Proper development of an organism requires the precise regulation of many genetic factors. Mis-regulation of gene expression often results in human diseases such as cancer. This can be caused by mutations in key classes of genes required for cellular development. Tumor suppressor genes are one class of genes known to play a role in cancer prevention. In mammals, retinoblastoma (Rb) is a tumor suppressor gene that, when inactivated, promotes cancer. We are using C. elegans as a model to study Rb function. We performed an RNAi screen to identify suppressors of lin-35, the C. elegans Rb homolog.
Our study builds directly on an RNAi suppressor screen previously published by Polley and Fay (2012). Polley and Fay exploited the genetic interaction between lin-35 and slr-2. lin-35 and slr-2 act redundantly in the intestine and as a result, simultaneous mutations in both genes result in a synthetic lethal phenotype. In order to circumvent the synthetic lethality and to maintain the strain, Polley and Fay expressed a wild-type copy of lin-35 in the double mutant as an extrachromosomal array with a GFP marker. Worms that contain the array, and therefore a wild-type copy of lin-35, can live past the larval stage. This array is lost in about 30% of offspring resulting in non-green offspring that would die as young L1 larvae, unless the lethality is suppressed by RNAi knockdown of another gene.
This screen is exceptionally well-suited for teaching genetics concepts and techniques. As a teaching tool, the preliminary screen was performed in a genetics lab course at Ithaca College. In Spring 2015, the genetics class screened the supplemental X chromosome Ahringer RNAi library. They identified 171 out of 371 genes as potential suppressors. In the Lo lab, through additional experiments, we have identified 50 of the strongest suppressors of the lin-35 lethal phenotype. For each experiment, we included known suppressors of lin-35 as positive controls to ensure all reagents and techniques functioned properly. RNAi bacteria that did not target a gene was included in each experiment as a negative control.
Ongoing research including additional viability and larval arrest experiments will further identify the most relevant suppressors. Ultimately, these studies will help identify components of the Rb tumor suppressor pathway and better inform future human disease treatments.