgenic mice.

As noted above, the murine WAP promoter was employed to ensure the site-specific mammary gland expression of human alpha-1, 2-glycosyltransferase. The precipitate of insoluble milk protein of ethanol, obtained from the mice, as described above in Example 3, was resuspended in sodium dodecylsulfate (SDS) containing pH regulator of polyacrylamide gel electrophoresis (PAGE). The volume of pH regulator SDS-PAGE used to resuspend the protein pellet was exactly equal to that of the original volume of the milk sample. The reconstituted samples were analyzed for the presence of alpha-1, 2-fucosyltransferase. This presence was determined using immunoblot technology, as described below. More specifically, Western Blots were used. Five microliter samples of the protein pellet resuspended in SDS-PAGE were electrophoresed in a 12.5% polyacrylamide gel. Electrophoresis was performed at 150 volts. After electrophoresis, the resolved proteins were transferred to nitrocellulose membranes. The transmigration was carried out for one hour at 100 volts, using pH regulator of Tris-HCL 12.5 mM, pH 7.5, containing 96 mM of glycine, 20% of methanol and 0.01% of SDS. After transfer, the remaining unbound, reactive groups on the nitrocellulose membranes were blocked by incubation in a pH buffer of 50 mM Tris-HCL, pH 7.5, containing 0.5 M NaCl and 2% gelatin, hereinafter referred to as TBS. Next, the membranes were washed three times in TBS containing 0.05% Tween-20. The membranes were incubated for 18 hours at 1% gelatin / TBS containing a 1: 500 dilution of polyclonal rabbit antibody having specific character against alpha-1,2-fucosyltransferase. This polyclonal antibody was obtained as described in Example 2. Following growth with TBS-Tween, the membrane was then incubated with a solution of 1% gelatin-TBS containing goat anti-rabbit IgG previously conjugated to horseradish peroxidase. . The membrane was then washed with TBS-Tween. The presence and position of the proteins on the nitrocellulose membrane were visualized by incubating the membrane in a volume of 50 ml of TBS containing 0.018% hydrogen peroxide and 10 ml of methanol containing 30 mg of 4-chloro-naphthol. Figure 5 shows the result of this experiment for milk samples obtained from a control animal (non-transgenic) and two transgenic animals. The transgenic animals are referred to in Figure 5 as 28-89 and 29-1 19. The non-transgenic animals are referred to in Figure 5 as the control. Figure 5 indicates that a very intense band is clearly present in the milk samples obtained from the two transgenic animals, but is absent from the milk obtained from the non-transgenic control animal. The intense bands are clearly present at a relative molecular weight of approximately 46 kilodaltons corresponding to the predicted molecular weight of alpha-1,2-fucosyltransferase. Also, the intense bands are present in positions corresponding to lower relative molecular weights in the range of approximately 30-25 kilodaltons. these bands are absent in the sample of milk derived from the non-transgenic sample. Without being limited to the inventors, it is speculated that these lower molecular weight bands probably correspond to Fuc-T fragments. These results prove that the milk samples, of the transgenic samples, contain Fuc-T, while the milk samples, of the non-transgenic animal, do not contain Fuc-T.

EXAMPLE 5 Analysis to Test the Production of Secondary Gene Products Heterologous, Specific, in the Milk of Transgenic Mammals, that are not Human Beings. This example proves the feasible character to obtain secondary gene products, heterologous in the milk of transgenic mammals, which are not human beings. More specifically, this example demonstrates the ability to obtain the secondary gene product of Fuc-T in the milk of an animal that is not a human being. More specifically, the presence of the secondary gene product, 2'-fucosyl lactose, was demonstrated in transgenic milk. The non-transgenic mouse control milk does not contain 2'-fucosyl lactose. Evaporated oligosaccharide extracts, obtained as described in Example 3, were analyzed and separated using a liquid chromatography and high pressure ion exchange composition in a Dionex apparatus, as previously described by Reddy and Bush (Analytical Biochemestry , Volume 1 98, pp. 139-147, 1991). These techniques are well known in normal practice.

The specific points of the experimental establishment, elution profiles and conditions for the separation and analysis of the oligosaccharide extracts, were as follows: the Dionex apparatus was equipped with a degasser to remove C02 from the elution pH regulators, a suppressor of ions to remove ions from the eluents in the column and an in-line conductivity meter to ensure the removal of the ions by the ion suppressor. The chromatographic parameters were as follows: Operating Time: 45 minutes Peak Width: 50 seconds Peak Peak: 0.500 Peak Area Rejection: 500 Volume of I njection: 20 L Flow Rate: 1 .0 ml / min.

