CMMB 461 Lecture Notes - Lecture 18: Lentivirus, Histone H3, Short Hairpin Rna
18 Nov 2018
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Phenotypic assays for rnai screens in mammalian cells. Knockdown gene, see if promoter is turned on or off. See if modification is made when kinase is knocked down or not. This is driven by fluorescence microscopy, using fluorescent markers. Automated microscope for acquisition of thousands of cell images. Cells in well, objective lens goes to first well, takes multiple pictures. Lens moves to next well, and takes pictures. Software helps determine cell size and other properties of the cell. Automated image analysis with software to find cells and carry out assays including cell size measurements, morphology recognition and fluorescence intensity of biomarkers. Require specialized hardware and software for handling terabytes of data. Issue: because you take so many pictures, you need a big computer to cluster, because it requires a lot of memory for storage. Lentiviral rnai library of human genes applied to high-throughput screening. Lentiviral shrna library targeting 12 000 human genes with 5 shrna constructs/gene.
