Abstract
We have tested the feasibility of producing large quantities of human serum albumin (HSA) in the milk of transgenic livestock by generating transgenic mice as a model system. The sheep β-lactoglobulin (BLG) 5′-regulatory promoter sequences were used to support expression of BLG or HSA in transgenic mice. Transgenic animals generated from the entire BLG gene including 3, 5.5 or 10.8 kb of 5′-sequences demonstrated that 3 kb of 5′-sequences were sufficient to support high levels of expression of BLG, and that the longer 5′-sequences did not improve upon the levels of expression. As such, the 3 kb 5′-sequences were used to drive expression of HSA in BLG-HSA constructs. HSA was not detectably expressed in eight transgenic lines generated from a BLG-HSA construct containing the HSA cDNA. Two transgenic lines of 26 generated, using five different constructs, with an HSA minigene possessing the first intron expressed HSA in their milk. One of these expressed HSA at high levels (2.5 mg ml−1) and has stably transmitted this ability to its progeny. A high percentage of transgenic mouse lines (four of six) generated from a vector containing an HSA minigene possessing introns 1 and 2 expressed HSA in their milk at levels which ranged from 1 to 35 μg ml−1. In a similar trend, levels of expression of HSA by transfected tissue culture cells from BLG-HSA vectors containing an introduced SV40 enhancer were low with the HSA cDNA, increased with the HSA minigene with intron 1 and increased further with the minigene containing introns 1 and 2. This study demonstrates that high levels of HSA can be expressed in the milk of transgenic animals, that introns of the HSA gene play a role in its expression and that transfected cell lines may be used to quickly evaluate the relative expression efficiencies of various vector constructs intended for future transgenic evaluation.
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References
Archibald, A.L., McClenaghan, M., Hornsey, V., Simons, J.P. and Clark, A.J. (1990) High-level expression of biologically active α-antitrypsin in the milk of transgenic mice.Proc. Natl Acad. Sci. USA 87, 5178–82.
Auffray, C. and Rougeon, F. (1980) Purification of immunoglobulin heavy chain mRNA from total myeloma tumor RNA.Eur. J. Biochem. 107, 303–13.
Brinster, R.L., Allen, J.M., Behringer, R.R., Gelinas, R.E. and Palmiter, R.D. (1988) Introns increase transcriptional efficiency in transgenic mice.Proc. Natl Acad. Sci. USA 84, 836–40.
Buhler, T.A., Bruyere, T., Went, D.F., Stranzinger, G. and Burki, K. (1990) Rabbit β-casein promoter directs secretion of human IL2 into the milk of transgenic rabbits.Bio/Technology 8, 140–3.
Clark, A.J., Simons, P., Wilmunt, I. and Lathe, R. (1987) Pharmaceuticals from transgenic livestock.Trends Biotechnol. 5, 20–4.
Clark, A.J., Bessos, H., Bishop, J.O., Brown, P., Harris, S., Lathe, R., McClenaghan, M., Prowse, C., Simons, J.P., Whitelow, C.B.A. and Wilmut, I. (1989) Expression of human anti-hemophilic factor IX in the milk of transgenic sheep.Bio/Technology 7, 487–92.
Denman, J., Hayes, M., O’Day, C., Edmunds, T., Bartlett, C., Hirani, S., Ebert, K.M., Gordon, K. and McPherson, J.M. (1991) Transgenic expression of a variant of human tissue-type plasminogen activator in goat milk: purification and characterization of the recombinant enzyme.Bio/Technology 9, 839–43.
Ebert, K.M., Selgrath, J.P., DiTullio, P., Denman, J., Smith, T.E., Memon, M.A., Schindler, J.E., Monastersky, G.M., Vitale, J.A. and Gordon, K. (1991) Transgenic production of a variant of human tissue-type plasminogen activator in goat milk: generation of transgenic goats and analysis of expression.Bio/Technology 9, 835–8.
Etcheverry, T., Forredter, W. and Hitzeman, R. (1986) Regulation of the chelatin promoter during the expression of human serum albumin or yeast phosphogylcerate kinase in yeast.Bio/Technology 4, 729–30.
Fleer, R., Yeh, P., Amellal, N., Maury, I., Fournier, A., Bacchetta, F., Baduel, P., Jung, G., L’Hôte, H., Becquart, J. Fukuhara, H. and Mayaux, J.F. (1991) Stable multicopy vectors for high-level secretion of recombinant human serum albumin byKluyveromyces yeasts.Bio/Technology 9, 968–75.
Fraker, P.J. and Speck, J.C. (1978) Protein and cell membrane iodinations with a sparingly soluble chloroamide 1,3,4,6,-tetrachloro-3a, 6-adiphenylglycouril.Biochem. Biophys. Res. Comm. 80, 849–57.
Gordon, K., Lee, E., Vitale, J.A., Smith, A.E., Westphal, H. and Hennighausen, L. (1987) Production of human tissue plasminogen activator in transgenic mouse milk.Bio/Technology 5, 1183–7.
Gorman, C.M., Moffat, L.F. and Howard, B.H. (1982) Recombinant genomes which express chloramphenicol acetyl-transferase in mammalian cells.Mol. Cell. Biol. 2, 1044–51.
Harris, S., McClenaghan, M., Simons, J.P., Ali, S., and Clark, A.J. (1990) Gene expression in the mammary gland.J. Reprod. Fert. 88, 707–15.
Hogan, B., Costantini, F. and Lacy, E. (1986)Methods of Manipulating the Mouse Embryo. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory.
Huang, M.T.F. and Gorman, C.M. (1990) Intervening sequences increase efficiency of RNA 3′ processing and accumulation of cytoplasmic RNA.Nucl. Acids Res. 18, 937–47.