The gradient elution program, presented in Table 3, comprised the following eluents: Eluent 1: Sodium Acetate 600 mM in Sodium Hydroxide 100 mM Eluent 2: Water Milli-Q NanoPure Eluent 3: Sodium Hydroxide 200 mM TABLE 3 ELDER GRADIENT PROGRAM Time (minijit) Flow (ml / min)% # 1% # 2% # 3 0.0 1.0 0 50 50 12.0 1.0 0 50 50 12.1 1.0 7 46 47 20.0 1.0 7 46 47 20.1 1.0 10 45 45 27.0 1.0 10 45 45 27.1 1.0 50 25 25 32.0 1.0 50 25 25 32.1 1.0 0 50 50 45.0 1.0 0 50 50 90.0 0.1 0 50 50 The eluted fractions were collected every 0.5 minutes. The chromatographic profiles of the milk samples obtained from the two control mice and from four transgenic animals, expressing alpha-1,2-fucosyltransferase, are shown in Figures 6A to 6F. It was determined that 2'-fucosyl lactose (which is the oligosaccharide product synthesized by the enzyme encoded by the transgene) is eluted later than lactose. The review of the profiles revealed that only the transgenic animals produce milk containing a carbohydrate that co-elutes with the normal 2'-fucosyl-lactose. Based on the chromatographic peak areas, it was possible to calculate the concentrations of 2'-fucosyl lactose present in the milk samples of transgenic animals using normal techniques. The data is summarized in Table 4.

TABLE 4 CONCENTRATION OF 2'-FUCOSIL-LACTOSE IN VARIOUS SAMPLES OF NON-HUMAN MILK Donor Concentration of 2'-fucosiol-lactose (mg / L) 1 . Control (non-transgenic) 0 2. Transgenic 28-29 71 1 29- 1 1 9 468 34-34 686 72-66 338 These data prove, in accordance with the present invention, that a secondary gene product, mainly a 2 ' -Fucosil-lactose, can be produced in the milk of transgenic mammals, which are not human beings. To further characterize the oligosaccharide, a different method was used for carbohydrate analysis. Flouroforo Assisted Carbohydrate Electrophoresis (FACE) is a technology first described by P. Jackson, J. Chromatography, Volume 270, pgs. 705-713, 1990. The FACE technique was used to unequivocally demonstrate that carbohydrate co-eluted with 2'-fucosyl lactose has the same mobility as normal 2'-fucosyl lactose in an electrophoresis system. This provides additional confirmation that the identity of the oligosaccharide, content in the sample of transgenic milk is 2'-fucosil-lactose. To conduct FACE experiments with putative 2'-fucosyl lactose, separate fractions were deposited during Dionex-HPLC chromatography. The fractions between the arrows (indicated on the abscissa) in Figures 6A to 6F were desposited from each sample. The portions of each sample (1/8) obtained from the control and 2 transgenic mice were labeled for 3 hours at 45 ° C using 8-aminonaphthalen-2, 3,6-trisulfonic acid (ANTS) from Glyki Inc. (Novato, California). Dry samples were resuspended in 5 μl of labeling reagent in 5 μl of reduction reagent solution (sodium cyanoborohydride) and incubated at 45 ° C for 3 hours. The resulting labeled samples were dried and resuspended in 6 μl of deionized water. From this solution, a 2 μl aliquot was transferred to a fresh microfuge tube. 2 μl of charge p H regulator containing glycerol was added, and the mixture was vigorously combined. Then, the total mixture (4 μl) was subjected to electrophoresis in an "O-linked oligosaccharide gel" (Glyko). Electrophoresis was conducted at a constant current of 20 milli amp. , and 1 5 ° C. The profile of the migrated gel pattern, thus obtained, was imaged using a Millipore imaging apparatus. Figure 7 shows the image of the gel obtained in this way. The sample from a control mouse (lane 2) illustrates a single band that migrates at the position of a normal lactose marker. Samples obtained from transgenic mice (lanes 3 and 4) contained an additional band of higher molecular weight. This band, indicated in the figure with an arrow, em igra in the position of a normal 2'-fucosyl lactose (lane 6). Another characterization of the oligosaccharide was performed by incubating aliquots equivalent to 1/8 of the deposits illustrated in Figures 6A to 6F in the presence of the enzyme fucosidase which is cleaved specifically in alpha-1,2-fucose ligations. This enzyme used was derived from Corynebacterium sp. and was purchased at Panvera Corp., Madison, Wisconsin. Overnight oligosaccharides were incubated in the presence of 20 μl of sodium phosphate pH regulator, pH 6.0, containing 20 milliunities of the enzyme at 37 ° C. Then, the collections were labeled with ANTS and subjected to electrophoresis, as described above. The gels in Fig. 8 show the results of this experiment. It is readily apparent that the material that co-elutes with 2'-fucosyl lactose in HPS-H PLS chromatography and co-migrates with the same molecule after ANTS labeling and electrophoresis, is also susceptible to the action of the hydrolysis enzyme, specific, alpha-1,2-fucosidase. 3'-fucosyl lactose (which is the most similar isomer to 2'-fucosyl lactose) is unaffected by the enzyme. This experiment further confirms the identity of the oligosaccharide in the transgenic milk sample being 2'-fucosyl lactose. In contrast, milk samples obtained from non-transgenic control animals (lanes 6 and 14) after hydrolysis, produce only one band (lanes 7 and 15) that migrate to the normal galactose position.

