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Phenotypic screen of early-developing larvae of the blood fluke, schistosoma mansoni, using RNA interference.

Mourão MM, de Moraes Mourão M, Dinguirard N, Franco GR, Yoshino TP - PLoS Negl Trop Dis (2009)

Bottom Line: The size-reducing phenotype was observed in 11 of the 33 (33%) dsRNA treatment groups, and of these 11 phenotype-associated genes (superoxide dismutase, Smad1, RHO2, Smad2, Cav2A, ring box, GST26, calcineurin B, Smad4, lactate dehydrogenase and EF1alpha), only 6 demonstrated a significant and consistent knockdown of specific transcript expression.Of those, 6 genes exhibited consistent reductions in steady-state transcript levels, while expression level for the rest remained unchanged.Although RNAi holds great promise as a functional genomics tool for larval schistosomes, our finding of potential off-target or nonspecific effects of some dsRNA treatments and variable efficiencies in specific gene knockdown indicate a critical need for gene-specific testing and optimization as an essential part of experimental design, execution and data interpretation.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

ABSTRACT
RNA interference (RNAi) represents the only method currently available for manipulating gene-specific expression in Schistosoma spp., although application of this technology as a functional genomic profiling tool has yet to be explored. In the present study 32 genes, including antioxidants, transcription factors, cell signaling molecules and metabolic enzymes, were selected to determine if gene knockdown by RNAi was associated with morphologically definable phenotypic changes in early intramolluscan larval development. Transcript selection was based on their high expression in in vitro cultured S. mansoni primary sporocysts and/or their potential involvement in developmental processes. Miracidia were allowed to transform to sporocysts in the presence of synthesized double-stranded RNAs (dsRNAs) and cultivated for 7 days, during which time developing larvae were closely observed for phenotypic changes including failure/delay in transformation, loss of motility, altered growth and death. Of the phenotypes evaluated, only one was consistently detected; namely a reduction in sporocyst size based on length measurements. The size-reducing phenotype was observed in 11 of the 33 (33%) dsRNA treatment groups, and of these 11 phenotype-associated genes (superoxide dismutase, Smad1, RHO2, Smad2, Cav2A, ring box, GST26, calcineurin B, Smad4, lactate dehydrogenase and EF1alpha), only 6 demonstrated a significant and consistent knockdown of specific transcript expression. Unexpectedly one phenotype-linked gene, superoxide dismutase (SOD), was highly induced ( approximately 1600-fold) upon dsRNA exposure. Variation in dsRNA-mediated silencing effects also was evident in the group of sporocysts that lacked any definable phenotype. Out of 22 nonphenotype-expressing dsRNA treatments (myosin, PKCB, HEXBP, calcium channel, Sma2, RHO1, PKC receptor, DHHC, PepcK, calreticulin, calpain, Smeg, 14.3.3, K5, SPO1, SmZF1, fibrillarin, GST28, GPx, TPx1, TPx2 and TPx2/TPx1), 12 were assessed for the transcript levels. Of those, 6 genes exhibited consistent reductions in steady-state transcript levels, while expression level for the rest remained unchanged. Results demonstrate that the efficacy of dsRNA-treatment in producing consistent phenotypic changes and/or altered gene expression levels in S. mansoni sporocysts is highly dependent on the selected gene (or the specific dsRNA sequence used) and the timing of evaluation after treatment. Although RNAi holds great promise as a functional genomics tool for larval schistosomes, our finding of potential off-target or nonspecific effects of some dsRNA treatments and variable efficiencies in specific gene knockdown indicate a critical need for gene-specific testing and optimization as an essential part of experimental design, execution and data interpretation.

No MeSH data available.


Related in: MedlinePlus

In vitro cultured S. mansoni larvae 7 days post-dsRNA treatments.Brightfield photomicrographs of in vitro cultured Schistosoma mansoni sporocysts after 7 days of treatments with a specific GST26-dsRNA (A) compared to the control GFP-dsRNA (B), illustrating the effects of exposure to phenotype-inducing GST26-dsRNA on sporocyst lengths. Arrows indicate examples of shortened (white arrows) and normal elongate (black arrows) sporocysts measured in both treatments. Asterisks indicate rounded ciliated epidermal plates that were shed from the miracidial surface after transformation.
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pntd-0000502-g001: In vitro cultured S. mansoni larvae 7 days post-dsRNA treatments.Brightfield photomicrographs of in vitro cultured Schistosoma mansoni sporocysts after 7 days of treatments with a specific GST26-dsRNA (A) compared to the control GFP-dsRNA (B), illustrating the effects of exposure to phenotype-inducing GST26-dsRNA on sporocyst lengths. Arrows indicate examples of shortened (white arrows) and normal elongate (black arrows) sporocysts measured in both treatments. Asterisks indicate rounded ciliated epidermal plates that were shed from the miracidial surface after transformation.

