Sigma-Aldrich helps siRNA N-TER cells
with the release of its N-TER nanoparticle system for
hard-to-transfect cell lines.
siRNAs (small interfering RNAs) are a class of short (20 to 25 nucleotide) double-stranded RNA molecules that have variety of biological roles, most notably in interfering with the expression of a specific gene, causing that gene to be unable to produce the proteins that it codes for. The new N-TER system allows researchers to easily and reproducibly interrogate gene function and the effects of gene knockdown on drug efficacy in cell lines that have previously been considered unamenable to more traditional lipid-based siRNA delivery reagents. With pharmaceutical companies wanting to increase the accuracy of their in vitro experiments, the ability to study the effects of gene knockdown in cell lines that closely mimic the in vivo environment is increasingly in demand. This desire has led to increased interest from suppliers to provide the best possible transfection methods, with Cellectricon having shipped its first high-throughput (HT) RNAi screening system and Amaxa gaining a €2.75m EU grant to develop HT transfection systems based on its Nucleofector technology. In addition, Thermo Fisher Scientific has just announced a collaboration with Lentigen to use lentivrial particles, much like those used in Sigma-Aldrich's Mission LentiExpress, to deliver RNAi (RNA interference) agents into a cells genome (see below). Unfortunately, those cell lines that are most 'life-like' are often the ones that are hardest to transfect using traditional liposomal delivery approaches. This is particularly noticeable for primary, neuronal and non-dividing cell lines. In addition, liposomal delivery approaches often lead to high cell death rates due to disruption of the cell wall. This latest release avoids these limitations by utilising a nanoparticle delivery system that quickly enters into a cell's cytoplasm to deliver its siRNA payload without disrupting the cell wall. "The N-TER system has been specifically designed to deliver synthetic siRNA into a cell's cytoplasm to trigger the RNAi mechanism, while leaving the cell alive so that you can study the effects" said Phil Simmons, product manager for the N-TER system at Sigma-Aldrich's Functional Genomics department. "The problem with the traditional lipid-based transfection approach is that you can't get into a huge variety of cells, and those that you can get the siRNA into often experience high levels of cell death." Simmons continued by explaining that the nanoparticles are very easy to generate and are formed simply by mixing the siRNA molecules of choice with the N-TER peptide. "One of the key advantages of the system is that the nanoparticle solutions are very, very stable, and we have results from stability studies that show the nanoparticles are stable in the freezer for up to two years," said Simmons. "This enables researchers to make up a stock solution of the nanoparticles and use it over a course of months or years and this leads to upfront time savings as well as increasing efficiency and reproducibility." According to Simmons, researchers usually buy in the siRNA molecules from oligomer suppliers, such as Sigma-Aldrich, that use extensive computer algorithms to validate that the siRNAs will target specific transcripts and give the best knockdown effects. Customers will often order multiple siRNAs so that they can validate the effects are due to the specificity of the siRNA rather than an off-target effect. These synthetic siRNAs deliver a short-lived knockdown of around a week, unlike systems that deliver knockdown agents into the genome such as the company's recently released Mission LentiExpress viral delivery system that delivers short-hairpin RNA for long-term gene knockdown experiments. Simmons envisages that these two approaches will be used in tandem if researchers need to validate difficult targets. "Now that we know what the majority of genes are, the whole race is trying to discover what each one does," said Simmons. Thermo Fisher Scientific joins the Lentiviral RNAi game After having successfully launched a HT RNAi screening services laboratory earlier this year Thermo Fisher Scientific has launched a new RNAi system that combines its gene-silencing technologies with Lentigen's viral delivery systems. The new system, named Thermo Scientific Dharmacon SMARTvector shRNA Lentiviral Particles, aims to permanently silence genes in a wide range of cell types using short hairpin RNA (shRNA). "With the SMARTvector advancement, we can now effectively deliver stably expressed gene silencing sequences into a broad range of cell types, including those that are difficult to work with, such as primary cells, non-dividing cells, stem cells and neuronal cell lines," said Dr Ian Jardine, vice president of global research and development for Thermo Fisher Scientific. "Thermo Fisher Scientific has developed a specialised shRNA design algorithm that greatly enhances the ability to select highly functional gene targeting sequences. Alternative technologies frequently produce shRNA sequences that are ineffective at silencing the targeted genes, while causing high levels of unintended gene silencing that can lead to flawed experimental results."