Hurwitz, D.R., Hodges, R., Drohan, W. and Sarver, N. (1987) Optimizing gene expression in BPV-transformed cells: effects of cell type on enhancer/promoter interactions.Nucl. Acids Res. 15, 7137–53.
Krimpenfort, P., Rademakers, A., Eyestone, W., van der Schans, A., van den Broek, S., Kooiman, P., Kootwijk, E., Platenburg, G., Pieper, F., Strijker, R. and de Boer, H. (1991) Generation of transgenic dairy cattle using ‘in vitro’ embryo production.Bio/Technology,9, 844–7.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–5.
Latta, M., Knapp, M., Sarmientos, P., Brefort, G., Becquart, B., Guerrier, L., Jungm, G. and Mauyaux, J.-F. (1987) Synthesis and purification of mature serum albumin fromE. coli.Bio/Technology 5, 1309–14.
Lee, K.F., DeMayo, F.J., Atiee, S.H. and Rosen, J.M. (1988) Tissue specific expression of the rat β-casein gene in transgenic mice.Nucl. Acids Res. 16, 1027–41.
Meade, H., Gates, L., Lacy, E. and Lonberg, N. (1990) Bovine αS1 casein gene sequences direct high level expression of active human urokinase in mouse milk.Bio/Technology 8, 443–6.
Minghetti, P.P., Ruffner, D.E., Kuang, W.J., Dennison, O.E., Hawkins, J.W., Beattie, E.G. and Dugaiczyk, A. (1986) Molecular structure of the human albumin gene is revealed by nucleotide sequence within q11-22 of chromosome 4.J. Biol. Chem. 261, 6747–57.
Mulligan, R.C. and Berg, P. (1980) Expression of a bacterial gene in mammalian cells.Science 209, 1422–7.
Nahon, J.-L., Tratner, I., Poliard, A., Presse, F., Poiret, M., Gal, A., Sala-Trepat, J.M., Legres, L., Feldman, G. and Bernuau, D. (1988) Albumin and α-fetoprotein gene expression in various nonhepatic rat tissues.J. Biol. Chem. 263, 11436–42.
Peters Jr, T. (1975) Serum albumin. In Putman, F.W. ed.,The Plasma Proteins; Structure, Function and Genetic Control, pp. 133–73. San Diego: Academic Press.
Pittius, C.W., Hennighausen, L., Lee, E., Westphal, H., Nicols, E., Vitale, J. and Gordon, K. (1988) A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice.Proc. Natl Acad. Sci. USA 85, 5874–8.
Quirk, A.V., Geisow, M.J., Woodrow, J.R., Burton, S.J., Wood, P.C., Sutton, A.D., Johnson, R.A. and Dodsworth, N. (1989) Production of recombinant human serum albumin fromSaccharomyces cerevisiae.Biotec. Appl. Biochem. 11, 273–87.
Sassoon, D., Lyons, G., Wright, W.E., Lin, V., Lassar, A., Weintraub, H. and Buckingham, M. (1989) Expression of two myogenic regulatory factors myogenin and MyoDl during mouse embryogenesis.Nature 341, 303–7.
Shani, M. (1985) Tissue specific expression of rat myosin light chain 2 gene in transgenic mice.Nature 314, 283–6.
Shani, M. (1986) Tissue specific and developmentally regulated expression of a chimeric actin/globin gene in transgenic mice.Mol. Cell. Biol. 6, 2624–31.
Simons, J.P., McClenaghan, M., and Clark, A.J. (1987) Alteration of the quality of milk by expression of sheep β-lactoglobulin in transgenic mice.Nature 328, 530–2.
Simons, J.P., Wilmut, I., Clark, A.J., Archibald, A.L., Bishop, J.O. and Lathe, R. (1988) Gene transfer into sheep.Bio/Technology 6, 179–83.
Sleep, D., Belfield, G.P. and Goodey, A.R. (1990) The secretion of human serum albumin from the yeastSaccharomyces cerevisiae using five different leader sequences.Bio/Technology 8, 42–6.
Tomasetto, C., Wolf, C., Rio, M.-C., Mehtali, M., LeMear, M., Gerlinger, P., Chambon, P. and Lathe, R. (1989) Breast cancer protein PS2 synthesis in mammary gland of transgenic mice and secretion into milk.Mol. Endocrinol. 3, 1579–84.
Van Brunt, J. (1988) Molecular farming: transgenic animals as bioreactors.Bio/Technology 6, 1149–54.
Vilotte, J.L., Solulier, S., Stinnakre, M.G., Massoud, M. and Mercier, J.C. (1989) Efficient tissue specific expression of bovine α-lactalbumin in transgenic mice.Eur. J. Biochem. 186, 43–8.
Wall, R.J., Parsel, V.G., Shamay, A., McKnight, R.A., Pittius, C.W. and Hennighausen, L. (1991) High-level synthesis of a heterologous milk protein in the mammary glands of transgenic swine.Proc. Natl Acad. Sci. USA 88, 1696–700.
Wright, G., Carver, A., Cottom, D., Reeves, D., Scott, A., Simons, P., Wilmut, I., Garner, I. and Colman, A. (1991) High level expression of active human alpha-1-antitrypsin in the milk of transgenic sheep.Bio/Technology 9, 830–4.
Yu, S.-H., Deen, K.C., Lee, E., Henninghausen, L., Sweet, R.W., Rosenberg, M. and Westphal, H. (1989) Functional human CD4 protein produced in milk of transgenic mice.Mol. Biol. Med. 6, 255–61.
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Shani, M., Barash, I., Nathan, M. et al. Expression of human serum albumin in the milk of transgenic mice. Transgenic Research 1, 195–208 (1992). https://doi.org/10.1007/BF02524750
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DOI: https://doi.org/10.1007/BF02524750