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The Mammary Gland as a Bioreactor: Expression, Processing, and Production of Recombinant Proteins

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Abstract

A variety of transgenic animal species are beingused to produce recombinant proteins. The generalapproach is to target the expression of the desiredprotein to the mammary gland using regulatory elements derived from a milk protein gene and thencollect and purify the product from milk. Promotersequences from a number of different milk protein geneshave been used to target expression to the mammarygland, although significant problems remain withregard to achieving transgene expression levelsconsistent with commercial exploitation. The mammarygland appears to be capable of carrying out the complexposttranslational modifications, such as glycosylation andγ-carboxylation required for the biologicalactivity and stability of specific proteins. Effectivepurification protocols have been established andproducts produced by this route have now enteredclinical trials.

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References

  1. R. L. Palmiter, R. D. Brinster, R. E. Hammer, et al. (1982). Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300: 611-615.

    Google Scholar 

  2. A. J. Clark, J. P. Simons, I. Wilmut, and R. Lathe (1987). Pharmaceutical s from transgenic livestock. Trends Biotech. 5: 20-24.

    Google Scholar 

  3. J. Gordon and F. Ruddle (1981). Integration and stable germline transmission of genes injected into mouse pronuclei. Science 214: 1244-1246.

    Google Scholar 

  4. K. Gordon, E. Lee, J. Vitale, A. Smith, H. Westphal, and L. Hennighausen (1987). Production of human tissue plasmnogen activator in transgenic mouse milk. Biotechnology 5: 1183-1187.

    Google Scholar 

  5. J. P. Simons, I. Wilmut, A. J. Clark, A. L. Archibald, J. O. Bishop, and R. Lathe (1988). Gene transfer into sheep. Biotechnology 6: 179-183.

    Google Scholar 

  6. K. Ebert, J. Selgrath, P. DiTullio, J. Denman, T. Smith, M. Memon, J. Schindler, G. Monastersky, and K. Gordon (1991). Transgenic production of a variant of human tissue plasminogen activator in goat milk: Generation of transgenic goats and the analysis of expression. Biotechnology 9: 835-838.

    Google Scholar 

  7. R. E. Hammer, V. G. Pursel, C. Rexroad, et al. (1985). Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315: 680-683.

    Google Scholar 

  8. P. Krimpenfort, A. Rademakers, W. Eyestone, et al. (1991). Generation of transgenic dairy cattle by in vitro embryo production. Biotechnology 9: 844-847.

    Google Scholar 

  9. T. A. Buhler, T. Bruyere, D. F. Went, G. Stranzinger, and K. Burki (1990). Rabbit β-casein promoter directs secretion of interleukin-2 into the milk of transgenic rabbits. Biotechnology 8: 140-143.

    Google Scholar 

  10. A. J. Clark, J. P. Simons, and I. Wilmut (1992). Germline manipulation; applications in agriculture and biotechnology. In F. Grosveld and G. Kollias (eds.), Transgenic Mice in Biology and Medicine Academic Press, London, pp. 247-269.

    Google Scholar 

  11. R. L. Page, S. P. Butler, A. Subramanian, F. C. Gwazdauskas, J. L. Johnson, and W. H Velander (1995). Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures. Trans. Res. 4: 353-360.

    Google Scholar 

  12. S. L. Stice (1998). Opportunities and challenges in domestic animal embryonic stem cell research. In, A. J. Clark (ed.), Animal Breeding: Technology for the 21st Century Harwood Academic Publishers, Chur 7000, Switzerland, pp. 64-71.

    Google Scholar 

  13. K. H. S. Campbell, J. McWhir, W. A. Ritchie, and I. Wilmut (1996). Sheep cloned by nuclear transfer from a cultured cell line. Nature 380: 64-66.

    Google Scholar 

  14. I. Wilmut, A. E. Schnieke, J. McWhir, A. J. Kind, and K. H. S. Campbell (1997). Viable offspring derived from fetal and adult mammalian cells. Nature 385: 810-813.

    Google Scholar 

  15. A. E. Schnieke, A. J. Kind, W. A. Ritchie, et al. (1997). Factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278: 2130-2133.

    Google Scholar 

  16. A. J. Clark, P. Bissinger, D. W. Bullock, et al. (1994). Reprod. Fert. Dev. 6: 589-598.

    Google Scholar 

  17. J-C. Mercier and J-L. Vilotte (1993). Structure and function of milk protein genes. J. Dairy Sci. 76: 3079-3098.

    Google Scholar 

  18. M. A. Persuy, M-G Stinnakre, C. Printz, M-F. Mahe, and J.-C. Mercier (1992). High expression of the caprine β-casein gene in transgenic mice. Eur. J. Biochem 205: 887-893.

    Google Scholar 

  19. J. P. Simons, M. McClenaghan, and A. J. Clark (1987). Alteration of the quality of milk by expression of sheep β-lactoglobulin in transgenic mice. Nature 328: 530-532.

