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.

Keywords: RNA interference; fatty acid desaturase.

Figure 1

Determination of desaturation index. RNAi efficiency was measured by the ability to knock down the fatty acid desaturase genes fat-1 and fat-4 with RNAi by feeding. We calculated the desaturation index for each treatment, defined as the ratio of fatty acid desaturation products (red) to their substrates (blue) (A and C). Representative chromatographs reveal that in RNAi competent worms (control strain), treatment with RNAi against either fat-1 (B) or fat-4 (D) causes accumulation of substrates (blue) and depletion of products (red) leading to a low desaturation index. Example chromatographs of an RNAi “resistant strain” show a similar desaturation indices as the empty vector control treated worms, while chromatographs of the “partially resistant” strain show intermediate activity.

Figure 2

Desaturation indices for FAT -1 and FAT-4 of control (N2) and RNAi-deficient strains FAT-1 and FAT-4 desaturation indices. (A) rde-1 (ne219) and rde-1 (ne300) compared to control (N2) reveal that rde-1 (ne219) is partially resistant to feeding RNAi and rde-1 (ne300) is strongly RNAi resistant. (B) Germline-specific RNAi deficient (ppw-1) and somatic-specific RNAi deficient (rrf-1) strains both show nearly wild-type levels of RNAi efficiency. (C) Screen of RNAi-deficient strains showing RNAi deficiency (sid-1 and rde-4), partial deficiency (MAGO and rde-3) and nearly normal RNAi efficiency (rde-10 and rde-11) when treated with fat-1 and fat-4 RNAi by feeding. Comparisons of RNAi treatment to empty vector treatment showed statistically significant differences (P values reported in Table S1), except for comparisons of the strains depicted on the graphs with NS, not significant. The strains with no significant difference in desaturation index are considered to be completely RNAi resistant.

Figure 3

New strains for intestine- and epidermis-specific RNAi knockdown. (A) The intestine-specific RNAi strain shows developmental arrest on act-5(RNAi) like the wild-type strain, while rde-1 null (rde-1(ne300)) and the adult skin-specific RNAi strain reach adult 48 h after transferring synchronized L1 larvae onto RNAi plates at 25°C. (B and C) Similar to the wild-type, the adult skin-specific RNAi strain becomes shorter (B) and presents a high expression of nlp-29 in the epidermis (C) on dpy-7(RNAi), compared to rde-1 null and intestine-specific RNAi strain; the phenotype is observed as in (A) 48 h after transferring synchronized L1 larvae onto RNAi plates at 25°C. Individual data points are shown, with bars depicting the mean and SEM.

Figure 4

Desaturation indices for wild type, RNAi-deficient (rde-1(ne300)) and tissue-specific RNAi strains. For both fat-1 RNAi (A) and fat-4 RNAi (B), the greatest reduction of RNAi activity occurred in the intestine-specific RNAi strain, revealing intestine as the major tissue of fatty acid desaturation. Comparisons of RNAi treatment to empty vector treatment showed statistically significant differences (P values reported in Table S1), except for comparisons of the strains depicted on the graphs with NS, not significant.