We developed a book knockdown strategy to examine cell-specific gene function

We developed a book knockdown strategy to examine cell-specific gene function in gene encoding the pan-neuronally expressed G-protein subunit GOA-1 with a degradation-tagged transgene. can cause global knockdown effects (Jose 2009 2011 In addition extrachromosomal transgenes typically used to drive expression of dsRNAs are randomly lost during cell division leading to mosaic knockdown effects within individual animals of a population (Stinchcomb 1985). We have developed a method to knock down the expression of any gene in any cell type in that is both cell autonomous Rabbit Polyclonal to CAD (phospho-Thr456). and genetically stable. In this strategy an endogenous gene is replaced by an integrated single-copy transgene containing the endogenous gene’s promoter and coding sequence tagged with a unique 3′-UTR that targets transgene mRNA for destruction by the host cell’s nonsense-mediated decay (NMD) machinery. In NMD-defective animals the tagged transgene is expressed at levels comparable to that of the endogenous gene and is able to restore wild-type gene function. Spatial control of knockdown is achieved by cell-specific restoration of NMD activity. Using appropriate cell-specific promoters to control NMD activity one can restrict the knockdown of transgene expression to individual cell types in the animal without affecting its expression in any other cells. To demonstrate the utility of this strategy we used a tagged transgene to investigate the cell-specific function of the G-protein subunit GOA-1 and discovered that selective removal of GOA-1 from both hermaphrodite-specific neurons (HSNs) (however not from additional cells from the organism) was adequate to trigger null egg-laying DCC-2036 problems. Therefore GOA-1 acts cell in the HSNs to inhibit egg-laying behavior autonomously. This cell-autonomous approach to gene knockdown may be used to examine the cell-specific function of any proteins removing the confounding results due to the global reduced amount of proteins function normal of additional knockdown strategies. Strategies and Components Transgenes To create tagged transgenes 4188 bp of series was amplified DCC-2036 from pPD118.60 (L3808 Addgene) using primers promoter and mCherry coding series was amplified from pGH8 using primers coding area was amplified from genomic DNA using primers goa-1-pro-coding area was amplified from genomic DNA using primers unc-4 pro-genomic series and 3′-UTR was amplified from genomic DNA using primers smg-5-promoter series was amplified using primers unc-17-series was amplified using primers goa-1-pro-promoter series was amplified using primers tph-1-pro-promoter series was amplified using the primers unc-4-pro-transgenes were made as described using 2912 bp of promoter (Esposito 2007). The sense promoter used primers unc-4-pro-fusion-sense-R and unc-4-pro-outer-F. The antisense promoter used primers unc-4-pro-fusion-R-AS and unc-4-pro-outer-F. The RNAi target sequence was amplified using unc-4-ex6-outer-R and unc-4-seq-exon-4-F primers. Using these DNAs as template the fusion feeling transgene was produced using primers unc-4-pro-inner-F and unc-4-former mate6-inner-R as well as the fusion antisense transgene DCC-2036 was produced using primers unc-4-pro-inner-F and unc-4-former mate4-inner-F to create 3525 bp items. PCR transgenes had been purified before shot. strains Worm strains had been generated and taken care of using standard strategies and circumstances (Brenner 1974). The wild-type stress was Bristol N2. The limitations of the deletion mutation were determined to be 5′-AGAACAATATAGAAGTAGTGCTTAG-ACGCAACTTTTCCAATTGGC-3′ resulting in a 15 217 bp deletion that removed the entire coding sequence of I; I; I; IV; I KO98 I XP447 I; II XP466 I XP467 I; II. Figure 2 Expression of a DCC-2036 degradation-tagged transgene rescues endogenous gene function in DCC-2036 NMD-defective but not NMD-competent animals. (A-C) Quantification of (A) locomotion rate (B) spontaneous reversal frequency and (C) egg-lying behavior. Genotypes … Figure 3: N2 KO98 I XP467 I; II XP469 I; II; IV XP468 I; II; IV. Figure 3 NMD-dependent knockdown of tagged transgenes is robust and can be restricted to individual cell types. (A-C) Quantification of (A) locomotion rate (B) spontaneous reversal frequency and (C) egg-laying behavior. Genotypes and transgenes expressed … Figure 4: N2 KO98 I XP467 I; II XP469 I; II; IV. Figure 4 Levels of expression from tagged transgenes are similar to those of wild-type animals and are dramatically reduced by NMD. (A) Quantification of mRNA levels by qRT-PCR in wild-type and transgenic animals. All values represent the average from … Table 1: N2 VC1453 II XP470 I; II XP481 I;.