EXAMPLE 6 Analysis to Test the Identity of the Oligosaccharide Produced in the Milk of Transgenic Mammals, which are not Human Beings. This experiment evaluated the monosaccharide units comprising the oligosaccharide. For this purpose, samples of deposited milk, obtained from control and transgenic mice, were thoroughly treated with a mixture of glycosidases. Aliquots (1/8 of the total in 20 μl of water) of the deposits illustrated in Figures 6A to 6F were dried by evaporation in conical tubes. The dried contents were resuspended in 20 μl of a solution containing 20 milliunits of alpha-1,2-fucosidase (Panvera, Madison, Wisconsin) and 20 μl of a suspension containing 30 units of E. coli β-galactosidase (Boehringer Mannheim, Indianapolis , Indiana). The resulting suspensions were incubated for 1 8 hours at 37 ° C under an atmosphere of toluene. In this way, only the oligosaccharides susceptible to the sequential actions of fucosidase and β-galactosidase were hydrolyzed to their corresponding monosaccharide units. After incubation, the mixtures were dried in a Speed Vac concentrator. The oligosaccharides resulting from this hydrolysis were labeled as described above in Example 5. The labeled monosaccharides were subjected to electrophoresis in a "Monosaccharide Gel" (Glyko). Electrophoresis was performed at 30 milliamps, constant voltage for 1 hour and 10 minutes. Figure 9 shows the results of this experiment. The samples of milk obtained from transgenic animals (lanes 2 and 4) contain 3 bands corresponding to fucose, galactose and glucose. The monosaccharides released from a normal 2'-fucosyl lactose (lane 1) are identical to the monosaccharides released from the oligosaccharide deposits obtained from two transgenic animals (lanes 2 and 4). 3'-Fucosyl lactose is unaffected by the enzymatic action of glycosidases (lane 3). This result unequivocally establishes the identity of the oligosaccharide in the transgenic milk as being 2-p-yl-lactose. Collectively, these previously discussed findings prove that the invention, as described and claimed, allows the production of secondary gene products in the milk of transgenic animals. More specifically, the experimental data provide the feasible character to obtain oligosaccharides in the milk of transgenic animals that contain a transgene composed, in part, of DNA encoding glycosyltransferases. To further corroborate the invention, it was decided to demonstrate the presence of other glycoconjugates, such as glycoproteins in the milk of the transgenic animals. These glycoproteins are covalent adducts of protein and oligosaccharide, wherein the oligosaccharide is the product of the glycosyltransferase. The oligosaccharide is covalently linked to the protein by the glycosyltransferase.

