HT shRNA Validation Technology
To avoid the excessive labor and cost of carrying out large-scale gene-by-gene shRNA validation, 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.
Fig. 1. 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.
View our Frequently Asked Questions (FAQs) page for detailed information on our pooled lentiviral shRNA libraries. For relevant references and literature, visit the Articles page under Resources.
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