Evaluation of Promoters for Use in Tissue-Specific Gene Delivery

  • Changyu Zheng
  • Bruce J. Baum
Part of the Methods in Molecular Biology™ book series (MIMB, volume 434)


Vectors used in gene therapy require an expression cassette. The expression cassette consists of three important components: promoter, therapeutic gene and polyadenylation signal. The promoter is essential to control expression of the therapeutic gene. A tissue-specific promoter is a promoter that has activity in only certain cell types. Use of a tissue-specific promoter in the expression cassette can restrict unwanted transgene expression as well as facilitate persistent transgene expression. Therefore, choosing the correct promoter, especially a tissue-specific promoter, is a major step toward achieving successful therapeutic transgene expression. Ideally, the elements of the natural promoter region, necessary for obtaining the required level of the gene expression while retaining tissue-specificity, should be known. Also, it is important to understand whether interactions occur between the promoter region and the rest of the vector genome that could affect promoter activity and specificity. To assess this, it is helpful to select a suitable vector system that will be used in further gene therapy studies. Second, have one or several candidate tissue-specific promoters available for use. Third, ideally have an in vitro cell model suitable to evaluate tissue-specificity. Fourth, have a convenient in vivo animal model to use. Fifth, select a good reporter gene system. Next, using conventional recombinant DNA techniques create different promoter constructs with the selected vector system. Lastly, have a suitable transfection method to test the plasmid constructs in both the in vitro and the in vivo models.

Key Words

Tissue-specific promoter transcription expression cassette gene therapy vector 



We thank Drs. Biman Paria and Gabor Racz for their careful reading of, and helpful comments on, an earlier version of this manuscript. Our research is supported by the Division of Intramural Research, National Institute of Dental and Craniofacial Research NIH.


