Successful RNAi-mediated knock-down requires efficient delivery of the payload. Traditional methods (e.g., lipid-based reagents) may not work well for certain cell types such as neurons and hematopoietic cells. Viral vectors offer an alternative system for delivery.
“Viral biology can be engineered to deliver genetic material into cells very effectively and modulate gene expression for both short- and long-term functional studies,” explained Devin Leake, Ph.D., global director of R&D genomics, Thermo Fisher Scientific.
According to Dr. Leake, a viral vector system needs to address three key parameters. “It must be able to effectively deliver genomic content, have a very broad tropism, and allow for the long-term expression of that content. This requires the careful design of the short hairpin RNA (shRNA), the vector backbone, and choosing the best promoter for the particular cell type utilized.”
In creating the Thermo Scientific SMARTvector2.0® Lentiviral shRNA platform, the company designed a microRNA-adapted expression scaffold to maximize accurate processing of the silencing sequence while minimizing the potential for off-target effects.
“We rigorously tested a panel of miRNAs and selected one that could consistently and efficiently be processed by the endogenous RNAi-silencing machinery. It is also necessary to have rationally designed highly functional gene targeting sequences. The SMARTvector2.0 algorithm selects optimal shRNA targeting sequences and includes seed-based filters to reduce toxicity.”
Another important feature of a lentiviral vector system is the promoter. Although the company initially included the promoter from cytomegalovirus (CMV), it will soon be releasing an a-la-carte series of promoters that investigators may choose.
“The CMV promoter may be too weak in some cell types. There is no universally optimal promoter, so we have developed a series of SMARTvector backbones with the option of seven different promoters, called the Thermo Scientific SMARTchoice system, which will provide researchers with greater flexibility and more choices for successful RNAi experiments.”
Thermo Fisher Scientific custom-builds the lentiviral system using the requested promoter and the appropriate sequence for the client’s target of interest. It plans to release the system in the first quarter of 2012.