The most widely employed method of achieving post-transcriptional gene silencing in use today is RNA interference, or RNAi. Methodologies to carry out protein knockdown via RNAi are quite varied and, thus, amenable to a wide range of experimental protocols. RNAi can be transient or stable, and can be accomplished by the introduction of double-stranded RNA or plasmid vectors into cultured cells. In response to the tremendous popularity of these methods, Santa Cruz Biotechnology has expanded their catalog of RNAi reagents to include >99% of all putative human and mouse genes.
In addition to the more standard plasmid vectors containing small interfering RNA (siRNA) coding sequences, Santa Cruz also offers lentiviral-based reagents for the stable expression of short-hairpin RNA (shRNA) molecules. Once expressed, these shRNAs will be cleaved into siRNAs by cellular enzymes. Since no transfection agent is required with the lentiviral system, it is not necessary to optimize the transfection protocol with the aim of achieving high transfection efficiency and low cellular mortality. You simply add the virus particles to the culture and, 24 to 48 hours later, add the selection reagent (puromycin in this case). Once the untransfected control cells have all died, which means that only successfully transfected cells are alive, you can either proceed to culture puromycin-resistant clones from the transfected cells, or work with the entire resistant population.
I recently used Santa Cruz’s lentiviral-based system to knockdown a secreted protein in a lung cancer cell line. Since I wanted to develop cell populations expressing different amounts of the protein of interest, I isolated small clusters of clones using cloning cylinders. After allowing the clones to grow to a sufficient density in duplicate wells of a 96-well tray, I performed RT-PCR on the RNA from the cells in one well and used the remaining well to expand the cultures further. As shown in the figure, the endpoint RT-PCR showed that the clones expressed variable amounts of the transcript of interest.
The higher molecular weight band in the figure is a GAPDH amplicon and the lower band is an amplified region of the transcript of interest. I chose 10 of the most promising clones to expand, collected conditioned medium, and performed an immunoblot for the secreted protein I had theoretically knocked down. The immunoblot showed that strikingly variable quantities of the protein were being secreted from the cell clones. In some, the protein was undetectable. The lentiviral transfection had worked flawlessly, and on the first try.
shRNA Lentiviral Particles from Santa Cruz Biotechnology
An extremely straightforward method of preparing stable knockdowns via shRNA. Transfection with lentiviral particles resulted in approximately 90% of the cells acquiring puromycin resistance.
There’s a price associated with convenience. I paid $595 for 10<sup>6</sup> lentiviral particles. This is enough for several transfections, provided you start with a relatively small number of cells since Santa Cruz recommends an MOI of 5 to 20. Also, getting authorization at your institution to use these virus particles, even though they are self-inactivating and replication incompetent, can be quite involved.
The Bottom Line
One of the simplest methods to create stable knockdowns, although the expense of the viral particles and effort associated with institutional authorization are significant concerns.