Mentions: In our initial phenotype analysis of 24 different dsRNA treatments, no differences between specific dsRNA-treated larvae and controls (GFP dsRNA-treated and untreated sporocysts) were noted in miracidial transformation rates, larval motility or mortality during the in vitro cultivation period. The only notable phenotype observed in treated 7-day cultured sporocysts was an apparent greater number of small-sized or shortened sporocysts possibly involving a growth-related defect(s) (Fig. 1A, 1B). However, because sporocysts in a given culture population typically represented a range of sizes, live sporocyst images were captured, from which larval lengths were measured and digitally-analyzed using Metamorph software. Within each biological replicate, such measurements were taken for the dsRNA treatment groups and statistically compared to both the GFP dsRNA-treated and no treatment (blank) groups. For a given dsRNA to be identified as having a putative dsRNA-mediated effect, the median larval length had to significantly differ (P≤0.05) from both the GFP dsRNA and the blank controls in each of the biological replicates. Using these criteria, we observed significant decreases in parasite lengths in 9 of 24 dsRNAs in the first screening trial: Smad4, lactate dehydrogenase (LDH), Smad2, Cav2A, elongation factor 1α (EF1α), Smad1, RHO2, calcineurin B, and ring box (Fig. 2). Similar results were found in a second experimental series, which included nine additional dsRNA treatments. In this case, using the same criteria for significance, 2 of the 9 dsRNAs treatments (GST26 and SOD) exhibited a consistent size-related phenotype effect when compared to controls (Fig. 3). As before, greater frequency of shortened larvae was the only observable dsRNA-associated phenotype.


Phenotypic screen of early-developing larvae of the blood fluke, schistosoma mansoni, using RNA interference.

Mourão MM, de Moraes Mourão M, Dinguirard N, Franco GR, Yoshino TP - PLoS Negl Trop Dis (2009)

In vitro cultured S. mansoni larvae 7 days post-dsRNA treatments.Brightfield photomicrographs of in vitro cultured Schistosoma mansoni sporocysts after 7 days of treatments with a specific GST26-dsRNA (A) compared to the control GFP-dsRNA (B), illustrating the effects of exposure to phenotype-inducing GST26-dsRNA on sporocyst lengths. Arrows indicate examples of shortened (white arrows) and normal elongate (black arrows) sporocysts measured in both treatments. Asterisks indicate rounded ciliated epidermal plates that were shed from the miracidial surface after transformation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2719580&req=5

pntd-0000502-g001: In vitro cultured S. mansoni larvae 7 days post-dsRNA treatments.Brightfield photomicrographs of in vitro cultured Schistosoma mansoni sporocysts after 7 days of treatments with a specific GST26-dsRNA (A) compared to the control GFP-dsRNA (B), illustrating the effects of exposure to phenotype-inducing GST26-dsRNA on sporocyst lengths. Arrows indicate examples of shortened (white arrows) and normal elongate (black arrows) sporocysts measured in both treatments. Asterisks indicate rounded ciliated epidermal plates that were shed from the miracidial surface after transformation.
Mentions: In our initial phenotype analysis of 24 different dsRNA treatments, no differences between specific dsRNA-treated larvae and controls (GFP dsRNA-treated and untreated sporocysts) were noted in miracidial transformation rates, larval motility or mortality during the in vitro cultivation period. The only notable phenotype observed in treated 7-day cultured sporocysts was an apparent greater number of small-sized or shortened sporocysts possibly involving a growth-related defect(s) (Fig. 1A, 1B). However, because sporocysts in a given culture population typically represented a range of sizes, live sporocyst images were captured, from which larval lengths were measured and digitally-analyzed using Metamorph software. Within each biological replicate, such measurements were taken for the dsRNA treatment groups and statistically compared to both the GFP dsRNA-treated and no treatment (blank) groups. For a given dsRNA to be identified as having a putative dsRNA-mediated effect, the median larval length had to significantly differ (P≤0.05) from both the GFP dsRNA and the blank controls in each of the biological replicates. Using these criteria, we observed significant decreases in parasite lengths in 9 of 24 dsRNAs in the first screening trial: Smad4, lactate dehydrogenase (LDH), Smad2, Cav2A, elongation factor 1α (EF1α), Smad1, RHO2, calcineurin B, and ring box (Fig. 2). Similar results were found in a second experimental series, which included nine additional dsRNA treatments. In this case, using the same criteria for significance, 2 of the 9 dsRNAs treatments (GST26 and SOD) exhibited a consistent size-related phenotype effect when compared to controls (Fig. 3). As before, greater frequency of shortened larvae was the only observable dsRNA-associated phenotype.

