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Cancer Research (Fach) / V.5 High throughput screens (Lektion)

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• Nennen Sie vier Charakteristika eines guten Screenings. 1) Inexpensive 2) Easy to perform 3) Reliable (consistent) 4) Valid (distinguishes hits and non-hits)
• Sie wollen einen neuen Regulator der Apoptose identifizieren. Würden Sie CRISPR/Cas9 oder shRNA vewenden? Schreiben Sie ihre Gedanken dazu auf insbesondere bezüglich der Spezifität und KD? Effizienz. CRISPR/Cas9 is a good method for identification of regulators through knockdown of the presumable regulators, which functions very precise. Knockouts made with CRISPR/Cas9 are effective in completely blocking protein expression and have a high specifity. On the other hand, shRNA allows targeting of sequences with limited complementarity and even verification of the phenotypic effect, despite the known reversible nature of knockdowns. For an optimal result, combining data from both methods would be beneficial. If only one method has to be used, CRISPR/Cas9 would the first choice, as it is precise and cost-effective.
• What are the activities of Cas9? -cut DNA to generate knockouts -histone modifications -DNA base editing -transcriptional repression -transcriptional activation -DNA methylation or demethylation
• What are the pro´s of barcodes? 1) short enough to be quickly sequenced 2) easily identified in all species of organisms 3) variable enough to provide a unique sequence for each species 4) size of sequence: 600-700bp 5) universitality
• How is it possible to track clonal populations of cells over time or through different treatments? Through introduction of a packaged lentiviral library encoding a highly heterogenous population of barcodes into a population of founder cells. After selection, treatment, or differentiation, barcode representation in cells providesdata on which clones from founder cells survived, thrived, or died out.
• Define ROC curves. ROC curves (Receiver Operating Characteristic Curves) provide means of defining the criterion of positivity, that maximizes test accuracy.
• What does the abbreviation DPCP stands for? detectable pre-clinical phase = time interval between possible detection by screening and later detection after symptoms
• What are the 4 principles of Screening? 1) choice of disease 2) nature of the screening test 3) availability of a treatment 4) relative cost of screening
• Which types of screening do exist? 1) Biochemical screening (measure enzyme activity)      --> reporter assays, small molecule microarrays, drug screens 2) Molecular screening (analysis of the DNA genetic code)     --> shRNA screens, CRISPR/Cas, DNA barcoding

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