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RNA interference in Schistosoma mansoni schistosomula: selectivity, sensitivity and operation for larger-scale screening.

Stefanić S, Dvořák J, Horn M, Braschi S, Sojka D, Ruelas DS, Suzuki B, Lim KC, Hopkins SD, McKerrow JH, Caffrey CR - PLoS Negl Trop Dis (2010)

Bottom Line: Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets.Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi.RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days); electroporation provided no added benefit.

View Article: PubMed Central - PubMed

Affiliation: Sandler Center for Drug Discovery, California Institute for Quantitative Biosciences (QB3), University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT

Background: The possible emergence of resistance to the only available drug for schistosomiasis spurs drug discovery that has been recently incentivized by the availability of improved transcriptome and genome sequence information. Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets. To date, RNAi studies in schistosome stages infecting humans have focused on single (or up to 3) genes of interest. Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi.

Methodology/principal findings: We investigated in vitro the sensitivity and selectivity of RNAi using double-stranded (ds)RNA (approximately 500 bp) designed to target 11 Schistosoma mansoni genes that are expressed in different tissues; the gut, tegument and otherwise. Among the genes investigated were 5 that had been previously predicted to be essential for parasite survival. We employed mechanically transformed schistosomula that are relevant to parasitism in humans, amenable to screen automation and easier to obtain in greater numbers than adult parasites. The operational parameters investigated included defined culture media for optimal parasite maintenance, transfection strategy, time- and dose-dependency of RNAi, and dosing limits. Of 7 defined culture media tested, Basch Medium 169 was optimal for parasite maintenance. RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days); electroporation provided no added benefit. RNAi, including interference of more than one transcript, was selective to the gene target(s) within the pools of transcripts representative of each tissue. Concentrations of dsRNA above 90 µg/ml were directly toxic. RNAi efficiency was transcript-dependent (from 40 to >75% knockdown relative to controls) and this may have contributed to the lack of obvious phenotypes observed, even after prolonged incubations of 3 weeks. Within minutes of their mechanical preparation from cercariae, schistosomula accumulated fluorescent macromolecules in the gut indicating that the gut is an important route through which RNAi is expedited in the developing parasite.

Conclusions: Transient RNAi operates gene-selectively in S. mansoni newly transformed schistosomula yet the sensitivity of individual gene targets varies. These findings and the operational parameters defined will facilitate larger RNAi screens.

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Related in: MedlinePlus

RNAi of SmCC is accompanied by decreased translation product measured as proteolytic activity.(A) Activity in extracts of schistosomula that had been co-incubated for 6 days with 30 µg/ml mCherry (Ch) or SmCC (CC) dsRNA was measured with the cathepsin C-selective peptidyl substrate, GR-AMC. Worm extracts were normalized to 0.4 µg of protein per reaction and the proteolytic activity in extracts from parasites exposed to mCherry dsRNA was set to 100%. (B) Reactivity with the fluorescent cathepsin C-selective activity-based probe (bodipy-labeled FY01; [91]) as assessed by SDS-PAGE. Worm extracts were normalized to 2 µg per labeling reaction. The fluorescent signal at 24 kDa is lost subsequent to exposure to CC-specific dsRNA. Data in (A) are the average of triplicate values (SD values were less the 5% of the mean). Representative data from two experiments are shown.
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pntd-0000850-g006: RNAi of SmCC is accompanied by decreased translation product measured as proteolytic activity.(A) Activity in extracts of schistosomula that had been co-incubated for 6 days with 30 µg/ml mCherry (Ch) or SmCC (CC) dsRNA was measured with the cathepsin C-selective peptidyl substrate, GR-AMC. Worm extracts were normalized to 0.4 µg of protein per reaction and the proteolytic activity in extracts from parasites exposed to mCherry dsRNA was set to 100%. (B) Reactivity with the fluorescent cathepsin C-selective activity-based probe (bodipy-labeled FY01; [91]) as assessed by SDS-PAGE. Worm extracts were normalized to 2 µg per labeling reaction. The fluorescent signal at 24 kDa is lost subsequent to exposure to CC-specific dsRNA. Data in (A) are the average of triplicate values (SD values were less the 5% of the mean). Representative data from two experiments are shown.

Mentions: Confirmation of RNAi at the protein level was sought for CC (Figure 6). A 6-day co-incubation of schistosomula and dsRNA targeting CC realized a knockdown of the translation product, measured either as an 80% decrease in the peptidolytic cleavage of the cathepsin C-selective substrate, GR-AMC (Figure 6A) or as a loss of the proteolytic signal (at approximately 24 kDa) using the specific active site-directed probe, bodipy-labeled FY01 (Figure 6B). Activity in the presence of GR-AMC was not inhibited by the general cysteine protease inhibitor, K11777, or by CA-074 and Z-Phe-Phe-DMK that target cathepsins B and L, respectively. This indicates that only cathepsin C activity was being measured. For CB1, a similar absence of protein was recorded after dsRNA application using immunoblotting with monospecific antibodies (not shown).


