RNAi Genetic Screening with Pooled shRNA Libraries
The basis of Cellecta's RNAi genetic screening technology is the stable suppression of specific genes on a large-scale using pooled shRNA libraries, allowing for loss-of-function screens in mammalian cell systems. Genetic screens with shRNA libraries can be utilized to investigate any aspect of biology that can be recapitulated in a cell culture model. As opposed to expressing and assaying the functional effects of an individual siRNA molecule, the development of complex shRNA libraries allows for simultaneous screening of thousands of different shRNA molecules on a target population. In general, genetic screens represent an unbiased approach to identify genes that act in specific cellular pathways.
HT RNAi genetic screens have been proven to be an extremely potent and versatile tool to explore the molecular basis of cancer development and progression, and to discover genes essential for viability in cancer cells that can be used as targets for anticancer drug development.
The screening process introduces a packaged lentiviral library encoding a highly heterogeneous population of bar-coded shRNA constructs 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 HT sequencing of shRNA-specific bar-codes.
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,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.
Viability Screening Results for 7K shRNA Library Targeting 1,200
genes.
DU145 Cells were transduced with a 7K shRNA Library Targeting 1,200 annotated
prostate-specific genes. The shRNA oligo library was cloned under the control
of an inducible promoter that responds to the tetracycline-analog doxycycline
(dox). After plating, shRNA were induced to express by the additional
of dox. Dox was not added to control cells. shRNA targeting vital
genes required for cell growth and proliferation caused some cells to die
when expressed. These shRNA then declined in the treated population. When
both treated and untreated cells were harvested, genomic DNA isolated, and
shRNA bar-codes amplified and sequenced, the shRNA sequences that induced
cell death can be readily identified dropping out of the main population (green,
yellow, and red points below the diagonal.
For this screening, 210 shRNAs targeting 142 genes were identified. 33 genes were identified by multiple shRNAs (1 gene by 6 shRNAs, 4 genes by 5 shRNAs, 5 genes by 4 shRNAs, 9 genes by 3 shRNAs, 14 genes by 2 shRNAs).
Review Cellecta's RNAi Genetic Screening Service.
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