Meeting Abstract
We report here on the efficacy of transient RNAi against the sallimus (sls) gene in the fruit fly, Drosophila melanogaster. The sallimus gene encodes a number of giant sarcomere associated protein (gSAP) isoforms, several of which are expressed in larval muscle. We designed a transient construct that utilizes the temperature sensitive yeast transcription enzyme Gal80ts to regulate Gal4-UAS promotion of dsRNA against sls. Although we have reported previously on the physiologic outcomes of this construct, this is the first time we present thorough transcriptional and translational evidence of its efficacy. We used qPCR, western blot of protein isoforms, and immunohistochemistry to probe complementary sites along the sls gene / protein. Since sls is the longest gene in the fly (>1 Mbp), as titin is the longest gene in mammals with which we are familiar, the action of the RNAi construct was somewhat different than anticipated in two main ways. (1) The construct required exposure to restrictive temperatures (31° C) for substantially longer than originally anticipated (~16 hours). (2) The reduction in expression of sls was not uniform across its length or isoforms; exons nearest the site of dsRNA were reduced significantly (p < 0.01) whereas those furthest from it were not. Nevertheless, expression of exon regions of interest such as the putative N2A analog were significantly reduced, permitting useful physiologic tools for the investigation of gSAP / titin physiology. We hope others will adopt this model for future mechanistic investigations of titin as well as comparative work to better understand the role gSAP molecules like titin play across Animalia.