RNA interference in Schistosoma mansoni schistosomula: selectivity, sensitivity and operation for larger-scale screening.

Stefanić S, Dvořák J, Horn M, Braschi S, Sojka D, Ruelas DS, Suzuki B, Lim KC, Hopkins SD, McKerrow JH, Caffrey CR - PLoS Negl Trop Dis (2010)

RNAi of SmCC is accompanied by decreased translation product measured as proteolytic activity.(A) Activity in extracts of schistosomula that had been co-incubated for 6 days with 30 µg/ml mCherry (Ch) or SmCC (CC) dsRNA was measured with the cathepsin C-selective peptidyl substrate, GR-AMC. Worm extracts were normalized to 0.4 µg of protein per reaction and the proteolytic activity in extracts from parasites exposed to mCherry dsRNA was set to 100%. (B) Reactivity with the fluorescent cathepsin C-selective activity-based probe (bodipy-labeled FY01; [91]) as assessed by SDS-PAGE. Worm extracts were normalized to 2 µg per labeling reaction. The fluorescent signal at 24 kDa is lost subsequent to exposure to CC-specific dsRNA. Data in (A) are the average of triplicate values (SD values were less the 5% of the mean). Representative data from two experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2957409&req=5

pntd-0000850-g006: RNAi of SmCC is accompanied by decreased translation product measured as proteolytic activity.(A) Activity in extracts of schistosomula that had been co-incubated for 6 days with 30 µg/ml mCherry (Ch) or SmCC (CC) dsRNA was measured with the cathepsin C-selective peptidyl substrate, GR-AMC. Worm extracts were normalized to 0.4 µg of protein per reaction and the proteolytic activity in extracts from parasites exposed to mCherry dsRNA was set to 100%. (B) Reactivity with the fluorescent cathepsin C-selective activity-based probe (bodipy-labeled FY01; [91]) as assessed by SDS-PAGE. Worm extracts were normalized to 2 µg per labeling reaction. The fluorescent signal at 24 kDa is lost subsequent to exposure to CC-specific dsRNA. Data in (A) are the average of triplicate values (SD values were less the 5% of the mean). Representative data from two experiments are shown.
Mentions: Confirmation of RNAi at the protein level was sought for CC (Figure 6). A 6-day co-incubation of schistosomula and dsRNA targeting CC realized a knockdown of the translation product, measured either as an 80% decrease in the peptidolytic cleavage of the cathepsin C-selective substrate, GR-AMC (Figure 6A) or as a loss of the proteolytic signal (at approximately 24 kDa) using the specific active site-directed probe, bodipy-labeled FY01 (Figure 6B). Activity in the presence of GR-AMC was not inhibited by the general cysteine protease inhibitor, K11777, or by CA-074 and Z-Phe-Phe-DMK that target cathepsins B and L, respectively. This indicates that only cathepsin C activity was being measured. For CB1, a similar absence of protein was recorded after dsRNA application using immunoblotting with monospecific antibodies (not shown).

Bottom Line: Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets.Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi.RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days); electroporation provided no added benefit.

View Article: PubMed Central - PubMed

Affiliation: Sandler Center for Drug Discovery, California Institute for Quantitative Biosciences (QB3), University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT

Background: The possible emergence of resistance to the only available drug for schistosomiasis spurs drug discovery that has been recently incentivized by the availability of improved transcriptome and genome sequence information. Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets. To date, RNAi studies in schistosome stages infecting humans have focused on single (or up to 3) genes of interest. Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi.

Methodology/principal findings: We investigated in vitro the sensitivity and selectivity of RNAi using double-stranded (ds)RNA (approximately 500 bp) designed to target 11 Schistosoma mansoni genes that are expressed in different tissues; the gut, tegument and otherwise. Among the genes investigated were 5 that had been previously predicted to be essential for parasite survival. We employed mechanically transformed schistosomula that are relevant to parasitism in humans, amenable to screen automation and easier to obtain in greater numbers than adult parasites. The operational parameters investigated included defined culture media for optimal parasite maintenance, transfection strategy, time- and dose-dependency of RNAi, and dosing limits. Of 7 defined culture media tested, Basch Medium 169 was optimal for parasite maintenance. RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days); electroporation provided no added benefit. RNAi, including interference of more than one transcript, was selective to the gene target(s) within the pools of transcripts representative of each tissue. Concentrations of dsRNA above 90 µg/ml were directly toxic. RNAi efficiency was transcript-dependent (from 40 to >75% knockdown relative to controls) and this may have contributed to the lack of obvious phenotypes observed, even after prolonged incubations of 3 weeks. Within minutes of their mechanical preparation from cercariae, schistosomula accumulated fluorescent macromolecules in the gut indicating that the gut is an important route through which RNAi is expedited in the developing parasite.

Conclusions: Transient RNAi operates gene-selectively in S. mansoni newly transformed schistosomula yet the sensitivity of individual gene targets varies. These findings and the operational parameters defined will facilitate larger RNAi screens.

Show MeSH
Related in: MedlinePlus