RNAi
RNA interference (RNAi) is a process through which cells control the activity and expression of particular genes. Small RNA molecules such as siRNA and miRNA are vital for inducing the RNAi process. In this mechanism, siRNAs and miRNAs bind to their complimentary RNAs and regulate their activity by preventing messenger RNA (mRNA) from engaging in protein production. This process plays an important role in the cell’s ability to defend itself against viruses, and may help researchers and scientists develop gene expression systems that can prevent or cure many disease-related processes.
Gene regulation or gene knockdown is useful for many research applications, including gene target discovery, validation of gene targets, functional analysis of genes, assay development, and screening of therapeutic compounds. Further research into the role of gene regulation has the promise to elucidate cellular mechanisms and treat diseases that are resultant of incorrect regulation of proteins and viral diseases. The use of RNAi-inducing species in vivo for research purposes and in therapeutic development is under investigation by a number of biotechnology companies. One hurdle they have encountered is that nucleic acid based products, including siRNA and gene constructs to manufacture shRNA, are notoriously difficult to deliver, and often excreted by the cell prior to providing effectiveness. DNA is vulnerable to nucleases in both blood and the gastrointestinal system, with solutions to bioavailability being a productive area of investigation. Improvements of delivery include using liposomal formulations, nanoparticles, viral vectors, and applying modifications to the nucleic acid mimic.
Commercial Services
A typical RNAi research laboratory offers cloning of short hairpin RNA (shRNA) into a suitable vector backbone, and then creates a stable expressing cell line in which the plasmid construct integrates into the genome and expresses an antibiotic resistance gene for clonal selection. The resultant cell line can then be utilized in functional RNAi validation experiments. Generating stable cell lines provide scientists with genetically homogenous and clonal populations. Stable cell lines will be able to grow for long time periods while continually expressing the transgene at a steady rate. Laboratories offering these services have specific protocols to maximize the efficiency and stability of the cell lines.
Commercial RNAi services often include the option to express double-stranded RNA (dsRNA) in vitro and in vivo to stimulate cellular processes or actions that reduce the expression of specific genes (such as oncogenes). The ability to inhibit or knockdown gene expression in cells is achieved using the process of plasmid DNA or through the transfection of small interfering RNA (siRNA). In either case, one must be careful to not induce an antiviral response, which is an essential consideration in the development of novel therapeutics and treatment protocols.
The advances in stable cell line creation have enabled scientists and researchers to accelerate stable cell line creation to roughly 4-5 weeks, whereas previously it took 30 or 40 weeks to complete. RNAi gene silencing experiments rely heavily on high transfection efficiencies as well as high cell viability. Various bioscience providers throughout the world offer RNAi services, along with pre-optimized siRNA/DNA transfection reagents.
Commercial RNAi services often include the option to express double-stranded RNA (dsRNA) in vitro and in vivo to stimulate cellular processes or actions that reduce the expression of specific genes (such as oncogenes). The ability to inhibit or knockdown gene expression in cells is achieved using the process of plasmid DNA or through the transfection of small interfering RNA (siRNA). siRNAs can be transfected into cells, injected into mice, or injected into plants to participate in RNA interference. They can also be endogenously expressed from siRNA expression vectors or from products of PCR in transgenic animals. In any case, one must be careful to not induce an antiviral response. dsRNA that are longer than 30 base pairs usually elicits this, which is why siRNA’s (20-25 nucleotides) are frequently used. Preventing immune responses is an essential consideration in the development of novel therapeutics and treatment protocols.