Vector-Based RNAi

Vector-based RNAi involves the use of plasmids or viral vectors to deliver small hairpin RNAs (shRNAs) instead of synthetic short interfering RNAs (siRNAs) into cells, enabling stable and long-term gene silencing. This technique offers significant advantages for studying gene function, validating gene targets, and conducting functional genomics research. You can use RNAi vector technologies to regulate gene inhibition with inducible RNAi expression, select for a pure population of cells stably expressing an RNAi sequence, and control gene expression in vivo with tissue-specific promoters.

To help you achieve successful and efficient gene knockdown in your experiments, we offer our own RNAi vectors along with protocols and workflow recommendations. Benefits of using RNAi vectors include:

• Stable or transient target knockdown
• Lentiviral or adenoviral delivery for hard-to-transfect, primary, and non-dividing cells
• Co-expression of a fluorescent reporter for visualization

Pol II miR RNAi and shRNA vectors are both tools used for gene silencing via RNAi, but they operate through slightly different mechanisms and offer distinct advantages.

Pol II miR RNAi vectors are designed to express miRNA mimics under the control of a Pol II promoter, which allows for regulated expression in a tissue-specific or inducible manner. These vectors harness the natural miRNA processing pathway, leading to efficient and precise gene silencing.

In contrast, shRNA vectors express shRNAs under the control of either Pol III or Pol II promoters. The shRNAs are processed into siRNAs by cellular machinery, leading to targeted mRNA degradation and gene silencing.

While both systems are effective for stable gene knockdown, the choice between them depends on the specific requirements of the experiment. Pol II miR RNAi vectors offer more flexibility in expression control and mimic natural miRNA pathways, whereas shRNA vectors can provide robust and direct knockdown of target genes.

Highly efficient delivery of Pol II miR RNAi expression vectors or shRNA vectors helps achieve significant levels of knockdown. Optimizing transfection or transduction, controlling for experimental variability, and using a powerful transfection reagent vastly improves the chances for RNAi success. The use of positive and negative controls also helps in the assessment of your experiments. Here are some tips to help optimize delivery conditions for RNAi vector success:

1. Start with the cell-type specific protocol and transfection reagent most suitable for your experiment
2. Use a DNA plasmid that expresses a fluorescent protein, such as GFP, to monitor that the plasmid has entered the cell
3. Apply a viability indicator such as Ethidium Homodimer-1 (EthD-1) to monitor cell viability related to transfection conditions
4. Utilize Hoechst nuclei stain to measure the percent of transfected and/or dead cells to the total cell population