9. Analysis of RNA Interference of the unc-22 Muscle Gene in Caenorhabditis elegans
Goolla, Sai & Khan, Mishal.
College of Science and Mathematics
Professor: Dr. Rachel Hopp
ABSTRACT:
RNAi silencing induces down regulation of the unc-22 muscle gene that codes for twitchin protein in the nematode worm, Caenorhabditis elegans, resulting in a mutant muscular twitching phenotype. C. elegans unc-22 gene has been a tractable role model for studying the human homologue, titin, a sarcomeric regulatory protein necessary for the contraction-relaxation cycles in muscle movement. The mutant twitching phenotype exemplifies abnormal muscular characteristics, reflective of that in human disorders such as muscular dystrophy and cardiomyopathy. Introduction of an RNAi through feeding bacteria prohibits the translation of the unc-22 transcript in the worm, ultimately suppressing its locomotion and altering its natural muscular morphology. In this experiment, N2 wild type nematode worms were fed with the E.coli HT115 (DE3)/L4440 feeding bacteria, containing the plasmid with the appropriate RNAi insert. The bacteria were grown in NGM agar containing IPTG/ Carbenicillin (selective marker) plates to ensure positive RNAi silencing of unc-22 and incorporation of the L4440 recombinant plasmid, respectively. By varying the IPTG concentrations, the number of twitches per minute for 3 minutes among experimental worm groups and control worm groups (positive unc-22 worms) will be observed via the dissecting microscope. The results will be reported as experimental mean + standard deviation twitches per minute. Understanding RNAi in C. elegans unc-22 may provide better perspective on how silencing genes that make up defective titin could possibly induce proper muscle contraction in muscular disorders.
