Skip to main content

Advertisement

SpringerLink
Log in

One-Step Construction of Lentiviral Reporter Using Red-Mediated Recombination

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Current approaches to generate lentiviral vectors, which have been used extensively for gene therapy, are time consuming and require a large expenditure. Here, we directly clone the full length myosin light chain kinase cDNA into enhanced green fluorescence protein (EGFP)-fused pLenti6/V5 expression vector in just one step with the use of Red-mediated recombination system, allowing for rapid and effective cloning of lentiviral expression vectors. In addition, the simultaneous expression of EGFP reporter provides a convenient monitoring mean for host cell infection and for localization of the target proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Naldini, L. (1998). Lentiviruses as gene transfer agents for delivery to non-dividing cells. Current Opinion in Biotechnology, 9, 457–463.

    Article  CAS  Google Scholar 

  2. Escors, D., & Breckpot, K. (2010). Lentiviral vectors in gene therapy: their current status and future potential. Archivum Immunologiae et therapiae Experimentalis, 58, 107–119.

    Article  CAS  Google Scholar 

  3. Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D., et al. (1998). A third-generation lentivirus vector with a conditional packaging system. Journal of Virology, 72, 8463–8471.

    CAS  Google Scholar 

  4. Landy, A. (1989). Dynamic, Structural, and regulatory aspects of lambda-site-specific recombination. Annual Review of Biochemistry, 58, 913–949.

    Article  CAS  Google Scholar 

  5. He, W. Q., Peng, Y. J., Zhang, W. C., Lv, N., Tang, J., Chen, C., et al. (2008). Myosin light chain kinase is central to smooth muscle contraction and required for gastrointestinal motility in mice. Gastroenterology, 135, 610–620.

    Article  CAS  Google Scholar 

  6. Totsukawa, G., Wu, Y., Sasaki, Y., Hartshorne, D. J., Yamakita, Y., Yamashiro, S., et al. (2004). Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts. Journal of Cell Biology, 164, 427–439.

    Article  CAS  Google Scholar 

  7. Schmidt, J. T., Morgan, P., Dowell, N., & Leu, B. (2002). Myosin light chain phosphorylation and growth cone motility. Journal of Neurobiology, 52, 175–188.

    Article  CAS  Google Scholar 

  8. Zhang, W. C., Peng, Y. J., He, W. Q., Lv, N., Chen, C., Zhi, G., et al. (2008). Identification and functional characterization of an aggregation domain in long myosin light chain kinase. FEBS Journal, 275, 2489–2500.

    Article  CAS  Google Scholar 

  9. Poperechnaya, A., Varlamova, O., Lin, P., Stull, J. T., & Bresnick, A. R. (2000). Localization and activity of myosin light chain kinase isoforms during the cell cycle. Journal of Cell Biology, 151, 697–707.

    Article  CAS  Google Scholar 

  10. Poteete, A. R. (2001). What makes the bacteriophage lambda Red system useful for genetic engineering: molecular mechanism and biological function. FEMS Microbiology Letters, 201, 9–14.

    CAS  Google Scholar 

  11. Liu, P. T., Jenkins, N. A., & Copeland, N. G. (2003). A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Research, 13, 476–484.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by National Basic Research Program of China (973) (2009CB942602; 2005CB522501; 2007CB947100), National Natural Science Funding of China (30570911). We thank Dr. N. Copeland (Institute of Molecular and Cell Biology, National University of Singapore) for providing materials of Red-mediated recombination system and Dr. Dong Kong (Department of Medicine Beth Israel Deaconess Medical Center and Harvard Medical School) for providing pLenti-EGFP plasmid.

Conflict of interest

None.

Author information

Authors and Affiliations

  1. Department of Geratology, The First Hospital Affiliated to Nanjing Medical University, Nanjing, 210029, China

    Juanmin Zha & Guoxian Ding

  2. Model Animal Research Center and MOE Key Laboratory of Model Animal for Disease Study, Nanjing University, 12 Xuefu Road, Nanjing, 210061, China

    Xin Chen, Chaojun Li, Minsheng Zhu & Weiqi He

  3. The School of Medicine, Nanjing University, Nanjing, China

    Chaojun Li & Weiqi He

Authors
  1. Juanmin Zha
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xin Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chaojun Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Minsheng Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Guoxian Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Weiqi He
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Guoxian Ding or Weiqi He.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zha, J., Chen, X., Li, C. et al. One-Step Construction of Lentiviral Reporter Using Red-Mediated Recombination. Mol Biotechnol 49, 278–282 (2011). https://doi.org/10.1007/s12033-011-9405-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-011-9405-7

Keywords

Search

Navigation