A novel shRNA vector that enables rapid selection and identification of knockdown cells.
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 microg/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.
Nagasaki
Akira
A
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 4, Tsukuba, Ibaraki 305-8562, Japan. a-nagasaki@aist.go.jp
Kanada
Masamitsu
M
Uyeda
Taro Q P
TQ
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002459:Cell Fusion; D003587:Cytomegalovirus / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D020868:Gene Silencing / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D009705:Nucleosides / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D012641:Selection, Genetic; D014162:Transfection
D000900:Anti-Bacterial Agents; C489850:ECT2 protein, human; D009705:Nucleosides; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; C004500:blasticidin S