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

@article{citeulike:1677816, 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 10mug/ml blasticidin S rapidly killed untransfected cells, so that after 24h >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.}, address = {Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 4, Tsukuba, Ibaraki 305-8562, Japan.}, author = {Nagasaki, A. and Kanada, M. and Uyeda, T. Q. }, citeulike-article-id = {1677816}, doi = {10.1016/j.plasmid.2007.01.006}, issn = {0147-619X}, journal = {Plasmid}, keywords = {rnai}, month = {September}, number = {2}, pages = {190--194}, posted-at = {2007-09-20 02:31:16}, priority = {2}, title = {A novel shRNA vector that enables rapid selection and identification of knockdown cells.}, url = {http://dx.doi.org/10.1016/j.plasmid.2007.01.006}, volume = {58}, year = {2007} }

TY - JOUR ID - citeulike:1677816 L3 - citeulike-article-id:1677816 TI - A novel shRNA vector that enables rapid selection and identification of knockdown cells. JF - Plasmid VL - 58 IS - 2 SP - 190 EP - 194 AD - Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 4, Tsukuba, Ibaraki 305-8562, Japan. SN - 0147-619X N2 - 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 10mug/ml blasticidin S rapidly killed untransfected cells, so that after 24h >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. KW - rnai AU - Nagasaki, A AU - Kanada, M AU - Uyeda, TQ PY - 2007/09// UR - http://dx.doi.org/10.1016/j.plasmid.2007.01.006 ER -