A novel shRNA vector that enables rapid selection and identification of knockdown cells

Akira Nagasaki*, Masamitsu Kanada, Taro Q.P. Uyeda

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)

Abstract

Small interference RNA (siRNA) is a powerful tool for disrupting expression of specific genes in a variety of cells. We have developed a vector, piMARK, which mediates expression of both small hairpin RNA (shRNA) and the blasticidin resistance (Bsr) protein fused with enhanced green fluorescent protein (EGFP), enabling rapid selection and identification of knockdown cells. Using this vector, we targeted Ect2, a gene encoding a guanine nucleotide exchange factor for several small GTPases, in human cell lines. Incubation in the presence of 10 μg/ml blasticidin S rapidly killed untransfected cells, so that after 24 h >90% of surviving HeLa S3 cells emitted green fluorescence and >70% were binucleate as a result of the frequent failure of cell division. The GFP-Bsr fluorescence enabled easy identification of individual knockdown cells under a fluorescence microscope, which in turn enabled unambiguous assessment of the morphological consequences of silencing Ect2. Moreover, because untransfected cells rapidly died and detached from the substrate, they were easily removed by simply rinsing the culture dishes. It thus should be possible to analyze the biochemical consequences of gene silencing en masse in the absence of a background of untransfected cells.

Original languageEnglish
Pages (from-to)190-194
Number of pages5
JournalPlasmid
Volume58
Issue number2
DOIs
Publication statusPublished - 2007 Sep
Externally publishedYes

Keywords

  • Blasticidin
  • Ect2
  • Enhanced green fluorescent protein
  • RNA interference
  • Short hairpin RNA

ASJC Scopus subject areas

  • Molecular Biology

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