EXAMPLE 7 Analysis to Test the Production of Glycoconjugates in the Milk of Transgenic Mammals, which are not Human Beings. This example demonstrates the feasible character to obtain glycoproteins in the milk of transgenic animals, which are not human beings. The oligosaccharide portion is the same oligosaccharide produced as a result of the activity of the primary gene product, glycosyltransferase. The resulting glycosylated protein is an example of a secondary gene product. Western blots of the milk proteins of transgenic and control animals were prepared in the manner described in Example 4. If, however, instead of incubating the transferred membrane with polyclonal rabbit antibodies, the membrane was incubated with the Ulex lectin. europaeus agglutinin I (U EA I). This lectin specifically binds to the fucose ligation, alpha-1, 2. For this purpose, the protein pellets described in Example 3, were centrifuged at 13,000 xg for 10 minutes, the supernatant (excess ethanol and water) was removed and the resulting pellets were resuspended in a sample regulator volume of SDS-PAGE equal to that of the original milk volume . 5 μl of these extracts were electrophoresed on 12.5% polyacrylamide-SDS-PAG E, as described in detail in Example 3. Following the electrophoresis, the proteins were transferred to nitrocellulose membranes for 1 hour at 1 00 volts in 12.5 mM Tris-HCI, 96 mM glycine, 20% methanol, 0.01% SDS, pH 7.5. The nitrocellulose membranes were blocked for 1 hour with 2% gelatin in TBS (50 mM Tris-HCl, pH 7.5, 0.5 M NaCl) and washed 3 x 5 minutes, in TBS containing 0.05% Tween-20. Then, the membranes were incubated for 18 hours, in 1% gelatin / TBS containing a dilution of 1: 500 of UEA-1 labeled with peroxidase (Sigma, St. Louis Mo.). The resulting membrane was then washed and the proteins visualized by incubating in a mixture of 50 ml of TBS containing 0.01 8% hydrogen peroxide and 10 ml of methanol containing 30 mg of 4-chloro-naphthol (Bio Rad, Richmond, California) . Figure 10 shows a photograph of the proteins visualized using this technique. It is evident that only transgenic animals produced milk containing fucosylated proteins, especially recognized by the lectin UEA-1. These proteins migrated with a relative molecular weight of 35-40 kilodaltons and are thought to be with casein. These results indicate that the glycoproteins carrying the oligosaccharide 2'-fucosyl lactose (H antigen) have been produced in the milk of transgenic animals carrying a transgene encoding alpha-1,2-fucosyltransferase. The milk of the non-transgenic control animals had no glycoproteins carrying 2'-fucosyl lactose. Examples 3-7 have proven that it is possible to produce transgenic mammals, which are not human beings, capable of synthesizing, in their milk, secondary gene products. More specifically, it is possible to produce transgenic mammals, which are not human beings, that express human glycosyltransferases in breast tissue resulting in the presence of human oligosaccharides and glycosylated glycoconjugates in the milk of these animals.

Industrial Application The invention, as described and claimed herein, solves a great need to provide means for obtaining large quantities of desired oligosaccharides and glycoconjugates.

