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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

The gut of newly transformed schistosomula is active and likely facilitates RNAi.After their preparation from cercariae, schistosomula were incubated with (A) 30 µg/ml Cy5-linked dsRNA targeting SmCB1, (B) 200 µg/ml recombinant mCherry protein or (C) 5 µl washed and packed human erythrocytes. For each image, the anterior end of the parasite is leftmost and times stated are post-preparation from cercariae. Both the Cy5-linked dsRNA and mCherry emit a strong fluorescent signal that quickly accumulates in the gut including in the two terminal cecal chambers. The arrow and arrowheads indicate the acetabular ‘gland ducts’ and acetabular ‘glands’, both being distinct from the developing gut. In (C), brown material, derived from the digestion of hemoglobin, highlights the parasite gut. Images were captured using: (A) a Zeiss LSM 510 META confocal microscope and a 633 nm laser for excitation; (B) a Zeiss Axiovert 40 CFL inverted microscope using the Filter Set 20 (ex-546; em-575-640) and connected to a Zeiss AxioCam HRc digital camera and (C) a Zeiss Axiovert 40 C inverted microscope connected to a Zeiss AxioCam MRc. Both AxioCAms were operated by AxioVision 40, version 4.5.0.0, software. Scale bars represent 20 µm.
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pntd-0000850-g010: The gut of newly transformed schistosomula is active and likely facilitates RNAi.After their preparation from cercariae, schistosomula were incubated with (A) 30 µg/ml Cy5-linked dsRNA targeting SmCB1, (B) 200 µg/ml recombinant mCherry protein or (C) 5 µl washed and packed human erythrocytes. For each image, the anterior end of the parasite is leftmost and times stated are post-preparation from cercariae. Both the Cy5-linked dsRNA and mCherry emit a strong fluorescent signal that quickly accumulates in the gut including in the two terminal cecal chambers. The arrow and arrowheads indicate the acetabular ‘gland ducts’ and acetabular ‘glands’, both being distinct from the developing gut. In (C), brown material, derived from the digestion of hemoglobin, highlights the parasite gut. Images were captured using: (A) a Zeiss LSM 510 META confocal microscope and a 633 nm laser for excitation; (B) a Zeiss Axiovert 40 CFL inverted microscope using the Filter Set 20 (ex-546; em-575-640) and connected to a Zeiss AxioCam HRc digital camera and (C) a Zeiss Axiovert 40 C inverted microscope connected to a Zeiss AxioCam MRc. Both AxioCAms were operated by AxioVision 40, version 4.5.0.0, software. Scale bars represent 20 µm.

Mentions: Given the above data suggesting that simple co-incubation of newly transformed parasites and dsRNA is sufficient to mediate RNAi, we then asked the question whether the gut serves as a route of entry for dsRNA into the schistosome. The question is relevant in view of previous discussions to the effect that the parasite mouth is not open until about day 7 of the incubation (i.e., indicative of a non-functioning gut) and, therefore, cannot facilitate RNAi in younger schistosomula [96]. Using Cy5-labeled dsRNA to CB1, CC or mCherry, we demonstrate that the gut of schistosomula takes up and concentrates exogenous material within minutes of their mechanical preparation (Figure 10; results with CB1 dsRNA shown). Accumulation of the dye was evident along the gut and in the two terminal cecal chambers by 90 min post-transformation (Figure 10A). At the same time-point, a fluorescent signal from accumulated mCherry protein could be detected (Figure 10B). For both probes, the signal remained visible during the incubation period of 6 days illuminating the gut ceca as they elongated posteriorly over time. Consistent with this rapid onset of gut activity, we could already measure RNAi of CB1 at between 2 and 4 h post-incubation with dsRNA, an effect that increased as a function of time (Figure 3B). Finally, we could detect hemoglobin-derived pigment in the gut of 2 day-old schistosomula co-incubated with erythrocytes (Figure 10C). Taken together, the results suggest that the schistosomulum gut is functionally active within minutes of their preparation from cercariae and seems a likely route through which RNAi is mediated.


