Downregulation of Human Cdc6 Protein Using a Lentivirus RNA Interference Expression Vector

  • Feng Luo
  • Jiing-Kuan Yee
  • Sheng-He Huang
  • Ling-Tao Wu
  • Ambrose Y. Jong
Part of the Methods in Molecular Biology™ book series (MIMB, volume 342)


Eukaryotic CDC6 gene function is required for the initiation of DNA replication and is a key regulatory protein during cell cycle progression. The human CDC6 gene is not expressed in most normal tissues, in contrast with its marked expression in proliferating cancer cells. An effective way to explore the gene functions of CDC6 is to knock-down the CDC6 messenger RNA (mRNA) and examine the phenotypic consequences. In this chapter, we describe the construction of a lentivirus vector to express a CDC6 DNA segment. The transcript is able to fold by itself because the sense and antisense regions are complementary. There is a 9-nucleotide (nt) loop region allowing for the short hairpin RNA (shRNA) to form. Cellular ribonucleases process the shRNA into a functional short interfering RNA (siRNA). Down-regulation of Cdc6 protein is confirmed by Western blots.

Key Words

Lentivirus vector shRNA siRNA Cdc6 cell cycle progression 


  1. 1.
    Williams, R. S., Shohet, R. V., and Stillman, B. (1997) A human protein related to yeast Cdc6p. Proc. Natl. Acad. Sci. USA 94, 142–147.CrossRefPubMedGoogle Scholar
  2. 2.
    Prasanth, S. G., Mendez, J., Prasanth, K. V., and Stillman, B. (2004) Dynamics of pre-replication complex proteins during the cell division cycle. Philos. Trans. R. Soc. Lond. B Biol. Sci. 359, 7–16.CrossRefPubMedGoogle Scholar
  3. 3.
    Petersen, B. O., Wagener, C., Marinoni, F., et al. (2000) Cell cycle-and cell growth-regulated proteolysis of mammalian CDC6 is dependent on APC-CDH1. Genes Devel. 14, 2330–2343.CrossRefPubMedGoogle Scholar
  4. 4.
    Ohta, S., Koide, M., Tokuyama, T., Yokota, N., Nishizawa, S., and Namba H. (2001) Cdc6 expression as a marker of proliferative activity in brain tumors. Oncol. Rep. 8, 1063–1066.PubMedGoogle Scholar
  5. 5.
    Williams, G. H., Romanowski, P., Morris, L., et al. (1998) Improved cervical smear assess-ment using antibodies against proteins that regulate DNA replication. Proc. Natl. Acad. Sci. USA 95, 14,932–14,937.CrossRefPubMedGoogle Scholar
  6. 6.
    Feng, D., Tu, Z., Wu, W., and Liang, C. (2003) Inhibiting the expression of DNA replica-tion-initiation proteins induces apoptosis in human cancer cells. Cancer Res. 63, 7356–7364.PubMedGoogle Scholar
  7. 7.
    Gilmore, I. R., Fox, S. P., Hollins, A. J., Sohail, M., and Akhtar, S. (2004) The design and exogenous delivery of siRNA for post-transcriptional gene silencing. J. Drug Target. 12, 315–340.CrossRefPubMedGoogle Scholar
  8. 8.
    Yee, J. K., Friedmann, T., and Burns, J. C. (1994) Generation of high-titer pseudotyped retroviral vectors with very broad host range. Methods Cell Biol. 43(Pt A), 99–112.CrossRefPubMedGoogle Scholar
  9. 9.
    Yam, P. Y., Li, S. Wu, J., Hu J., Zaia J. A., and Yee J. K. (2002) Design of HIV vectors for efficient gene delivery into human hematopoietic cells. Mol. Ther. 5, 479–484.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Feng Luo
    • 1
  • Jiing-Kuan Yee
    • 2
  • Sheng-He Huang
    • 3
  • Ling-Tao Wu
    • 4
  • Ambrose Y. Jong
    • 5
  1. 1.Center for Craniofacial Molecular Biology and Graduate Program in Craniofacial BiologySchool of Dentistry, University of Southern CaliforniaLos Angeles
  2. 2.Department of PathologyCity of HopeDuarte
  3. 3.Department of PathologyUniversity of Southern California, Children‖s Hospital Los AngelesLos Angeles
  4. 4.Division of Infectious Diseases, Department of PediatricsUniversity of Southern California, Children‖s Hospital Los AngelesLos Angeles
  5. 5.Division of Hematology-Oncology, Department of PediatricsUniversity of Southern California, Children‖s Hospital Los AngelesLos Angeles

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