DATE: December 13, 2016
TIME: 8:00 AM PT, 11:00 AM ET
Understanding physiological mechanisms regulating growth and nutrient deposition in rainbow trout is central to establish production practices that optimize these traits. Analyzing expression of numerous genes across several physiological mechanisms is essential to characterize a systemic treatment response. Therefore, three independent multiplex RT-PCR assays were designed using the GenomeLab GeXP™ Genetic Analysis System to measure expression of 90+ genes across various physiological and biochemical mechanisms, including 1) proteolytic pathways, 2) the growth hormone/insulin-like growth factor axis, 3) the TOR pathway, 4) fatty acid transport and binding proteins, 4) beta-oxidation, and 5) fatty acid synthesis. Data from these assays have directly advanced our knowledge of the roles of sex steroids in nutrient repartitioning during sexual maturation, effects of phytoestrogens on nutrient metabolism and growth signals, and mechanisms involved in the fasting and compensatory growth response in rainbow trout. The data have also been used to identify genes associated with catabolic responses which are promising candidates for functional knock-out studies using CRISPR/Cas9 technology. Mutant genotypes were characterized by PCR amplification of the target region using a fluorescently labeled primer followed by capillary electrophoresis on the Genetic Analysis System. Indels that result in at least a two base pair difference from the single wild type amplicon are detected, providing for identification of individuals with extensive mutagenesis and therefore a high potential for functional protein knockout. Genotypes of select individuals are further characterized by product cloning and Sanger sequencing. This experimental approach was utilized to characterize mutations in the tyrosinase gene for a proof-of-concept study that was the first to demonstrate successful gene knockout using CRISPR/Cas9 in rainbow trout.
Learning objective 1: Understand how multiplex XP-PCR using the GeXP platform is a valuable gene expression profiling tool with the ability to analyze more genes than real-time RT-PCR without the high cost and low through-put associated with microarray analysis
Learning Objective 2: Recognize how the diverse capacities of the GeXP platform can be exploited to evaluate systematic regulation of physiological mechanisms in rainbow trout and analyze genotypes of fish genetically modified using CRISPR/Cas9 technology