Cellecta - Pooled shRNA Library Technology

Pooled shRNA Library Technology

There have been many articles published in recent years which clearly demonstrate the power of HT RNAi functional screening with pooled shRNA libraries. Although difficult to perform, these screens offer an unprecedented approach to isolate genes involved in a specific pathway or response.

In the pooled shRNA library format strategy, a library encoding a pooled set of packaged viral shRNA constructs is introduced into a population of identical cells under conditions where each transduced cell will express only a single gene-specific siRNA. Cells exhibiting the desired phenotypic changes are isolated and the shRNA constructs, presumably inducing the phenotypes, are recovered by PCR and identified by sequence analysis or microarray hybridization using bar-coding strategy, or designing siRNAs complementary to existing commercial microarrays.

A major advantage of the packaged lentiviral shRNA libraries is efficient transduction of non-transfectable cell types and tissues, overcoming a major limitation of synthetic siRNA libraries. A pooled, lentiviral vector-based format enables long-term silencing of target genes, presenting the possibility of screening functions (senescence, differentiation, growth in soft agar, etc.) that require weeks rather than days in vitro or ex vivo.

However, a drawback of genetic screens using pooled shRNA libraries is the requirement for recipient cells with desired phenotypic changes to be selected from a pool of unaffected cells; for example, by selection based on cell survival, appearance of specific markers, induction of reporter constructs, changes in cell morphology or behavior, etc. The design of a selection strategy is the most critical arm of any genetic screen project, considering that, in most cases, the inhibitory effect of siRNAs is weaker than that of gene knockout. The selection conditions, therefore, should be designed with less stringency. Repeated rounds of selection may be necessary for either secondary validation or to reduce the number of false positives thereby increasing the percentage of positive hits.

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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