A pre- and co-knockdown of RNAseT enzyme, Eri-1, enhances the efficiency of RNAi induced gene silencing in Caenorhabditis elegans.
BACKGROUND
The approach of RNAi mediated gene knockdown, employing exogenous dsRNA, is being beneficially exploited in various fields of functional genomics. The immense utility of the approach came to fore from studies with model system C. elegans, but quickly became applicable with varied research models ranging from in vitro to various in vivo systems. Previously, there have been reports on the refractoriness of the neuronal cells to RNAi mediated gene silencing following which several modulators like eri-1 and lin-15 were described in C. elegans which, when present, would negatively impact the gene knockdown.


RESULTS
Taking a clue from these findings, we went on to screen hypothesis-driven- methodologies towards exploring the efficiency in the process of RNAi under various experimental conditions, wherein these genes would be knocked down preceding to, or concurrently with, the knocking down of a gene of interest. For determining the efficiency of gene knockdown, we chose to study visually stark phenotypes of uncoordinated movement, dumpy body morphology and blistered cuticle obtained by knocking down of genes unc-73, dpy-9 and bli-3 respectively, employing the RNAi-by-feeding protocol in model system C. elegans.


CONCLUSIONS
Our studies led to a very interesting outcome as the results reveal that amongst various methods tested, pre-incubation with eri-1 dsRNA synthesizing bacteria followed by co-incubation with eri-1 and gene-of-interest dsRNA synthesizing bacteria leads to the most efficient gene silencing as observed by the analysis of marker phenotypes. This provides an approach for effectively employing RNAi induced gene silencing while working with different genetic backgrounds including transgenic and mutant strains.
Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, New Delhi, India ; Laboratory of Functional Genomics and Molecular Toxicology, Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow, India.
Pooja
P
Jadiya
Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, New Delhi, India ; Laboratory of Functional Genomics and Molecular Toxicology, Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow, India.
Aamir
A
Nazir
D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D003094:Collagen / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005095:Exoribonucleases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D055785:Gene Knockdown Techniques / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D009419:Nerve Tissue Proteins; D012330:RNA, Double-Stranded; D012097:Repressor Proteins; C111263:UNC-73 protein, C elegans; C116485:lin-35 protein, C elegans; D003094:Collagen; C544706:Bli-3 protein, C elegans; D010088:Oxidoreductases; C482202:ERI-1 protein, C elegans; D005095:Exoribonucleases