    Google Scholar 

  20. E. A. Maga and J. D. Murray (1995). Mammary gland expression of transgenes and the potential for altering the properties of milk. Biotechnology 13: 1452-1457.

    Google Scholar 

  21. A. L. Archibald, M. McClenaghan, V. Hornsey, J. P. Simons, and A. J. Clark (1990). High level expression of biologically active human α1-antitrypsin in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 87: 5178-5182.

    Google Scholar 

  22. H. Meade, L. Gates, E. Lacy, and N. Lonberg (1990). Bovine αs1-casein gene sequences direct high level expression of active human urokinase in mouse milk. Biotechnology 8: 443-446.

    Google Scholar 

  23. C. B. A. Whitelaw, A. L. Archibald, S. Harris, M. McClenaghan, J. P. Simons, and A. J. Clark (1991). Targeting expression to the mammary gland; intronic sequences can enhance the efficiency of gene expression in transgenic mice. Trans. Res. 1: 3-13.

    Google Scholar 

  24. R. L. Brinster, J. M. Allen, R. R. Behringer, R. E. Gelinas, R. E. and R. D. Palmiter (1988). Introns increase transcriptional efficiency in transgenic mice. Proc. Natl. Acad Sci. U.S.A. 85: 836-840.

    Google Scholar 

  25. G. Wright, A. Carver, D. Cottom, et al. (1991). High level expression of active human alpha-1-antitrypsin in the milk of transgenic sheep. Biotechnology 9: 830-834.

    Google Scholar 

  26. F. Grosveld, G. van Assendelft, D. R. Greaves, and G. Kollias (1987). Position independent high level expression of the human beta-globin gene. Cell 51: 975-985.

    Google Scholar 

  27. R. A. McKnight, A. Shamay, L. Saakaron, R. J. Wall, and L. Hennighausen (1992). Matrix attachment regions can improve position independent regulation of a tissue-specific gene in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 89: 6943-6947.

    Google Scholar 

  28. S. K. Bronson, E. G. Plaehn, K. D. Kluckmam, J. R. Hagaman, N. Maeda, and O. Smithies (1996). Single copy transgenic mice with chosen site integration. Proc. Natl. Acad. Sci. U.S.A. 93: 9067-9072.

    Google Scholar 

  29. A. J. Clark, A. Cowper, R. Wallace, G. Wright, and J. P Simons (1992). Rescuing transgene expression by cointegration. Biotechnology 10: 1450-1454.

    Google Scholar 

  30. F. Yull, B. Binas, G. Harold, R. Wallace, and A. J. Clark (1997). Transgene rescue in the mammary gland requires expresion but not translation of BLG transgenes. Trans. Res. 6: 11-17.

    Google Scholar 

  31. R. Festenstein, M. Tolaini, P. Corbella, et al. (1996). Locus control region function and the heterochromatin induced position effect variegation. Science 271: 1123-1126.

    Google Scholar 

  32. K. W. Dobie, M. Lee, J. A. Fantes, et al. (1996). Variegated expression in mouse mammary gland is determined by the transgene integration locus. Proc. Natl. Acad. Sci. U.S.A. 93: 6659-6664.

    Google Scholar 

  33. A. P. Wolffe (1997). Transcriptional control: Represssed repeats express themselves Curr. Biol. 7: R796-R798.

    Google Scholar 

  34. A. J. Clark, G. Harold, and F. E. Yull (1997). Mammalian cDNA and CAT reporter constructs silence the expression of adjacent transgenes in transgenic mice. Nucl. Acids Res. 25: 1009-1014.

    Google Scholar 

  35. R. Breathnach and P. Chambon (1981). Eucaryotic split genes. Ann. Rev. Biochem. 50: 349-383.

    Google Scholar 

  36. G. Donofrio, E. Bignetti, A. J. Clark, and C. B. A. Whitelaw (1996). Comparable processing of β-lactoglobulin pre-mRNA in cell culture and transgenic mouse models. Mol. Gen. Genet. 252: 465-469.

    Google Scholar 

  37. F. Yull, G. Harold, A. Cowper, J. Percy, I. Cottingham, and A. J. Clark (1995). Fixing human factor IX (fIX): Correction of a cryptic RNA splice enables the production of biologically active human factor IX in the mammary gland. Proc. Natl. Acad. Sci. U.S.A. 92: 10899-10903.

    Google Scholar 

  38. S. Jallet, F. Perraud, V. Dalemans, et al. (1990). Characterization of recombinant human factor IX expressed in transgenic mice and in derived trans-immortalized hepatic cell lines. EMBO J. 9: 3295-3301.

    Google Scholar 

  39. A. Jacobson and S. W. Peltz (1996). Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells. Ann. Rev. Biochem. 65: 693-739.

    Google Scholar 

  40. W. A Guyette, R. J. Matusik, and J. M. Rosen (1979). Prolactin mediated transcriptional and post-transcript ional control of casein gene expression. Cell 17: 1013-1023.

