Positive Selection Screens (aka. Rescue Screens)
Find genes required to produce a response to added factors or compounds, for example, genes necessary for trigging apoptosis or cell death in response to FAS, PUMA or other effectors. Positive screens are also known as enrichment screens. Many positive screens use FACS to look for modulators of signaling molecules like NF-κB, p53, c-myc, HSF-1, HIF-1α using fluorescent reporter cell lines, or cells expressing specific antibody-detectable markers, such as specific receptors.
Length of the Screen
A positive screen involves a selection that eliminates most of the cells. With this sort of screen, the goal is to isolate a small population of cells with shRNAs that enable the cells to pass through the selection step. The critical factor here then is the nature of the selection, which ultimately determines the screen procedure. In most cases, it is advisable to wait about 1 week after library transduction before carrying out the selection step. The 1 week wait period is needed to allow for knockdown of genes encoding for long half-life mRNAs and proteins, and the development of the resistant phenotype before applying selection. Cells should then be harvested as soon as positive selection is completed. Growing and expanding clones after positive selection is not advised.
For a positive selection screen, the puromycin or other antibiotic selection of transduced cells it is not essential, but provides a way to reduce the total number of cells before positive selection and makes cell culture handling more manageable.
MOI of Transduction
A positive screen involves isolation of a small population of cells with shRNA sequences that will be over-represented or enriched when compared to the starting library shRNA counts. As with any screen, to ensure reproducible and reliable results, it is critical that you transduce enough cells to maintain sufficient representation of each shRNA construct present in the library. The number of cells stably transduced with the shRNA library at the time of transduction should exceed the complexity of the shRNA library by at least 200-fold. For a library with 27,500 shRNAs, the starting population should be at least 5.5 million infected cells, and for a library with 55,000 shRNAs, the starting population should be at least 11 million infected cells.
Maintenance of the Cells
A positive selection screen often involves the comparison of two types of samples: selected and unselected (control) samples. After transduction and before selection, it is best practice not to discard any cells. However, this is often not practical. If cells have to be discarded or split before selection, the number of remaining cells in each sample should always exceed the complexity of the library by at least 1,000-fold (e.g. keep at least 2.7 × 107 cells after every splitting step, for a 27K library).
After the selection step, all the cells in the selected samples should be collected for genomic DNA purification and barcode PCR amplification. For the control samples, follow the aforementioned 1,000-fold rule in the sense that you should collect enough cells to equal 1,000- fold the complexity of the library. Similarly, when amplifying barcodes from isolated DNA, you should always use all the genomic DNA recovered from cell samples, up to the amount corresponding to 1000X cells the library size. For diploid cells, 25-30 million cells ~150-180 µg of genomic DNA.
Baseline Controls for Positive Selection Screens
In order to calculate the enrichment-fold of the shRNA sequences present in the selected population, a baseline control is needed. Depending on the screen, the plasmid library itself can be used as baseline, or pre-selection cells, or mock-selected cells.
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