Sigma-Aldrich jump on the LentiExpress for RNAi screening
dramatically simplify and speed-up high-throughput (HT) RNAi
screening of the human kinome.
The new system, dubbed Mission LentiExpress eliminates reagent preparation and enables simple screening optimisation using pre-arrayed viral particles that let researchers simply add their cell lines of choice to the wells before beginning the screen. The plates even come with optimisation plates to help researchers figure out the correct concentrations to use when transecting a particular cell type or cell line. "One of the main benefits of the LentiExpress is that you can very rapidly do a screen whether you have sophisticated automation or not," said Dr Edward Weinstein, manager of operation for Functional Genomics. Traditional approaches to RNAi screening can be laborious as researchers need to first transfect their cells with the relevant siRNA (small interfering RNA) or shRNA (short hairpin RNA) and this can limit the cell types that can be screened due to difficulties with the transfection step. "One of the main things that people want to study cells that are as close as possible to an in vivo situation, and this lentiviral delivery method allows you to study primary and differentiated cells expanding the range of cells that can be studied," said Dr Supriya Shivakumar, global commercialisation marketing manager for Functional Genomics at Sigma-Aldrich. "This new LentiExpress technology has been specifically put together to study the human kinome gene set, and we hope to offer other gene family sets in this format in the near future." The protein kinases that make up the human kinome are among the largest and most studied gene families as they play an essential role in intracellular communication. This communication is controlled by the protein kinases by transferring a phosphate group from energy donor molecules such as ATP (Adenosine 5'-triphosphate) to a protein, thereby activating it. The LentiExpress uses Lentiviral particles to deliver the shRNA to even hard-to-transfect cells. Moreover, because the system uses shRNA rather than siRNA the gene knockdown effects are longer lasting. "If you want to create a long term stable gene knockdown and create a cell line with that gene knocked down and study it in a variety of different assays this method allows you to do that," said Dr Shivakumar. In addition, the fact that the gene knockdown is long lasting makes it easy to run replicate studies as cells can be readily split into multiple samples after transfection. This is in stark contrast to more traditional siRNA approaches where cells have to be separately transfected after plating into the individual replicate samples which can lead to errors if the efficiency of transfection is not equal. The panel consists of 41 ready-to-use 96 well plates that each contains 80 different kinase specific viruses. Each well contains approximately 5,000 viral particles in a single reaction volume of 30ul with each plate including negative controls to monitor transduction efficiency. "The panel contains over 3000 clones targeting 673 kinase genes, covering nearly the entire human kinome," said Stephanie Uder, global product manager for Functional Genomics at Sigma-Aldrich. The LentiExpress contains between three and five constructs that target different regions of each of the gene sequences, making it easier to weed-out false positives and validate the hits all within a single screen. "We've done some initial work using the LentiExpress technology that we will be publishing shortly looking at drug sensitivity very quickly assay the effect of knocking out each gene in a family on whether or not that will make a cell more sensitive to chemotherapy," said Dr Weinstein. This work parallels research published in the journal Nature by Professor Michael White that showed that by blocking certain genes the efficacy of the anticancer drug paclitaxel could be increased by a factor of 10,000. However, this work was somewhat limited by the number of cell lines that could be studied due to transfection issues. Dr Weinstein believes that this latest product will enable researchers to study more cell lines than ever before and let researchers study the effects of gene knockdowns in cell lines that would not have been amenable to traditional siRNA screening studies.
