Pooled Shrna Screening
Pooled Shrna Screening - Related Videos
09:12
MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells
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Cited by 11 •
2011
Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.
11:15
Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome
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Cited by 2 •
2018
We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.
14:51
Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype
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Cited by 2 •
2022
High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).
00:09
Pooled CRISPR-Based Genetic Screens in Mammalian Cells
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Cited by 14 •
2019
CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.
03:33
Generating Conditional Knockdown Cells Using a Tetracycline-Responsive Lentiviral shRNA Expression System
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2026
Source: Kubala, M. H. & DeClerck, Y. A. Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment. J. Vis. Exp. (2017)This video demonstrates the generation of conditional knockdown cancer cell lines using a Tet-inducible lentiviral shRNA system. It outlines the steps for viral transduction, antibiotic selection, and doxycycline-induced gene silencing.
FAQs
Each shRNA is processed into an RNA interference guide that directs the RNA-induced silencing complex, or RISC, toward a matching messenger RNA. This targeting promotes degradation or inhibition of that transcript, reducing production from the corresponding gene. Because different shRNAs target different genes, the resulting cell population contains many gene-suppression states for functional comparison.
Selection changes the representation of individual shRNAs within the surviving population. An enriched shRNA is associated with cells that persist under the applied condition, whereas a depleted shRNA is associated with cells that become less represented after selection. Comparing these patterns helps connect gene suppression with responses to drug treatment, nutrient stress, or another experimental challenge.
Viral vectors typically introduce the shRNA library into the cell population, allowing many distinct gene-suppression reagents to be tested in parallel. The library format links each perturbation to a recognizable shRNA sequence or barcode. That linkage makes it possible to track which gene-suppression constructs are represented before and after the experimental selection.
The method converts changes in cell survival under a defined condition into functional evidence about genes. Patterns across many suppressed genes can reveal gene functions and pathway interactions rather than focusing on one target at a time. In disease research, these results can also identify genes that merit consideration as potential therapeutic targets.
Researchers introduce an shRNA library, apply a selection condition such as drug treatment or nutrient stress, and examine the shRNA composition of the surviving cells. Barcode analysis or next-generation sequencing then identifies constructs that became enriched or depleted. Interpreting these changes links the selection outcome to the genes targeted by the corresponding shRNAs.
After selection, researchers determine which shRNAs remain represented in the cell population and in what relative pattern. Barcode analysis or next-generation sequencing provides the readout for this comparison, distinguishing enriched constructs from depleted ones. The resulting sequence-based profile supplies the evidence used to infer gene effects under the selected biological condition.