Initial client request
“Looking to validate a gene target identified via DepMap for potential druggability across a range of cancer types. The end goal being to test a compound series designed with an advance computational genomics platform against said target”
Proposed experimental approach
- Number of cell lines: 8
o2 x cell lines (1 predicted sensitive & 1 insensitive) of the following lineages:
Breast, Liver, Intestinal, and Lung.
- Number of genes of interest: 1
-Target gene
- Number of assay controls: 3
-Untransfected
-2 x non-targeting siRNA control
- Number of technical replicates: 3
-Triplicate cell culture wells
-Triplicate qPCR measurements per culture well
- Timepoint: 10 days of repetitive siRNA transfection
- Number of biological replicates: 1
- Read outs:
-A) Live cell imaging every day to monitor % confluence
-B) Metabolic activity of cells, by way of CellTiter Glo® after every passage
-C) Endpoint qPCR-mediated gene expression (normalisation to housekeeping gene)
Chronos Scoring- dependency on target gene
- Cell lineage pairings were proposed based on DepMap dependency scoring.
- Final pairings, highlighted in yellow, were chosen considering additional factors such as licencing requirements and availability.
Key assay considerations
Proliferation - establishing accurate semi-quantifiable method
Cell density calibration curve generated by way of a CellTiter-Glo® assay. Assay performed using pre-established densities of trypsinised cells (5,000-80,000). A total of 9 cell densities were quantified (n=3) to generate a calibration curve. From these graphs, estimates of cell counts at passage were obtained by interpolation of the raw luminescence values. This was then used to determine the volume required for the re-seeding of each condition, to achieve a similar cell density across all 4 test conditions.
Effect of Target gene siRNA transfection on cell proliferation and gene expression. Top panel) The proliferation of three cancer cell lines of different origin; breast, liver, and melanoma was assessed under repetitive siRNA Knockdown of the same target gene, or non-targeting control siRNA for 12-days. Cell confluency (%) was measured daily (n=3 average +/- SEM). Bottom panel) qPCR validation of target gene knockdown, by normalisation of CT against housekeeping gene HNRNPK. Fold-change determined by 2^delta delta Ct method against the Untransfected control. (n=3 average +/- SEM). qPCR performed in technical triplicates, from each culture triplicate.
Summary and next steps
- siRNA treatment resulted in a significant reduction in target gene mRNA expression (≤ 50%) across all 8 cell lines at day 10 of treatment, when compared to the relative untreated and non-targeting siRNA controls.
- siRNA Knockdown impacted cell proliferation in cell lines with predicted dependency, but less so those without.
- Drug candidates targeting the gene of interest will be tested in the same assay to determine activity and target specificity.
