Construction of a germline-specific RNAi tool in C. elegans

Abstract

Analysis of complex biological functions usually requires tissue-specific genetic manipulations in multicellular organisms. The C. elegans germline plays regulatory roles not only in reproduction, but also in metabolism, stress response and ageing. Previous studies have used mutants of rrf-1, which encodes an RNA-directed RNA polymerase, as a germline-specific RNAi tool. However, the rrf-1 mutants showed RNAi activities in somatic tissues. Here we constructed a germline-specific RNAi strain by combining an indel mutation of rde-1, which encodes an Argonaute protein that functions cell autonomously to ensure RNAi efficiency, and a single copy rde-1 transgene driven by the sun-1 germline-specific promoter. The germline RNAi efficiency and specificity are confirmed by RNAi phenocopy of known mutations, knockdown of GFP reporter expression, as well as quantitative RT-PCR measurement of tissue-specific mRNAs upon RNAi knockdown. The germline-specific RNAi strain shows no obvious deficiencies in reproduction, lipid accumulation, thermo-tolerance and life span compared to wild-type animals. By screening an RNAi sub-library of phosphatase genes, we identified novel regulators of thermo-tolerance. Together, we have created a useful tool that can facilitate the genetic analysis of germline-specific functions in C. elegans.

The ribosomal RNA m5C methyltransferase NSUN-1 modulates healthspan and oogenesis inCaenorhabditis elegans

“…3H), phenocopying nsun-1 depletion in wildtype animals after whole animal RNAi. Similarly, no effect of nsun-1 knockdown was evident in another germline-specific RNAi strain, which was recently developed to enhance germline specificity (Zou et al, 2019) (Figure 3–figure supplement 1C).…”

New strains for tissue-specific RNAi studies in Caenorhabditis elegans

“…We obtained muscle-specific and germline specific RNAi strains, both of which were made in the rde-1(null) background. The germline-specific strain DCL569 uses a single-copy insertion of a transgene expressing rde-1 under control of the sun-1 promoter (Zou et al 2019). The musclespecific strain WM118 expresses rde-1 under control of the myo-3 promoter.…”

“…M03D4.6(tm1144) IV; sago-1(tm1195) V; nels10 X; WM49, rde-4(ne301) III, HC196, sid-1(qt9) V; WM118 , rde-1(ne300) V, neIs9 [myo-3::HA::RDE-1 + role-6(su1006)]. The WM49, rde-1(ne300) V strain was provided by Craig Mello and the DCL569 strain was provided by Di Chen (Zou et al 2019).…”

CREB non-autonomously regulates Reproductive Aging through Hedgehog/Patched Signalling

“…Therefore, we tested how germline-specific RNAi-mediated knockdown of four of the Patched-related receptors that have especially elevated expression in oocytes (Stoeckius et al, 2014) and oogenic gonads (Ortiz et al, 2014) affects reproductive aging of worms with hypodermal wrt-10 overexpression (i.e., rde-1(mkc36);Psun-1∷rde-1;Pdpy-7∷wrt-10 + PY37A1B.5∷wrt-10 , see (Zou et al, 2019)). We used adult-only RNAi exposure to avoid developmental effects, and mated and maintained hermaphrodites on control vector from day 5 onward to preclude potential effects of RNAi on males.…”

“…(A) Adult-only, germline-specific knockdown of Patched-related receptors ptc-1 and ptr-2 worsens the late-mating success of worms overexpressing hypodermal wrt-10. In hypodermal wrt-10 overexpression worms that are specifically sensitive to RNAi in the germline ( rde-1(mkc36);Psun-1∷rde-1;Pdpy-7∷wrt-10 + PY37A1B.5∷wrt-10 , see (Zou et al, 2019)), adult-only (day 1 of adulthood onward) exposure to ptc-1 or ptr-2 RNAi caused a significant reduction in the capacity of worms to produce progeny when mated at day 5 of adulthood; exposure to ptc-2 or scp-1 RNAi did not have a pronounced effect on late-mating capacity. Bars represent population means, error bars represent 95% confidence intervals.…”