New Strains for Tissue-Specific RNAi Studies in Caenorhabditis elegans

Abstract

RNA interference is a powerful tool for dissecting gene function. In Caenorhabditis elegans, ingestion of double stranded RNA causes strong, systemic knockdown of target genes. Further insight into gene function can be revealed by tissue-specific RNAi techniques. Currently available tissue-specific C. elegans strains rely on rescue of RNAi function in a desired tissue or cell in an otherwise RNAi deficient genetic background. We attempted to assess the contribution of specific tissues to polyunsaturated fatty acid (PUFA) synthesis using currently available tissue-specific RNAi strains. We discovered that rde-1 (ne219), a commonly used RNAi-resistant mutant strain, retains considerable RNAi capacity against RNAi directed at PUFA synthesis genes. By measuring changes in the fatty acid products of the desaturase enzymes that synthesize PUFAs, we found that the before mentioned strain, rde-1 (ne219) and the reported germline only RNAi strain, rrf-1 (pk1417) are not appropriate genetic backgrounds for tissue-specific RNAi experiments. However, the knockout mutant rde-1 (ne300) was strongly resistant to dsRNA induced RNAi, and thus is more appropriate for construction of a robust tissue-specific RNAi strains. Using newly constructed strains in the rde-1(null) background, we found considerable desaturase activity in intestinal, epidermal, and germline tissues, but not in muscle. The RNAi-specific strains reported in this study will be useful tools for C. elegans researchers studying a variety of biological processes.

Meisosomes, folded membrane platforms, link the epidermis to the cuticle inC. elegans

“…pNP165 was obtained by insertion of the dpy-7 promoter, which leads to an epidermal specific expression, in front of VHA-5::GFP into the pNP154 vector. pNP154 was made from a vector containing the SEC cassette for single insertion on Chromosome II at the position of ttTi5605 (pAP087, kindly provided by Ari Pani) (Watts et al, 2020). Constructs were made using Gibson Assembly (NEB Inc., MA) and confirmed by sequencing.…”

Floxed exon (Flexon): A flexibly positioned stop cassette for recombinase-mediated conditional gene expression

“…Because most animals are unaffected, we infer that the rde-1(flexon) allele strongly abrogates rde-1 activity, but the low proportion of escapers suggests that it may not be a true null allele. While there may be some situations in which a low background of residual rde-1 activity may be problematic ( 39 ), we demonstrate below that it is eminently feasible to use rde-1(flexon) for tissue-specific RNAi.…”

“…In C. elegans, RNAi is usually performed by feeding worms with bacteria that express double-stranded RNA for a gene of interest ( 37 ). Tissue-specific RNAi has been accomplished by selectively expressing RDE-1/Argonaute in an rde-1 hypomorphic or null mutant background ( 38 , 39 ). As with any other transgenic method, rescue experiments are affected by the strength and specificity of available regulatory regions; for example, our laboratory has had difficulty achieving effective tissue-specific RNAi using ckb-3p or lin-31p to drive RDE-1 expression in an rde-1 null mutant background.…”

Antagonistic fungal enterotoxins intersect at multiple levels with host innate immune defences

“…When we knocked down sta-2 expression by RNAi in worms expressing GPA-12*, we observed the expected decrease in the expression of the AMP gene nlp-34 , but a significant increase in the expression of ifas-1 ( Fig 5A ). Similarly, upon infection, knocking down sta-2 specifically in epidermis (in strain IG1502 [ 52 ]) decreased expression of the AMP genes nlp-34 and cnc-2 , but provoked a significant increase in the expression of ifas-1 ( Fig 5B ). In vertebrates, as well as acting as positive regulators of gene expression, STAT proteins can directly repress the accessibility and transcription of specific loci [ 53 ].…”