    Google Scholar 

  41. A. Vassilakos, M. J. G. Hughes, and D. W. Andrews (1995). The 3′ untranslated region of bovine pre-prolactin contains a transferable nonpoly-(A) mRNA sequence that prolongs translation. FEBS Lett. 359: 206-210.

    Google Scholar 

  42. M. Kozak (1991). An analysis of vertebrate mRNA sequences: Intimations of translational control. J. Cell Biol. 115: 887-903.

    Google Scholar 

  43. R. L. Tangualy and D. R. Gallie (1996). Translational efficiency is regulated by the length of the 3′ untranslated region. Mol. Cell Biol. 16: 146-156.

    Google Scholar 

  44. D. C. James, R. B. Freedman, M. Hoare, et al. (1995). N-glycosylation of recombinant human interferon-γ produced in different animal expression systems. Biotechnology 13: 592-596.

    Google Scholar 

  45. J. Denman, M. Hayes, C. O'Day, et al. (1991). Transgenic expression of a variant of human tissue-type plasminogen activator in goat milk: Purification and characterization of the recombinant enzyme. Biotechnology 9: 839-843.

    Google Scholar 

  46. N. Nakata, K. Furukawa, D. E. Greenwalt, T. Sato, and A. Kobata (1993). Structural study of the sugar chains of CD36 purified from bovine mammary epithelial cells. Biochemistry 32: 4369-4383.

    Google Scholar 

  47. P. Kemp, R. B. Freedman, A. J. Clark, and N. Jenkins. The glycosylation of α1-antitrypsin expressed in transgenic mice. Proc. Jpn. Assoc. Animal Cell Tech. (in press).

  48. D. S. Anson, D. E. G. Austen, and G. G. Brownlee (1985). Efficient expression of active human factor IX from recombinant DNA clones in mammalian cells. Nature 315: 683-685.

    Google Scholar 

  49. W. Drohan, D. Zhang, R. K. Paleyanda, et al. (1994). Inefficient processing of protein C in the mouse mammary gland. Trans. Res. 3: 355-364.

    Google Scholar 

  50. W. H. Velander, J. L. Johnson, R. L. Page, et al. (1992). High level expression of a heterologous protein in the milk of transgenic swine using the cDNA to human protein C. Proc. Natl. Acad. Sci. U.S.A. 89: 12003-12007.

    Google Scholar 

  51. R. K Paleyanda, W. H. Velander, T. K. Lee, et al. (1997). Transgenic pigs produce functional hunan factor VIII in milk. Nature Biotech. 15: 971-975.

    Google Scholar 

  52. R. Drew, R. K. Paleyanda, T. K. Lee, et al. (1995). Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin. Proc. Natl. Acad. Sci. U.S.A. 92: 10462-10466.

    Google Scholar 

  53. I. Garner and A. Colman (1998). Therapeutic proteins from livestock In A. J. Clark (ed.), Animal Breeding: Technology for the 21st Century Harwood Academic Publishers, Chur 7000, Switzerland. pp. 64-71.

    Google Scholar 

  54. Genzyme Transgenics (1996). Production of Recombinant Antibodies in the Milk of Transgenic Animals Genzyme Transgenics, Framingham, MA 01701-9322.

  55. PPLTherapeutics (1996). Annual Report PPL Therapeutics, Roslin, Midlothian, Scotland.

  56. F. S. de Loos, F. Hengst, F. Pieper, and M. Saladdine (1996). Trans-vaginal oocyte recovery used for generation of bovine embryos for DNA microinjection. Theriogenology 45: 349.

    Google Scholar 

  57. A. S. Carver, M. A. Dalrymple. G. Wright, et al. (1993). Transgenic livestock as bioreactors: Stable expression of human α1-antitrypsin by a flock of transgenic sheep. Biotechnology 7: 487-492.

    Google Scholar 

  58. J. M. Rosen, C. Zahnow, A. Kazansky, and B. Raught (1997). Composite response elements mediate hormonal and developmental regulation of milk protein gene expression. Biochem. Soc. Symp. 63: 101-113.

    Google Scholar 

  59. G. Brem, P. Hartl, U. Besenfelder, E. Wolf, N. Zinivieva, and R. Pfaller (1994). Expression of synthetic cDNA sequence encoding human insulin-like growth factor-1 (IGF-1) in the mammary gland of transgenic rabbits. Gene 149: 351-355.

    Google Scholar 

  60. A. J. Clark, H. Bessos, J. O. Bishop, et al. (1989). Expression of human anti-hemophilic factor IX in the milk of transgenic sheep. Biotechnology 7: 487-492.

    Google Scholar 

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Clark, A.J. The Mammary Gland as a Bioreactor: Expression, Processing, and Production of Recombinant Proteins. J Mammary Gland Biol Neoplasia 3, 337–350 (1998). https://doi.org/10.1023/A:1018723712996

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