The desired oligosaccharides and glycoconjugates can be isolated from the milk of transgenic mammals and used in the preparation of pharmaceuticals, diagnostic kits, nutritional products, and the like. Whole transgenic milk can also be used to formulate nutritious products that provide special advantages. Transgenic milk can also be used in the production of specialized, enteric nutritious products. The invention, as described and claimed, avoids the laborious organic chemistry and immobilized enzymatic chemical synthesis of these very important materials that have been used in pharmaceutical, research, diagnostic, nutritional, and agricultural formulas. Having described the preferred embodiments of the present invention, it will be obvious to those skilled in the art that various modifications may be made to the embodiments described, and that said modifications are intended to be within the scope of the present invention.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Abbott Laboratories, ROSS Products Division (ii) TITLE OF THE INVENTION: Transgenic Production of Oligosaccharides and Glycoconjugates. (iii) NUMBER OF SEQUENCES: 1 (iv) ADDRESS OF CORRESPONDENCE: (A) RECIPIENT: Donald O. Nickey ROSS Products Division Abbott Laboratories (B) STREET: 625 Cleveland Avenue (C) CITY: Columbus (D) STATE: Ohio ( E) COUNTRY: USA (F) C.P. 43215 (v) LEGIBLE FORM THROUGH COMPUTER: (A) TYPE OF MEDIA: Diskette, 3.5 in., Storage 1.44 Mb (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: MS-DOS Version 6.21 (D) SOFTWARE: WordPerfect, Version 6.0a (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (vii) PREVIOUS INFORMATION OF THE APPLICATION: Not Applicable (ix) TELECOMMUNICATION INFORMATION: (A) ) TELEPHONE: (614) 624-7080 (B) TELEFAX: (614) 624-3074 (C) TELEX: None (2) INFORMATION FOR SEC ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1155 base pairs (B) TYPE: Nucleic acid (C) STRING STRUCTURE: Individual (D) TOPOLOGY: Unknown (ii) TYPE OF MOLECULE: cloned cDNA representing the product of a segment of human genomic DNA (A) DESCRIPTION: GDP- L-fucose-β-D-galactoside 2-alpha-fucosyltransferase (iii) HYPOTHETICAL: (iv) ANTI-SENSE: (v) TYPE OF FRAGMENT: Entire amino acid sequence provided . (vi) ORIGINAL SOURCE: Human Epidermal Carcinoma Cell Line (A) ORGANISM: (B) CLASS: (C) INDIVIDUAL ISOLATED: (D) DEVELOPMENT STAGE: (E) HAPLOTIPO (F) TYPE OF TISSUE: (G) TYPE OF CELL: (H) CELL LINE: (I) ORGANEL: (vii) IMMEDIATE SOURCE: Human Epidermal Carcinoma Cell Line (A) COLLECTION: (B) CLON: (viii) POSITION IN THE GENOME: (A) ) CHROMOSOME / SEGMENT: 19 (B) MAPPING POSITION: (C) UNITS: (ix) ASPECT: (A) NAME / KEY: (B) LOCATION: (C) IDENTIFICATION METHOD: Sequence Analysis and DNA Restriction ( D) OTHER INFORMATION: The encoded product of nucleotide SEQ ID NO: 1: is the enzyme, GDP-L-fucose-β-galactoside 2-alpha-fucosyltransferase, having the amino acid sequence described in SEQ ID NO: 1 :. This enzyme is responsible for the synthesis of 2'-fucosyl lactose. