Reporter Assay for HT Identification of Functional shRNA

Development of the HT Reporter Assay and determination of shRNA efficacy was funded in part by NIH grants NCRR RR024095 and NHGRI HG003355. Data produced using grant resources is being distributed based on our NIH Data and Resource Sharing Policy.

Designing effective shRNAs is essential for effective RNAi knockdown screening. In addition to choosing the optimal sequence, there are a number of structural factors that affect shRNA efficacy. In addition, the stem-loop hairpin structure of shRNA poses problems for library construction and amplification. To create representative quality shRNA libraries, we have focused significant effort to optimize effective of the shRNA inserts in our libraries. We have developed an in-house algorithm to predict the most effective shRNA sequences, and combine this with published data of validated sequences when constructing our libraries. We have also optimized the structure of shRNA for library construction.

diagram of shRNA validationTo efficiently test shRNA structure variations on a large-scale, we developed a high-throughput shRNA efficacy testing technology based on a reporter assay. It was previously demonstrated that a reporter gene (such as GFP) with a 3′ fusion to a cDNA fragment or short oligonucleotide from a target gene could be effectively used to monitor the efficacy of siRNA and shRNA constructs against this target gene. Based on these findings, we developed a lentiviral reporter vector for identification of functional shRNA constructs (Figure 1). Our proprietary shRNA testing reporter vector allows for cloning of both shRNA template downstream of the U6-Tet inducible promoter and target sequences at the 3′ end of the GFP reporter. When this construct is transduced into cells, transcription from the U6-Tet promoter produces shRNA in the presence of doxycycline, while transcription from the CMV promoter generates a GFP-sense target mRNA fusion transcript that can be used as a reporter for screening functional shRNAs.

For a list of human and mouse functional shRNA (~120,000) selected with the help of this technology, see the Software page.

Please visit the Cellecta website for additional information on Pooled shRNA Libraries, RNAi Screening, and related custom services offered.