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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

Direct toxicity by dsRNA to newly transformed schistosomula.Parasites were incubated in a total volume of 1 ml Basch Complete Medium with various concentrations of dsRNA (aliquots of 70 µl water) to Discosoma sp. mCherry [84]. At days 3 and 6, three photographic fields of view per dsRNA concentration were taken and those parasites displaying an obvious loss of shape and morphological integrity counted. The data shown in (A) are the averages of those counts expressed as a percentage of the total number of worms. SD values were less than 10% of the mean. (B) Images of parasites exposed to different concentrations of dsRNA by day 6 of the incubation: arrowheads indicate examples of distressed worms induced by 150 µg/ml dsRNA. Data are representative of two experiments. Similar results were observed with dsRNA to SmCB1. Scale bars represent 100 µm.
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pntd-0000850-g002: Direct toxicity by dsRNA to newly transformed schistosomula.Parasites were incubated in a total volume of 1 ml Basch Complete Medium with various concentrations of dsRNA (aliquots of 70 µl water) to Discosoma sp. mCherry [84]. At days 3 and 6, three photographic fields of view per dsRNA concentration were taken and those parasites displaying an obvious loss of shape and morphological integrity counted. The data shown in (A) are the averages of those counts expressed as a percentage of the total number of worms. SD values were less than 10% of the mean. (B) Images of parasites exposed to different concentrations of dsRNA by day 6 of the incubation: arrowheads indicate examples of distressed worms induced by 150 µg/ml dsRNA. Data are representative of two experiments. Similar results were observed with dsRNA to SmCB1. Scale bars represent 100 µm.

Mentions: Direct toxicity to schistosomula by dsRNA was evident as greater proportions of parasites that displayed abnormal phenotypes [76] such as rounding, darkening, slowed motility and outright death, or a combination thereof (Figure 2). Using dsRNA to mCherry (or CB1, not shown) and at the highest concentration tested of 210 µg/ml, more than 35% of the parasites appeared abnormal by days 3 and 6 of the incubation (Figure 2A). At a concentration of 150 µg/ml, a marked increase in the percentage of distressed parasites was measured between days 3 (12%) and 6 (35%; Figure 2A and 2B, lower panel). Concentrations below this (30 and 90 µg/ml) did not increase the percentage of abnormal parasites above the 12% background for the duration of the experiment. Thus, we interpret the data to suggest that the upper concentration limit for dsRNA, without incurring obvious parasite distress, is approximately 90 µg/ml. Operationally, we chose 30 µg/ml dsRNA to provide an increased margin of safety and based on the dsRNA dose-dependency tests for CB1 (Figure 3A), MetAP, NEC and PP2a (not shown) indicating that 30 µg/ml was sufficient for maximal RNAi. We chose a period of 6 days for co-incubating dsRNA and parasites as it was sufficient to register maximal transcriptional suppression of CB1, MetAP and PP2a (Figure 3B, C and D, respectively).


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)

Direct toxicity by dsRNA to newly transformed schistosomula.Parasites were incubated in a total volume of 1 ml Basch Complete Medium with various concentrations of dsRNA (aliquots of 70 µl water) to Discosoma sp. mCherry [84]. At days 3 and 6, three photographic fields of view per dsRNA concentration were taken and those parasites displaying an obvious loss of shape and morphological integrity counted. The data shown in (A) are the averages of those counts expressed as a percentage of the total number of worms. SD values were less than 10% of the mean. (B) Images of parasites exposed to different concentrations of dsRNA by day 6 of the incubation: arrowheads indicate examples of distressed worms induced by 150 µg/ml dsRNA. Data are representative of two experiments. Similar results were observed with dsRNA to SmCB1. Scale bars represent 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000850-g002: Direct toxicity by dsRNA to newly transformed schistosomula.Parasites were incubated in a total volume of 1 ml Basch Complete Medium with various concentrations of dsRNA (aliquots of 70 µl water) to Discosoma sp. mCherry [84]. At days 3 and 6, three photographic fields of view per dsRNA concentration were taken and those parasites displaying an obvious loss of shape and morphological integrity counted. The data shown in (A) are the averages of those counts expressed as a percentage of the total number of worms. SD values were less than 10% of the mean. (B) Images of parasites exposed to different concentrations of dsRNA by day 6 of the incubation: arrowheads indicate examples of distressed worms induced by 150 µg/ml dsRNA. Data are representative of two experiments. Similar results were observed with dsRNA to SmCB1. Scale bars represent 100 µm.
Mentions: Direct toxicity to schistosomula by dsRNA was evident as greater proportions of parasites that displayed abnormal phenotypes [76] such as rounding, darkening, slowed motility and outright death, or a combination thereof (Figure 2). Using dsRNA to mCherry (or CB1, not shown) and at the highest concentration tested of 210 µg/ml, more than 35% of the parasites appeared abnormal by days 3 and 6 of the incubation (Figure 2A). At a concentration of 150 µg/ml, a marked increase in the percentage of distressed parasites was measured between days 3 (12%) and 6 (35%; Figure 2A and 2B, lower panel). Concentrations below this (30 and 90 µg/ml) did not increase the percentage of abnormal parasites above the 12% background for the duration of the experiment. Thus, we interpret the data to suggest that the upper concentration limit for dsRNA, without incurring obvious parasite distress, is approximately 90 µg/ml. Operationally, we chose 30 µg/ml dsRNA to provide an increased margin of safety and based on the dsRNA dose-dependency tests for CB1 (Figure 3A), MetAP, NEC and PP2a (not shown) indicating that 30 µg/ml was sufficient for maximal RNAi. We chose a period of 6 days for co-incubating dsRNA and parasites as it was sufficient to register maximal transcriptional suppression of CB1, MetAP and PP2a (Figure 3B, C and D, respectively).

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