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.
Highlights
The nematode C. elegans serves as a great model organism in biology research largely due to the ease of genetic manipulations
The strategies usually involve tissue-specific promoters-driving transgene rescue of mutations that are essential for the RNAi machinery. rde-1, which encodes an Argonaute protein, functions cell autonomously to ensure RNAi capacity[11]
The germline serves as the reproductive tissue that produces gametes, and affects metabolism, stress response and life span through non-autonomous regulation of gene expression in distal tissues[12,13,14,15,16,17]
Summary
The nematode C. elegans serves as a great model organism in biology research largely due to the ease of genetic manipulations. Genome-wide RNAi screens have been performed by many labs since the construction of the whole genome RNAi libraries, which are collections of E. coli strains that produce dsRNAs against nearly every gene in the C. elegans genome[2]. A strain that carries the rde-1(ne219) mutation and a single copy rde-1 transgene driven by the mex-5 promoter was constructed for tissue-specific RNAi experiments. In order to facilitate the genetic analysis in the C. elegans germline, we set out to create a tissue-specific RNAi strain that allows RNAi to be effective only in the germline. Mos[1] transposon-based transgenic approaches, we constructed an indel mutation of rde-1 that carries a single copy rde-1 transgene driven by the sun-1 germline-specific promoter. We performed an RNAi sub-library screening of phosphatase genes in the germline-specific RNAi strain and identified novel regulators of thermo-tolerance. We have created a useful tool that will help to analyze gene functions in the C. elegans germline
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