  1. 1.
    Eloranta, J.J. and Goodbourn, S. (1996) Positive and negative regulation of RNA polymerase II transcription. In Eukaryotic Gene Transcription (Goodbourn, J.J., ed.), Oxford University Press, Walton Street, Oxford, pp. 1–33.Google Scholar
  2. 2.
    Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. andWalter, P. (2002) Molecular Biology of the Cell. GarlandScience, New York, NY.Google Scholar
  3. 3.
    Majumder, S. and DePamphilis, M.L. (1994) TATA-dependent enhancerstimulation of promoter activity in mice is developmentallyacquired. Mol. Cell. Biol. 14, 4258–4268.PubMedGoogle Scholar
  4. 4.
    Zheng, C., Hoque, A.T., Braddon, V.R., Baum, B.J., and O’Connell,B.C. (2001) Evaluation of salivary gland acinar and ductalcell-specific promoters in vivo with recombinant adenoviral vectors.Hum. Gene. Ther. 12, 2215–2223.CrossRefPubMedGoogle Scholar
  5. 5.
    Zheng, C. and Baum, B.J. (2005) Evaluation of viral and mammalianpromoters for use in gene delivery to salivary glands. Mol.Ther. 12, 528–536.CrossRefPubMedGoogle Scholar
  6. 6.
    Baum, B.J., Wellner, R.B., and Zheng, C. (2002) Gene transfer tosalivary gland. Int. Rev. Cytol. 213, 93–146.CrossRefPubMedGoogle Scholar
  7. 7.
    Mastrangeli, A., O’Connell, B., Aladib, W., Fox, P.C., Baum, B.J.,and Crystal, R.G. (1994) Direct in vivo adenovirus-mediated genetransfer to salivary glands. Am. J. Physiol. 266, G1146–G1155.PubMedGoogle Scholar
  8. 8.
    Delporte, C., Redman, R.S., and Baum, B.J. (1997) Relationshipbetween the cellular distribution of the alpha(v)beta3/5 integrinsand adenoviral infection in salivary glands. Lab. Invest. 77, 167–173.PubMedGoogle Scholar
  9. 9.
    Baum, B.J., Wang, S., Cukierman, E., Delporte, C., Kagami, H.,Marmary, Y., Fox, P.C., Mooney, D.J., and Yamada, K.M. (1999) Re-engineering the functions of a terminally differentiatedepithelial cell in vivo. Ann. N.Y. Acad. Sci. 875, 294–300.CrossRefPubMedGoogle Scholar
  10. 10.
    Moll, R., Franke, W.W., Schiller, D.L., Geiger, B., and Krepler, R. (1982) The catalog of human cytokeratins: patterns of expression innormal epithelia, tumors and cultured cells. Cell. 31, 11–24.CrossRefPubMedGoogle Scholar
  11. 11.
    Chow, Y.H., O’Brodovich, H., Plumb, J., Wen, Y., Sohn, K.J., Lu, Z.,Zhang, F., Lukacs, G.L., Tanswell, A.K., Hui, C.C., Buchwald, M.,and Hu, J. (1997) Development of an epithelium-specific expressioncassette with human DNA regulatory elements for transgene expressionin lung airways. Proc. Natl. Acad. Sci. U.S.A. 94, 14695–14700.CrossRefPubMedGoogle Scholar
  12. 12.
    Brembeck, F.H. and Rustgi, A.K. (2000) The tissue-dependent keratin19 gene transcription is regulated by GKLF/KLF4 and Sp1. J.Biol. Chem. 275, 28230–28239.PubMedGoogle Scholar
  13. 13.
    Kagaya, M., Kaneko, S., Ohno, H., Inamura, K., and Kobayashi, K. (2001) Cloning and characterization of the 5′;-flanking region ofhuman cytokeratin 19 gene in human cholangiocarcinoma cell line.J. Hepatol. 35, 504–511.CrossRefPubMedGoogle Scholar
  14. 14.
    Ting, C.N., Rosenberg, M.P., Snow, C.M., Samuelson, L.C., andMeisler, M.H. (1992) Endogenous retroviral sequences are requiredfor tissue-specific expression of a human salivary amylase gene.Genes Dev. 6, 1457–1465.CrossRefPubMedGoogle Scholar
  15. 15.
    He, X., Tse, C.M., Donowitz, M., Alper, S.L., Gabriel, S.E., andBaum, B.J. (1997) Polarized distribution of key membrane transportproteins in the rat submandibular gland. Pflugers Arch. 433, 260–268.CrossRefPubMedGoogle Scholar
  16. 16.
    Brown, A.M., Rusnock, E.J., Sciubba, J.J., and Baum, B.J. (1989) Establishment and characterization of an epithelial cell line fromthe rat submandibular gland. J. Oral. Pathol. Med. 18, 206–213.CrossRefPubMedGoogle Scholar
  17. 17.
    Graham, F.L., Smiley, J., Russell, W.C., and Nairn, R. (1977) Characteristics of a human cell line transformed by DNA from humanadenovirus type 5. J. Gen. Virol. 36, 59–74.CrossRefPubMedGoogle Scholar
  18. 18.
    Chen, X., Wu, J.M., Hornischer, K., Kel, A., and Wingender, E. (2006) TiProD: the tissue-specific promoter database. NucleicAcids Res. 34, D104–D107.CrossRefGoogle Scholar
  19. 19.
    Borok, Z., Li, X., Fernandes, V.F., Zhou, B., Ann, D.K., andCrandall, E.D. (2000) Differential regulation of rat aquaporin-5promoter/enhancer activities in lung and salivary epithelialcells. J. Biol. Chem. 275, 26507–26514.CrossRefPubMedGoogle Scholar
  20. 20.
    Henikoff, S. (1984) Unidirectional digestion with exonuclease IIIcreates targeted breakpoints for DNA sequencing. Gene. 28, 351–359.CrossRefPubMedGoogle Scholar
  21. 21.
    Putney, S.D., Benkovic, S.J., and Schimmel, P.R. (1981) A DNAfragment with an alpha-phosphorothioate nucleotide at one end isasymmetrically blocked from digestion by exonuclease III and can bereplicated in vivo. Proc. Natl. Acad. Sci. U.S.A. 78, 7350–7354.CrossRefPubMedGoogle Scholar
  22. 22.
    Sambrook, J. and Russell, D.W. (eds.) (2001) Molecular CloningA Laboratory Manual. Cold Spring Harbor Laboratory, New York.Google Scholar
  23. 23.
    Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Scidman,J.G., Smith, J.A., and Struhl, K. (eds.) (2006) CurrentProtocols in Molecular Biology. Harvard Medical School.John Wiley Sons, Inc.Google Scholar
  24. 24.
    McGrory, W.J., Bautista, D.S., and Graham, F.L. (1988) A simpletechnique for the rescue of early region I mutations into infectioushuman adenovirus type 5. Virology 163, 614–617.CrossRefPubMedGoogle Scholar
  25. 25.
    Bundschu, K., Gattenlohner, S., Knobeloch, K.P., Walter, U., andSchuh, K. (2006) Tissue-specific Spred-2 promoter activitycharacterized by a gene trap approach. Gene Expr. Patterns 6, 247–255.CrossRefPubMedGoogle Scholar
  26. 26.
    Bohwan, J., Seong, J.K., and Ryu, D.Y. (2005) Tissue-specific andDe Novo promoter methylation of the mouse glucose transporter2. Biol. Pham. Bull. 28, 2054–2057.CrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Changyu Zheng
    • 1
  • Bruce J. Baum
    • 1
  1. 1.Gene Therapy and Therapeutics BranchNIDCRDHHSMD

Personalised recommendations