Establishment and Characterization of a Novel Tissue-specific DNA Construct and Culture System with Potential for Avian Bioreactor Generation

  • Juan M. Pérez Sáez
  • Leonardo E. Bussmann
  • Ursula Agnes BussmannEmail author
Original paper


Transgenic chickens are of great interest for the production of recombinant proteins in their eggs. However, the use of constitutive strong promoters or the tissue-specific ovalbumin promoter for the generation of the transgenic chickens have different drawbacks that have to be overcome in order to make chicken bioreactor an efficient production system. This prompted us to investigate the use of an alternative tissue-specific promoter, the vitellogenin promoter, which could overcome the difficulties currently found in the generation of chicken bioreactors. In the present work we establish and characterize a DNA construct consisting of a fragment of the 5´-flanking region of the chicken vitellogenin II gene cloned in a reporter vector. This construct is capable of showing the ability of the promoter to drive expression of a reporting gene in a tissue-specific manner and in a way that closely resembles physiologic regulation of vitellogenin, making it an ideal candidate to be used in the future for generation of avian bioreactors. Besides, we validate an in vitro culture system to test the performance of the DNA construct under study that could be used as a practical tool before generating any transgenic chicken. These results are important since they provide the proof of concept for the use of the vitellogenin promoter for future genetic modification of chickens bioreactors with improved characteristics in terms of quality of the recombinant protein produced.


Vitellogenin promoter Avian bioreactors Recombinant proteins Tissue-specific Promoters Transgenic Chicken LMH cells Hepatocytes 



This work was supported by the ANPCYT and CONICET through grants PICT 2008-1175 and PIP 112-200801-03201, respectively. The authors gratefully acknowledge the generous contribution of Dr. M. Plano from Tres Arroyos Farm (Buenos Aires, Argentina), who kindly facilitated the fertilized eggs, and thank Fundación Williams (Argentina) and Fundación Rene Barón (Argentina) for their support.

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants performed by any of the authors. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.


