Pooled Lentiviral-Delivery Genetic Screens.

Area of Science:

• Functional Genomics
• Mammalian Cell Biology
• Genetic Screening Technologies

Background:

• Pooled genetic screens are powerful tools for large-scale gene function evaluation in mammalian systems.
• These screens utilize viral transduction to introduce barcoded genetic perturbations into cell populations.
• Identifying genes that modulate specific cellular phenotypes is crucial for understanding biological processes.

Purpose of the Study:

• To provide detailed experimental protocols for key steps in pooled cell-based genetic screens.
• To highlight critical factors for achieving well-powered and reproducible screening results.
• To facilitate systematic, genome-scale evaluation of gene function using various perturbation types.

Main Methods:

• Employing pooled libraries with barcoded genetic perturbations (e.g., CRISPR knockout, gene expression, RNAi).
• Utilizing viral transduction for efficient delivery of perturbations into mammalian cells.
• Selecting and isolating cells exhibiting a phenotype of interest, followed by barcode sequencing to identify causative genes.

Main Results:

• The described protocols cover essential procedures common to diverse pooled screening approaches.
• Key factors for optimizing screen power and reproducibility are identified.
• The methodology enables the identification of genes that promote or inhibit specific cellular phenotypes.

Conclusions:

• Detailed protocols and optimization strategies are presented for robust pooled genetic screening.
• This approach facilitates comprehensive functional genomics studies in mammalian cell models.
• The methods support systematic, genome-wide discovery of gene function and regulatory networks.