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)

The gut of newly transformed schistosomula is active and likely facilitates RNAi.After their preparation from cercariae, schistosomula were incubated with (A) 30 µg/ml Cy5-linked dsRNA targeting SmCB1, (B) 200 µg/ml recombinant mCherry protein or (C) 5 µl washed and packed human erythrocytes. For each image, the anterior end of the parasite is leftmost and times stated are post-preparation from cercariae. Both the Cy5-linked dsRNA and mCherry emit a strong fluorescent signal that quickly accumulates in the gut including in the two terminal cecal chambers. The arrow and arrowheads indicate the acetabular ‘gland ducts’ and acetabular ‘glands’, both being distinct from the developing gut. In (C), brown material, derived from the digestion of hemoglobin, highlights the parasite gut. Images were captured using: (A) a Zeiss LSM 510 META confocal microscope and a 633 nm laser for excitation; (B) a Zeiss Axiovert 40 CFL inverted microscope using the Filter Set 20 (ex-546; em-575-640) and connected to a Zeiss AxioCam HRc digital camera and (C) a Zeiss Axiovert 40 C inverted microscope connected to a Zeiss AxioCam MRc. Both AxioCAms were operated by AxioVision 40, version 4.5.0.0, software. Scale bars represent 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000850-g010: The gut of newly transformed schistosomula is active and likely facilitates RNAi.After their preparation from cercariae, schistosomula were incubated with (A) 30 µg/ml Cy5-linked dsRNA targeting SmCB1, (B) 200 µg/ml recombinant mCherry protein or (C) 5 µl washed and packed human erythrocytes. For each image, the anterior end of the parasite is leftmost and times stated are post-preparation from cercariae. Both the Cy5-linked dsRNA and mCherry emit a strong fluorescent signal that quickly accumulates in the gut including in the two terminal cecal chambers. The arrow and arrowheads indicate the acetabular ‘gland ducts’ and acetabular ‘glands’, both being distinct from the developing gut. In (C), brown material, derived from the digestion of hemoglobin, highlights the parasite gut. Images were captured using: (A) a Zeiss LSM 510 META confocal microscope and a 633 nm laser for excitation; (B) a Zeiss Axiovert 40 CFL inverted microscope using the Filter Set 20 (ex-546; em-575-640) and connected to a Zeiss AxioCam HRc digital camera and (C) a Zeiss Axiovert 40 C inverted microscope connected to a Zeiss AxioCam MRc. Both AxioCAms were operated by AxioVision 40, version 4.5.0.0, software. Scale bars represent 20 µm.
Mentions: Given the above data suggesting that simple co-incubation of newly transformed parasites and dsRNA is sufficient to mediate RNAi, we then asked the question whether the gut serves as a route of entry for dsRNA into the schistosome. The question is relevant in view of previous discussions to the effect that the parasite mouth is not open until about day 7 of the incubation (i.e., indicative of a non-functioning gut) and, therefore, cannot facilitate RNAi in younger schistosomula [96]. Using Cy5-labeled dsRNA to CB1, CC or mCherry, we demonstrate that the gut of schistosomula takes up and concentrates exogenous material within minutes of their mechanical preparation (Figure 10; results with CB1 dsRNA shown). Accumulation of the dye was evident along the gut and in the two terminal cecal chambers by 90 min post-transformation (Figure 10A). At the same time-point, a fluorescent signal from accumulated mCherry protein could be detected (Figure 10B). For both probes, the signal remained visible during the incubation period of 6 days illuminating the gut ceca as they elongated posteriorly over time. Consistent with this rapid onset of gut activity, we could already measure RNAi of CB1 at between 2 and 4 h post-incubation with dsRNA, an effect that increased as a function of time (Figure 3B). Finally, we could detect hemoglobin-derived pigment in the gut of 2 day-old schistosomula co-incubated with erythrocytes (Figure 10C). Taken together, the results suggest that the schistosomulum gut is functionally active within minutes of their preparation from cercariae and seems a likely route through which RNAi is mediated.

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