Searching Novel Clock Genes Using RNAi-Based Screening

• DOI: 10.1007/978-1-0716-0381-9_8
• Publication Date: 2020-12-07T15:36:28Z
• Authors: Bert Maier; Stephan Lorenzen; Anna-Marie Finger; Hanspeter Herzel; Achim Kramer
• Keywords: HEK293 Cells; Genes, Reporter; Genetic Vectors; Lentivirus; Animals; CLOCK Proteins; Humans; RNA Interference; Cloning, Molecular

RNA interference (RNAi) allows for the selective downregulation of gene expression by neutralizing targeted mRNA molecules and has frequently been used in high-throughput screening endeavors. Here, we describe a protocol for the highly parallel RNAi-mediated downregulation of gene expression in order to search for components involved in circadian rhythm generation. We use lentiviral gene transfer to deliver shRNA expressing plasmids into circadian reporter cells ensuring for efficient and stable knockdown. Circadian rhythms are monitored using live-cell bioluminescence recording of synchronized reporter cells over several days. In addition, we present a new software tool (ChronoStar) for efficient, parallel time-series analysis to extract rhythm parameters such as period, phase, amplitude, and damping.