Optimization of shRNA Design
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.
To 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 H1 promoter and target sequences at the 3' end of the GFP reporter. When this construct is transduced into cells, transcription from the H1 promoter produces shRNA, 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.
Map of shRNA-target lentiviral reporter construct integrated in genomic DNA and mechanism of knockdown of GFP-target reporter. The cells transduced with functional shRNA constructs will have low GFP mRNA levels, while non-functional shRNAs permit a high level of GFP mRNA expression.
To
optimize the structure of the shRNA, we constructed an shRNA-target
library in the shRNA validation vector for 150 shRNAs, with each
shRNA constructed in 40 different designs described in the literature.
We performed RNAi screens in CHO-TRex cells transduced with this 150x40 bar-coded shRNA-Target library and measured representation (in the library and transduced cells) and knockdown efficiency of each shRNA construct. Some representative data from this screening is shown below.
The ten best designs were selected based on those having the highest target knockdown activity, equal amplification efficiency of same-design pooled oligonucleotides, and equal representation of same-design shRNAs in the pooled RNAi libraries. The knockdown efficiency of three p53 shRNAs constructed with these 10 best designs in a lentiviral vector was further analyzed by RT-PCR after transduction into mouse fibroblast cells.
View our Frequently Asked Questions (FAQs) page for detailed information on our pooled lentiviral shRNA libraries.
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