The Expression of Therapeutic Proteins in Transgenic Animals

Paleyanda, Rekha K.; Young, Janet M.; Velander, William H.; Drohan, William N.

doi:10.1007/978-1-4615-3698-7_13

Cited by 6 publications

(4 citation statements)

References 47 publications

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“…The structure and composition of the free oligosaccharides in milk differ between mammals, but the human lactating mammary gland synthesizes the most complex mixture of free, reducing oligosaccharides, many of which contain fucose and other determinants for human blood groups, including the ABO and Lewis system. The structures of some of these oligosaccharides in humans are determined genetically by the expression of glycosyltransferases during lactation and the availability of sugar nucleotide substrates (Hill and Brew, 1975;Ginsburg, 1972;Smith et al 1987), but the precise mechanisms regulating differential expression of individual milk oligosaccharides are not known.Recent advances in transgenic technology have resulted in successful expression of transgene-encoded proteins in animal milk (Wilde et al, 1992;Paleyanda et al, 1991;Archer et al, 1994). Using this methodology, we have begun to explore factors regulating glycoconjugate biosynthesis.…”

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Remodeling of Mouse Milk Glycoconjugates by Transgenic Expression of a Human Glycosyltransferase

Prieto

Mukerji

Kelder

et al. 1995

Journal of Biological Chemistry

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The mammary gland is a unique biosynthetic tissue that produces a variety of species-specific glycoconjugates, but the factors regulating the production of specific glycoconjugates are not well understood. To explore the underlying regulation, a fusion gene containing a cDNA encoding the human ␣1,2-fucosyltransferase (␣1,2FT), which generates the H-blood group antigen, flanked by the murine whey acidic protein promoter and a polyadenylation signal, was introduced into mice. Milk samples from transgenic animals contained soluble forms of the ␣1,2FT, as revealed by Western blots of milk samples using an anti-␣1,2FT antiserum and by the demonstration of ␣1,2FT enzyme activity. Milk from transgenic animals also contained large quantities of 2 -fucosyllactose (Fuc␣1-2Gal␤1-4Glc) and modified glycoproteins containing the H-antigen, whereas milk from control animals lacked these glycoconjugates. Expression levels of 2 -fucosyllactose were high in most animals and represented 1 ⁄3 to nearly 1 ⁄2 of the total milk oligosaccharides. These results demonstrate that heterologous transgenic expression of a glycosyltransferase can result in the expression of both the transgene and its secondary gene products and that the structures of milk oligosaccharides can be remodeled depending on expression of the appropriate enzyme. Furthermore, these results suggest that the lactating mammary gland may be a unique biosynthetic reactor for the production of biologically active oligosaccharides and glycoconjugates.Despite the fact that animal oligosaccharides are important in many biological and pathological processes (Karlsson, 1989;Kornfeld, 1992; Drickamer and Taylor, 1993;McEver et al., 1995), little is known about the regulation of animal cell glycoconjugate biosynthesis. Biosynthesis is influenced by expression of appropriate glycosyltransferases, correct targeting of the enzymes to the Golgi apparatus, sugar nucleotide levels, competition between enzymes, residence time of glycoconjugates in intracellular compartments, acceptor specificity of the enzymes, and many other factors (Kornfeld and Kornfeld, 1985;Kobata and Takasaki, 1992;Cummings, 1992;Baenziger, 1994). Experimental modulation of these factors has been difficult. One approach that has proven somewhat successful in vitro is to attempt to alter oligosaccharide biosynthesis by genetic manipulation of glycosyltransferases (Lee et al., 1989;Smith et al., 1990;Lowe et al., 1990). This approach has so far been limited to cultured and immortalized cell lines and has been useful in producing neoglycoconjugates for biological studies .One major site for glycoconjugate biosynthesis in animals, and one that has been of enormous historical importance in the field of glycobiology, is the lactating mammary gland. Milk contains, in addition to lactose, numerous glycoproteins (Patton et al., 1990;Fiat and Jollès, 1989) and a variety of free oligosaccharides (Kobata et al., 1972). The structure and composition of the free oligosaccharides in milk differ between mammals, but the human lac...

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“…Recent advances in transgenic technology have resulted in successful expression of transgene-encoded proteins in animal milk (Wilde et al, 1992;Paleyanda et al, 1991;Archer et al, 1994). Using this methodology, we have begun to explore factors regulating glycoconjugate biosynthesis.…”

mentioning

confidence: 99%

Remodeling of Mouse Milk Glycoconjugates by Transgenic Expression of a Human Glycosyltransferase

Prieto

Mukerji

Kelder

et al. 1995

Journal of Biological Chemistry

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“…Estimates of annual US clinical need are now greatly in excess of the approximately 300 kg per 6 year which can be harvested on the total of 7 million liters of human plasma in the US (Paleyanda et al, 1991). Thus, the recombinant source must be capable of economically supplying about 1000 kg/yr in order to replace the maximum possible hfib obtainable from current plasma fractionation by cryoprecipitation.…”

Section: Transgenic Livestock As An Alternative Source Of Recombinantmentioning

confidence: 99%

Production and secretion of recombinant human fibrinogen by the transgenic murine mammary gland

Butler¹,

O'Sickey²,

Lord³

et al. 1999

Theriogenology

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The mammary gland of lactating transgenic animals has several advantages for production of heterologous proteins including a high cell density that results in high concentrations of secreted protein and the ability to perform several types of post-translational modifications. Transgenes were constructed from the 4.1 kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the A", B$ and ( fibrinogen chains to evaluate the requirements of the transgenic murine mammary gland for high level secretion of fully assembled human fibrinogen. After introducing the constructs into the murine zygotes by microinjection, secretion of fully assembled fibrinogen into milk was measured at concentrations between 10 :g/ml to 200 :g/ml.In one line of mice the total secretion of fibrinogen and unassembled subunits approached 700 :g/ml in milk. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the B$ and ( chains were rate limiting. Also, the subunit complexes ( 2 , A"( 2 and the individual subunits A", B$ and ( were found as secretion products. This is the first time that secretion of individual B$-subunits by any cell type has been reported and suggests the organization of the secretion pathway in mammary epithelia is different from that in liver. Glycosylated forms of individual B$-chain contained a complex saccharide with low mannose. Glycosylation of the (-chain was also observed. These results suggest the 4.1 mWAP promoter can drive expression of fibrinogen cDNAs to high levels and that the amount of fully assembled fibrinogen secreted is equal to the level of the lowest expressing chain.iii

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“…In addition to use as an hemostatic agent, new FS-applications include carrier mahix applications for the delivery of drugs and biologics (1)(2)(3)(4)(5). Estimates of annual US clinical need are now greatly in excess of the approximately 300 kg per year which can be harvested using the currently low yield, cryoprecipitation methods on the total of 7 million liters of human plasma available for fractionation in the US (6). Thus, the recombinant source mustbe capable of economically supplying about 1000 kg/yr in order to replace the maximum possible hfib obtainable from current plasma fractionation by cryoprecipitation.…”

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Current Progress in the Production of Recombinant Human Fibrinogen in the Milk of Transgenic Animals

et al. 1997

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The Expression of Therapeutic Proteins in Transgenic Animals

Cited by 6 publications

References 47 publications

Remodeling of Mouse Milk Glycoconjugates by Transgenic Expression of a Human Glycosyltransferase

Remodeling of Mouse Milk Glycoconjugates by Transgenic Expression of a Human Glycosyltransferase

Production and secretion of recombinant human fibrinogen by the transgenic murine mammary gland

Current Progress in the Production of Recombinant Human Fibrinogen in the Milk of Transgenic Animals

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