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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 gut-associated gene transcripts in newly transformed schistosomula is robust and selective.Parasites were incubated for 6 days with 30 µg/ml of each of the indicated dsRNA preparations. RNAi was measured by qRT-PCR and data expressed relative to those following parasite exposure to schistosome-unspecific mCherry dsRNA. A negative control (−ve) in which parasites had not been exposed to dsRNA was also included. For each of the targeted genes, (A) SmCB1, (B) SmCC and (C) SmCD, the RNAi effect was selective among the pool of gut-associated transcripts examined. Selective RNAi of SmCB1 was also achieved with 30 µg/ml Cy5-linked dsRNA. Each sample was tested in duplicate and representative data from two experiments are shown.
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pntd-0000850-g004: RNAi of gut-associated gene transcripts in newly transformed schistosomula is robust and selective.Parasites were incubated for 6 days with 30 µg/ml of each of the indicated dsRNA preparations. RNAi was measured by qRT-PCR and data expressed relative to those following parasite exposure to schistosome-unspecific mCherry dsRNA. A negative control (−ve) in which parasites had not been exposed to dsRNA was also included. For each of the targeted genes, (A) SmCB1, (B) SmCC and (C) SmCD, the RNAi effect was selective among the pool of gut-associated transcripts examined. Selective RNAi of SmCB1 was also achieved with 30 µg/ml Cy5-linked dsRNA. Each sample was tested in duplicate and representative data from two experiments are shown.

Mentions: Transcripts for the gut-associated proteins CB1, CC and CD are sensitive to RNAi and are robustly suppressed by >75% (Figure 4A, B and C, respectively). Importantly, the effect is selective for the intended mRNA within the pool of gut transcripts examined. Attachment of the fluorescent Cy5 label to CB1-(Figure 4A) or CC-(not shown) specific dsRNA does not interfere with suppression, or the degree of suppression, of the cognate mRNA. With non-labeled dsRNA targeting CB1, CC and CD, the RNAi effect was long-term, remaining constant out to 3 weeks of incubation without exchanging either culture medium or dsRNA (not shown). For CB1, long-term suppression (>30 days of incubation) of transcript has been previously noted for schistosomula exposed at 3 h [53] and 7 days post-transformation [96].


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 gut-associated gene transcripts in newly transformed schistosomula is robust and selective.Parasites were incubated for 6 days with 30 µg/ml of each of the indicated dsRNA preparations. RNAi was measured by qRT-PCR and data expressed relative to those following parasite exposure to schistosome-unspecific mCherry dsRNA. A negative control (−ve) in which parasites had not been exposed to dsRNA was also included. For each of the targeted genes, (A) SmCB1, (B) SmCC and (C) SmCD, the RNAi effect was selective among the pool of gut-associated transcripts examined. Selective RNAi of SmCB1 was also achieved with 30 µg/ml Cy5-linked dsRNA. Each sample was tested in duplicate and representative data from two experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000850-g004: RNAi of gut-associated gene transcripts in newly transformed schistosomula is robust and selective.Parasites were incubated for 6 days with 30 µg/ml of each of the indicated dsRNA preparations. RNAi was measured by qRT-PCR and data expressed relative to those following parasite exposure to schistosome-unspecific mCherry dsRNA. A negative control (−ve) in which parasites had not been exposed to dsRNA was also included. For each of the targeted genes, (A) SmCB1, (B) SmCC and (C) SmCD, the RNAi effect was selective among the pool of gut-associated transcripts examined. Selective RNAi of SmCB1 was also achieved with 30 µg/ml Cy5-linked dsRNA. Each sample was tested in duplicate and representative data from two experiments are shown.
Mentions: Transcripts for the gut-associated proteins CB1, CC and CD are sensitive to RNAi and are robustly suppressed by >75% (Figure 4A, B and C, respectively). Importantly, the effect is selective for the intended mRNA within the pool of gut transcripts examined. Attachment of the fluorescent Cy5 label to CB1-(Figure 4A) or CC-(not shown) specific dsRNA does not interfere with suppression, or the degree of suppression, of the cognate mRNA. With non-labeled dsRNA targeting CB1, CC and CD, the RNAi effect was long-term, remaining constant out to 3 weeks of incubation without exchanging either culture medium or dsRNA (not shown). For CB1, long-term suppression (>30 days of incubation) of transcript has been previously noted for schistosomula exposed at 3 h [53] and 7 days post-transformation [96].

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