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAATTCGGCT TATCTGCCAC CTGCAAGCAG CTCGGCC ATG TGG CTC CGG AGC CAT 55 Met Trp Leu Arg Ser His 1 5 CGT CAG CTC TGC CTG GCC TTC CTG CTA GTC TGT GTC CTC TCT GTA ATC 103 Arg Gln Leu Cys Leu Ala Phe Leu Leu Val Cys Val Leu Ser Val lie 10 15 20 TTC TTC CTC CAT ATC CAT CAA GAC AGC TTT CCA CAT GGC CTA GGC CTG 151 Phe Phe Leu His lie His Gln Asp Ser Phe Pro His Gly Leu Gly Leu 25 30 35 TCG ATC CTG TGT CCA GAC CGC CGC CTG GTG ACÁ CCC CCA GTG GCC ATC 199 Ser lie Leu Cys Pro Asp Arg Arg Leu Val Thr Pro Pro Val Ala lie 40 45 50 TTC TGC CTG CCG GGT ACT GCG ATG GGC CCC AAC GCC TCC TCT TCC TGT 247 Phe Cys Leu Pro Gly Thr Ala Met Gly Pro Asn Wing Being Ser Cys 55 60 65 70 CCC CAG CAC CCT GCT TCC CTC TCC GGC ACC TGG ACT GTC TAC CCC AAT 295 Pro Gln His Pro Wing Ser Leu Ser Gly Thr Trp Thr Val Tyr Pro Asn 75 80 85 GGC CGG TTT GGT AAT CAG ATG GGA CAG TAT GCC ACG CTG CTG GCT CTG 343 Gly Arg Phe Gly Asn Gln Met Gly Gln Tyr Ala Thr Leu Leu Ala Leu 90 95 100 GCC CAG CTC AAC GGC CGC CGG GCC TTT ATC CTG CCT GCC ATG CAT GCC 391 Ala Gln Leu Asn Gly Arg Arg Wing Phe lie Leu Pro Wing Met His Wing 105 110 115 GCC CTG GCC CCG GTA TTC CGC ATC ACC CTG CCC GTG CTG GCC CCA GAA 439 Ala Leu Ala Pro Val Phe Arg lie Thr Leu Pro Val Leu Ala Pro Glu 120 125 130 GTG GAC AGC CGC ACG CCG TGG CGG GAG CTG CAG CTT CAC GAC TGG ATG 487 Val Asp Ser Arg Thr Pro Trp Arg Glu Leu Gln Leu His Asp Trp Met 135 140 145 150 TCG GAG GAG TAC GCG GAC TTG AGA GAT CCT TTC CTG AAG CTC TCT GGC 535 Ser Glu Glu Tyr Wing Asp Leu Arg Asp Pro Phe Leu Lys Leu Ser Gly 155 160 165 TTC CCC TGC TCT ACT TTC TTC CTC CAT CTC CGG GAA CAG ATC CGC 583 Phe Pro Cys Ser Trp Thr Phe Phe His His Leu Arg Glu Gln lie Arg 170 175 180 AGA GAG TTC ACC CTG CAC GAC CAC CTT CGG GAA GAG GCG CAG AGT GTG 631 Arg Glu Phe Thr Leu His Asp His Leu Arg Glu Glu Ala Gln Ser Val 185 190 195 CTG GGT CAG CTC CGC CTG GGC CGC ACA GGG GAC CGC CCG CGC ACC TTT 679 Leu Gly Gln Leu Arg Leu Gly Arg Thr Gly Asp Arg Pro Arg Thr Phe 200 205 210 GTC GGC GTC CAC GTG CGC CGT GGG GAC TAT CTG CAG GTT ATG CCT CAG 727 Val Gly Val His Val Arg Arg Gly Asp Tyr Leu Gln Val Met Pro Gln 215 220 225 230 CGC TGG AAG GGT GTG GTG GGC GAC AGC GCC TAC CTC CGG CAG GCC ATG 775 Arg Trp Lys Gly Val Val Gly Asp Ser Ala Tyr Leu Arg Gln Wing Met 235 240 245 C TGG TTC CGG GCA CGG CAC GAA GCC CCC GTT TTC GTG GTC ACC AGC 823 AL Trp Phe Arg Wing Arg His Glu Wing Pro Val Phe Val Val Thr Ser 250 255 260 AAC GGC ATG GAG TGG TGT AAA GAA AAC ATC GAC ACC TCC CAG GGC GAT 871 Asn Gly Met Glu Trp Cys Lys Glu Asn Lie Asp Thr Ser Gln Gly Asp 265 270 275 GTG ACG TTT GCT GGC GAT GGA CAG GAG GCT ACÁ CCG TGG AAA GAC TTT 919 Val Thr Phe Wing Gly Asp Gly Gln Glu Wing Thr Pro Trp Lys Asp Phe 280 285 290 GCC CTG CTC ACA CAG TGC AAC CAC ACC ATT ATG ACC ATT GGC ACC TTC 967 Wing Leu Leu Thr Gln Cys Asn His Thr lie Met Thr lie Gly Thr Phe 295 300 305 310 <; * .- xxc TGG GCT GCC TAC CTG GCT GGC GAC ACT GTC TAC CTG GCC 1015 Gly Phe Trp Wing Wing Tyr Leu Wing Gly Gly Asp Thr Val Tyr Leu Wing 315 320 235 AAC TTC ACC CTG CCA GAC TCT GAG TTC CTG AAG ATC TTT AAG CCG GAG 1063 Asn Phe Thr Leu Pro Asp Ser Glu Phe Leu Lys lie Phe Lys Pro Glu 330 335 340 GCG GCC TTC CTG CCC GAG TGG GTG GGC ATT AAT GCA GAC TTG TCT CCA 1H1 Ala Ala Phe Leu Pro Glu Trp Val Gly lie Asn Ala Asp Leu Ser Pro 345 350 355 CTC TGG ACÁ TTG GCT AAG CCT TGAGAGCCAG GGAAGCCGAA TTC 1155 Leu Trp Thr Leu Wing Lys Pro 360 365