Bottom Line: The size-reducing phenotype was observed in 11 of the 33 (33%) dsRNA treatment groups, and of these 11 phenotype-associated genes (superoxide dismutase, Smad1, RHO2, Smad2, Cav2A, ring box, GST26, calcineurin B, Smad4, lactate dehydrogenase and EF1alpha), only 6 demonstrated a significant and consistent knockdown of specific transcript expression.Of those, 6 genes exhibited consistent reductions in steady-state transcript levels, while expression level for the rest remained unchanged.Although RNAi holds great promise as a functional genomics tool for larval schistosomes, our finding of potential off-target or nonspecific effects of some dsRNA treatments and variable efficiencies in specific gene knockdown indicate a critical need for gene-specific testing and optimization as an essential part of experimental design, execution and data interpretation.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

ABSTRACT
RNA interference (RNAi) represents the only method currently available for manipulating gene-specific expression in Schistosoma spp., although application of this technology as a functional genomic profiling tool has yet to be explored. In the present study 32 genes, including antioxidants, transcription factors, cell signaling molecules and metabolic enzymes, were selected to determine if gene knockdown by RNAi was associated with morphologically definable phenotypic changes in early intramolluscan larval development. Transcript selection was based on their high expression in in vitro cultured S. mansoni primary sporocysts and/or their potential involvement in developmental processes. Miracidia were allowed to transform to sporocysts in the presence of synthesized double-stranded RNAs (dsRNAs) and cultivated for 7 days, during which time developing larvae were closely observed for phenotypic changes including failure/delay in transformation, loss of motility, altered growth and death. Of the phenotypes evaluated, only one was consistently detected; namely a reduction in sporocyst size based on length measurements. The size-reducing phenotype was observed in 11 of the 33 (33%) dsRNA treatment groups, and of these 11 phenotype-associated genes (superoxide dismutase, Smad1, RHO2, Smad2, Cav2A, ring box, GST26, calcineurin B, Smad4, lactate dehydrogenase and EF1alpha), only 6 demonstrated a significant and consistent knockdown of specific transcript expression. Unexpectedly one phenotype-linked gene, superoxide dismutase (SOD), was highly induced ( approximately 1600-fold) upon dsRNA exposure. Variation in dsRNA-mediated silencing effects also was evident in the group of sporocysts that lacked any definable phenotype. Out of 22 nonphenotype-expressing dsRNA treatments (myosin, PKCB, HEXBP, calcium channel, Sma2, RHO1, PKC receptor, DHHC, PepcK, calreticulin, calpain, Smeg, 14.3.3, K5, SPO1, SmZF1, fibrillarin, GST28, GPx, TPx1, TPx2 and TPx2/TPx1), 12 were assessed for the transcript levels. Of those, 6 genes exhibited consistent reductions in steady-state transcript levels, while expression level for the rest remained unchanged. Results demonstrate that the efficacy of dsRNA-treatment in producing consistent phenotypic changes and/or altered gene expression levels in S. mansoni sporocysts is highly dependent on the selected gene (or the specific dsRNA sequence used) and the timing of evaluation after treatment. Although RNAi holds great promise as a functional genomics tool for larval schistosomes, our finding of potential off-target or nonspecific effects of some dsRNA treatments and variable efficiencies in specific gene knockdown indicate a critical need for gene-specific testing and optimization as an essential part of experimental design, execution and data interpretation.

No MeSH data available.


Related in: MedlinePlus