  1. 1.
    Demain, A. L., & Vaishnav, P. (2009). Production of recombinant proteins by microbes and higher organisms. Biotechnology Advances, 27, 297–306.CrossRefGoogle Scholar
  2. 2.
    Houdebine, L. M. (2009). Production of pharmaceutical proteins by transgenic animals. Comparative Immunology, Microbiology and Infectious Diseases, 32, 107–121.CrossRefGoogle Scholar
  3. 3.
    Maksimenko, O. G., Deykin, A. V., Khodarovich, Y. M., & Georgiev, P. G. (2013). Use of transgenic animals in biotechnology: prospects and problems. Acta Naturae, 5(1), 33–46.Google Scholar
  4. 4.
    Kesik-Brodacka, M. (2018). Progress in biopharmaceutical development. Biotechnology and Applied Biochemistry, 65(3), 306–322.CrossRefGoogle Scholar
  5. 5.
    Ivarie, R. (2006). Competitive bioreactor hens on the horizon. Trends in Biotechnology, 24(3), 99–101.CrossRefGoogle Scholar
  6. 6.
    Farzaneh, M., Hassani, S. N., Mozdziak, P. and Baharvand, H. (2017) Avian embryos and related cell lines: A convenient platform for recombinant proteins and vaccine production. Biotechnol J.
  7. 7.
    Raju, S. (2003). Glycosylation variations with expression systems. Bioprocess International, 1, 44–53.Google Scholar
  8. 8.
    van de Lavoir, M. C., Diamond, J. H., Leighton, P. A., Mather-Love, C., Heyer, B. S., Bradshaw, R., et al. (2006). Germline transmission of genetically modified primordial germ cells. Nature, 441, 766–769.CrossRefGoogle Scholar
  9. 9.
    Macdonald, J., Glover, J. D., Taylor, L., Sang, H. M., & McGrew, M. J. (2010). Characterization and germline transmission of cultured avian primordial germ cells (PGCs). PLoS ONE, 5(11), e15518. Scholar
  10. 10.
    Dimitrov, L., Pedersen, D., Ching, K. H., Yi, H., Collarini, E. J., Izquierdo, S., et al. (2016). Germline gene editing in chickens by efficient CRISPR-mediated homologous recombination in primordial germ cells. PLoS ONE, 11(4), e0154303. Scholar
  11. 11.
    Taylor, L., Carlson, D. F., Nandi, S., Sherman, A., Fahrenkrug, S. C., & McGrew, M. J. (2017). Efficient TALEN-mediated gene targeting of chicken primordial germ cells. Development, 144(5), 928–934.CrossRefGoogle Scholar
  12. 12.
    Oishi, I., Yoshii, K., Miyahara, D., & Tagami, T. (2018). Efficient production of human interferon beta in the white of eggs from ovalbumin gene-targeted hens. Scientific Reports, 8(1), 10203. Scholar
  13. 13.
    Kojima, Y., Mizutani, A., Okuzaki, Y., Nishijima, K., Kaneoka, H., Sasamoto, T., et al. (2015). Analyses of chicken sialyltransferases related to N-glycosylation. Journal of Bioscience and Bioengineering, 119(6), 623–628.CrossRefGoogle Scholar
  14. 14.
    van het Schip, F. D., Samallo, J., Broos, J., Ophuis, J., Mojet, M., Gruber, M., et al. (1987). Nucleotide sequence of a chicken vitellogenin gene and derived amino acid sequence of the encoded yolk precursor protein. Journal of Molecular Biology, 196, 245–260.CrossRefGoogle Scholar
  15. 15.
    Burch, J. B. E., Evans, M. I., Freidman, T. M., & O’Malley, P. J. (1988). Two functional estrogen responsive elements are located upstream of major chicken vitellogenin gene. Molecular and Cellular Biology, 8, 1123–1131.CrossRefGoogle Scholar
  16. 16.
    Scheidereit, C., Westphal, H. M., Carlson, C., Bosshard, H., & Beato, M. (1986). Molecular model of the interaction between the glucocorticoid receptor and the regulatory elements of inducible genes. DNA, 5, 383–391.CrossRefGoogle Scholar
  17. 17.
    Cato, A. C., Heitlinger, E., Ponta, H., Klein-Hitpass, L., Ryffel, G. U., Bailly, A., et al. (1988). Estrogen and progesterone receptor-binding sites on the chicken vitellogenin II gene: synergism of steroid hormone action. Molecular and Cellular Biology, 8, 5323–5330.CrossRefGoogle Scholar
  18. 18.
    Geiser, M., Mattaj, I. W., Wilks, A. F., Seldran, M., & Jost, J. P. (1983). Structure and sequence of the promoter area and of a 5’ upstream demethylation site of the estrogen regulated chicken vitellogenin II gene. Journal of Biological Chemistry, 258, 9024–9030.Google Scholar
  19. 19.
    Bussmann, U. A., Pérez Sáez, J. M., Bussmann, L. E., & Barañao, J. L. (2013). Aryl hydrocarbon receptor activation leads to impairment of estrogen-driven chicken vitellogenin promoter activity in LMH cells. Comparative Biochemistry and Physiology Part C, 157(2), 111–118.Google Scholar
  20. 20.
    Haché, R. J., Tam, S. P., Cochrane, A., Nesheim, M., & Deeley, R. G. (1987). Long-term effects of estrogen on avian liver: estrogen-inducible switch in expression of nuclear, hormone-binding proteins. Molecular and Cellular Biology, 7(10), 3538–3547.CrossRefGoogle Scholar
  21. 21.
    Zhou, C., & Zhang, C. (2005). Protective effects of antioxidant vitamins on Aroclor 1254-induced toxicity in cultured chicken embryo hepatocytes. Toxicology in Vitro, 19(5), 665–673.CrossRefGoogle Scholar
  22. 22.
    Di Croce, L., Vicent, G. P., Pecci, A., Bruscalupi, G., Trentalance, A., & Beato, M. (1999). The promoter of the rat 3-hydroxy-3-methylglutaryl coenzyme A reductase gene contains a tissue-specific estrogen-responsive region. Molecular Endocrinology, 13(8), 1225–1236.CrossRefGoogle Scholar
