Rapid, controlled and intensive lentiviral vector-based RNAi

Manuel Llano, Natassia Gaznick, Eric M. Poeschla

Research output: Chapter in Book/Report/Conference proceedingChapter

9 Scopus citations

Abstract

RNA interference (RNAi) is a powerful technology for studying the functional significance of genes. The technique is more accessible than gene knockout methods, and is directly applicable to diverse human cells. However, inadequate reductions in target mRNAs can reduce the utility of RNAi and insufficiently rigorous controls can lead to spurious conclusions. Optimally combining pol III promoters to drive short hairpin RNA expression with the gene transfer capabilities of lentiviral vectors has led to ways to perform especially effective and convincing RNAi, which we review here. We detail practical methods, including one-step vector construction. Deep, stable knockdowns to trace mRNA levels are readily achieved in T cell lines, which can then be subjected to comprehensive HIV challenge studies. Rescue of preknockdown phenotype by RNAi-resistant gene re-expression is a critical validating step. The methods can also be applied to primary T cells and macrophages. The time from thinking of a target to initial data read-out can be a few weeks.

Original languageEnglish (US)
Title of host publicationHIV Protocols
EditorsVinayaka R. Prasad, Ganjam V. Kalpana
Pages257-270
Number of pages14
DOIs
StatePublished - 2009

Publication series

NameMethods in Molecular Biology
Volume485
ISSN (Print)1064-3745

Keywords

  • HIV
  • Lentiviral vector
  • RNAi
  • Short hairpin RNA (shRNA)
  • T cell

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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    Llano, M., Gaznick, N., & Poeschla, E. M. (2009). Rapid, controlled and intensive lentiviral vector-based RNAi. In V. R. Prasad, & G. V. Kalpana (Eds.), HIV Protocols (pp. 257-270). (Methods in Molecular Biology; Vol. 485). https://doi.org/10.1007/978-1-59745-170-3_18