Claims (3)

1 - . 1 - Milk of a transgenic mammal, which is not a human being, said milk characterized in that it contains heterologous components produced as the secondary gene products of at least one heterologous gene contained in the genome of said transgenic mammal, which is not a human.

2 - The milk according to claim 1, wherein the heterologous gene encodes a catalytic entity. 3. The milk according to claim 1, wherein the catalytic entity is selected from the group consisting of enzymes and antibodies. 4. The milk according to claim 1, wherein the heterologous component is selected from the group consisting of oligosaccharides and glycoconjugates. 5. The milk according to claim 4, wherein the oligosaccharides are selected from the group consisting of lactose, 2-fucosyl lactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentase I, lacto-N-fucopentase II, lacto-N-fucopentase I II, lacto-N-difucopentaose I, sialyl-lactose,

3-sialyl-lactose, sialyltetrasaccharide a, sialyltetrasaccharide b, sialyltetrasaccharide c, disialyltetrasaccharide and sialyl-lacto-N-fucopenta . 6. The milk according to claim 4, wherein the glycoconjugates are selected from the group consisting of glycosylated homologous proteins, glycosylated heterologous proteins and glycosylated lipids. 7. The milk according to claim 6, wherein the glycosylated heterologous proteins are selected from the group consisting of human serum proteins and human milk proteins. 8. The milk according to claim 7, wherein the human milk proteins are selected from the group consisting of secretory immunoglobulins, lysozyme, lactoferrin, kapa-casein, alpha-lactalbumin, beta-lactalbumin, lactoperoxidase and lipase stimulated with bile salts. 9. The milk according to claim 1, wherein the transgenic mammal, which is not a human being, is selected from the group consisting of mice, rats, rabbits, pigs, goats, sheep, horses and cows. 10. The milk according to claim 9, wherein said mammal, which is not a human being, is a cow. 1. The milk according to claim 1, wherein the heterologous gene is selected from the group consisting of genes encoding human enzymes and human antibodies. 12. The milk according to claim 1, wherein the human enzymes are selected from the group consisting of glycosyltransferases, phosphorylases, hydroxylases, peptidases and sulfotransferases. 13. Milk according to claim 12, wherein the glycosyltransferases are selected from the group consisting of fucosyltransferase, galactosyltransferase, glucosyltransferase, xylosyltransferase, acetylases, glucuronyltransferases, glucuronylepimerases, sialyltransferases, mannosyltransferases, sulfotransferases, β-acetylgalactosaminyltransferase and N-acetyl -glucosaminyltransferases. 14. A product produced in the milk of transgenic mammals, which are not human beings, wherein said product results from the action of a catalytic entity selected from the group consisting of heterologous enzymes and heterologous antibodies, and wherein said transgenic mammal, that is not a human being, contains in its genome at least one heterologous gene that codes for said catalytic entity. 15. A substantially pure product according to claim 14, wherein said product is isolated from the milk of said transgenic mammal, which is not a human being. 16 - The product according to claim 14, wherein said product is selected from the group consisting of oligosaccharides and glycoconjugates. 17. The product according to claim 16, wherein the oligosaccharides are selected from the group consisting of lactose, 2-fucosyl lactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, Lacto-N-fucopentase II, lacto-N-fucopentase III, lacto-N-difucopentaose I, sialyl lactose, 3-sialyl lactose, sialyltetrasaccharide a, sialyltetrasaccharide b, sialyltetrasaccharide c, disialyltetrasaccharide and sialyl-lacto-N-fucopentase. 18. The product according to claim 16, wherein the glycoconjugates are selected from the group consisting of glycosylated homologous proteins, glycosylated heterologous proteins and glycosylated lipids. 19. The product according to claim 18, wherein the glycosylated heterologous proteins are selected from the group consisting of human serum proteins and human milk proteins. 20. - The product according to claim 19, wherein the human milk proteins are selected from the group consisting of secretory immunoglobulins, lysozyme, lactoferrin, kapa-casein, alpha-lactalbumin, beta-lactalbumin, lactoperoxidase and lipase stimulated with bile salts . 21. The product according to claim 14, wherein the human enzymes are selected from the group consisting of glycosyltransferases, phosphorylases, hydroxylases, peptidases and sulfotransferases. 22. The product according to claim 21, wherein the heterologous human glycosyltransferase is selected from the group consisting of fucosyltransferase, galactosyltransferase, glucosyltransferase, xylosyltransferase, acetylases, glucuronyltransferases, glucuronylepimerases, sialyltransferases, mannosyltransferases, sulfotransferases, β-acetylgalactosaminyltransferase and N-acetyl-glucosaminyltransferases. 23 - An enteric nutritive product useful in the nutritional maintenance of an animal, said enteric nutrient product containing the milk according to claim 1. 24. - A pharmaceutical product useful in the treatment of an animal, said pharmaceutical product containing the product according to claim 15. 25.- A medical diagnostic product useful in the diagnosis of an animal, said diagnostic product containing the product in accordance with claim 15. 26.- An agricultural product useful in the maintenance of grains, said agricultural product containing the product according to claim 14.