  23. 23.
    Bussmann, U. A., Bussmann, L. E., & Barañao, J. L. (2006). An aryl hydrocarbon receptor agonist amplifies the mitogenic actions of estradiol in granulosa cells: Evidence of involvement of the cognate receptors. Biology of Reproduction, 74(2), 417–426.CrossRefGoogle Scholar
  24. 24.
    Sokal, R. R., & Rohlf, F. J. (1995). Biometry. New York: WH Freemann Co.Google Scholar
  25. 25.
    Berkowitz, E. A., & Evans, M. I. (1992). Estrogen-dependent expression of the chicken very low density apolipoprotein II gene in serum-free cultures of LMH cells. In Vitro Cellular Developmental Biology, 28A(6), 391–396.CrossRefGoogle Scholar
  26. 26.
    Herron, L. R., Pridans, C., Turnbull, M. L., Smith, N., Lillico, S., Sherman, A., et al. (2018). A chicken bioreactor for efficient production of functional cytokines. BMC Biotechnology, 18(1), 82. Scholar
  27. 27.
    Kwon, M. S., Koo, B. C., Kim, D., Nam, Y. H., Cui, X. S., Kim, N. H., et al. (2018). Generation of transgenic chickens expressing the human erythropoietin (hEPO) gene in an oviduct-specific manner: Production of transgenic chicken eggs containing human erythropoietin in egg whites. PLoS ONE, 13(5), e0194721. Scholar
  28. 28.
    Morrison, S. L., Mohammed, M. S., Wims, L. A., Trinh, R., & Etches, R. (2001). Sequences in antibody molecules important for receptor-mediated transport into the chicken egg yolk. Molecular Immunology, 38(8), 619–625.CrossRefGoogle Scholar
  29. 29.
    Sato, M., Kawashima, T., Aosasa, M., Horiuchi, H., Furusawa, S., & Matsuda, H. (2005). Excision of foreign gene product with cathepsin D in chicken hepatoma cell line. Biochemical and Biophysical Research Communications, 330(2), 533–539.CrossRefGoogle Scholar
  30. 30.
    Lazier, C. B. (1978). Ontogeny of the vitellogenic response to oestradiol and of the soluble nuclear oestrogen receptor in embryonic-chick liver. Biochemical Journal, 174(1), 143–152.CrossRefGoogle Scholar
  31. 31.
    Shapiro, D. (1982). Steroid hormone regulation of vitellogenin gene expression. Critical Reviews Biochemistry, 12(3), 187–203.CrossRefGoogle Scholar
  32. 32.
    Pinto, P. I., Singh, P. B., Condeça, J. B., Teodósio, H. R., Power, D. M., & Canário, A. V. (2006). ICI 182,780 has agonistic effects and synergizes with estradiol-17 beta in fish liver, but not in testis. Reproductive Biology Endocrinology, 4, 67.CrossRefGoogle Scholar
  33. 33.
    Ankenbauer, W., Strähle, U. and Schütz, G. (1988). Synergistic action of glucocorticoid and estradiol responsive elements. Proceedings National Academy Sciences USA 85(20), 7526-7530.Google Scholar
  34. 34.
    Slater, E. P., Redeuilh, G., & Beato, M. (1991). Hormonal regulation of vitellogenin genes: an estrogen-responsive element in the Xenopus A2 gene and a multihormonal regulatory region in the chicken II gene. Molecular Endocrinology, 5(3), 386–396.CrossRefGoogle Scholar
  35. 35.
    Prisco, M., Valiante, S., Maddalena Di Fiore, M., Raucci, F., Del Giudice, G., Romano, M., et al. (2008). Effect of 17beta-estradiol and progesterone on vitellogenesis in the spotted ray Torpedo marmorata Risso 1810 (Elasmobranchii: Torpediniformes): studies on females and on estrogen-treated males. General and Comparative Endocrinology, 157(2), 125–132.CrossRefGoogle Scholar
  36. 36.
    Coccia, E., De Lisa, E., Di Cristo, C., Di Cosmo, A., & Paolucci, M. (2010). Effects of estradiol and progesterone on the reproduction of the freshwater crayfish Cherax albidus. Biological Bulletin, 218(1), 36–47.CrossRefGoogle Scholar
  37. 37.
    Gupta, S., & Kanungo, M. S. (1996). Modulation of vitellogenin II gene by estradiol and progesterone in the Japanese quail. Biochemical and Biophysical Research Communications, 222(1), 181–185.CrossRefGoogle Scholar
  38. 38.
    Wangh, L. J. (1982). Glucocorticoids act together with estrogens and thyroid hormones in regulating the synthesis and secretion of Xenopus vitellogenin, serum albumin, and fibrinogen. Developmental Biology, 89(2), 294–329.CrossRefGoogle Scholar
  39. 39.
    Lethimonier, C., Flouriot, G., Valotaire, Y., Kah, O., & Ducouret, B. (2000). Transcriptional interference between glucocorticoid receptor and estradiol receptor mediates the inhibitory effect of cortisol on fish vitellogenesis. Biology of Reproduction, 62(6), 1763–1771.CrossRefGoogle Scholar
  40. 40.
    Binder, R., MacDonald, C. C., Burch, J. B., Lazier, C. B., & Williams, D. L. (1990). Expression of endogenous and transfected apolipoprotein II and vitellogenin II genes in an estrogen responsive chicken liver cell line. Molecular Endocrinology, 4, 201–208.CrossRefGoogle Scholar
  41. 41.
    Dierich, A., Gaub, M. P., LePennec, J. P., Astinotti, D., & Chambon, P. (1987). Cell-specificity of the chicken ovalbumin and conalbumin promoters. EMBO Journal, 6(8), 2305–2312.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Juan M. Pérez Sáez
    • 1
  • Leonardo E. Bussmann
    • 1
  • Ursula Agnes Bussmann
    • 1
    Email author
  1. 1.Instituto de Biología y Medicina Experimental-CONICETBuenos AiresArgentina

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