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Updated: Jun 19, 2026

Lentivirus Production
11:42

Lentivirus Production

Published on: October 2, 2009

Lentivirus production.

Xiaoyin Wang1, Michael McManus

  • 1Diabetes Center, University of California, San Francisco, USA.

Journal of Visualized Experiments : Jove
|October 6, 2009
PubMed
Summary
This summary is machine-generated.

This study details lentivirus packaging for RNA interference (RNAi) gene silencing. The described method enables efficient RNAi in various cell types by producing functional lentiviruses for gene expression studies.

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Area of Science:

  • Molecular Biology
  • Virology
  • Genetics

Background:

  • RNA interference (RNAi) is a natural gene silencing mechanism.
  • Short interfering RNAs (siRNA) mediate post-transcriptional gene silencing.
  • Short hairpin RNAs (shRNA) are precursors to siRNA and can be delivered via lentivirus.

Purpose of the Study:

  • To describe a procedure for packaging lentiviruses for RNAi applications.
  • To explain the lentiviral vector production system and its safety features.
  • To outline the steps for generating and titering lentiviral vectors.

Main Methods:

  • Lentiviral vector production using a split system with four separate plasmid constructs.
  • Transient transfection of human 293 cells with an shRNA vector and three packaging vectors (p-VSVG, pRSV, pMDL).
  • Harvesting and concentrating virus-containing supernatant after a 48-hour incubation.
  • Determining virus titer via reporter gene expression using flow cytometry.

Main Results:

  • Successful packaging of lentiviruses capable of delivering shRNA for gene silencing.
  • The split packaging system minimizes the risk of replication-competent lentivirus formation.
  • Quantification of viral titer using fluorescent reporter expression.

Conclusions:

  • Lentivirus packaging is a viable method for achieving RNAi in diverse cell types.
  • The described protocol provides a reliable way to produce functional lentiviral vectors.
  • This technique facilitates gene silencing studies and therapeutic applications.