The use of small-interfering RNAs (siRNAs) has become a popular and powerful way to modulate gene expression. These siRNAs act by inducing RNA interference, a mechanism that suppresses gene expression by inducing the degradation of mRNA in a gene specific manner. Recently, our lab has been using the GeneSuppressor System from IMGENEX to generate siRNAs for our RNAi studies in mammalian cells. The GeneSuppressor System is a plasmid-based system for introducing siRNAs into mammalian cells, a process that is more effective than transfecting synthetic siRNAs. This is because using an siRNA expression plasmid results in a more long term inhibition of mRNA compared to transfecting with synthetic siRNAs. The system works by inserting the DNA of interest into the GeneSuppressor plasmid under the control of the U6 promoter, thus allowing the gene of interest to be transcribed by RNA Pol III. The expressed RNAs must contain fold-back stem-loops, which are then processed into siRNAs. The Neomycin resistant gene in the pSuppressor plasmid allows for the generation of permanent GeneSuppressed cell lines using G418 selection. The kit comes with the necessary reagents for transfection and a specific antibody for verifying gene knockdown. This kit also includes the linearized pSuppressorNeo plasmid, sequencing primer (for 20 reactions), circular control plasmid, transfection reagent, deionized water, and annealing buffer. The reagents required but not included in the kit are restriction enzymes and T4 DNA ligase.
For this system to work, the two oligonucleotide inserts or primers for making the hairpin RNA must correspond to the coding region of the gene of interest (sense and antisense). The target region of the gene should be 50-100 nts downstream of the start codon of the gene. The primers are designed and cloned into the SalI and XbaI sites in the pSuppressor plasmid (Neo/Kan) by annealing and ligation of the primers into the linearized vector (digested with SalI and XbaI) with annealing buffer and T4 DNA ligase provided in the kit. Enough of the pSuppressor plasmid is provided for cloning 20 inserts, but it is recommended that you amplify the plasmid before starting the cloning. It is important that one should have a control ligation without the insert. The ligated plasmid with and without insert is transformed into competent DH5alpha bacterial cells. Our constructs with the inserts were verified by sequencing. It is imperative to check your constructs by sequencing because a mutation may affect the suppression of your gene of interest. We transfected the correct constructs into our cells with the lipid-based transfection reagent provided in the kit. The transfection reagent provided is enough for 10 wells of a 6-well plate, which was not enough for us. But we were able to order more from IMGENEX. Permanent cell lines were created by using G418 in the cell culture medium.
Overall, the IMGENEX GeneSuppressor kit has been very easy to use, and most of the necessary reagents are provided in the kit (including transfection reagent, annealing reagent, ligation reagent, and sequencing primer). This makes the generation of siRNAs very cost effective, one of biggest advantages of using this kit. Also, the fact that the functional neomycin resistant gene allows for the generation of permanent GeneSuppressed cell lines is another big plus.
Hee Chul Lee
Dept. of Biochemistry
NYU School of Medicine