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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

Transcript levels in S. mansoni sporocysts at different times post-dsRNA treatment.Time-course of steady-state transcript levels was assessed in sporocysts treated with dsRNAs under culture conditions. Sporocysts were treated with dsRNA-EF1α, -calcineurin B, -SOD, -lactate dehydrogenase, -RHO2, Smad2, -Smad4, -myosin and -ring box for 2 days (stippled bars) or 4 days (gray bars), and compared to 7 day dsRNA treatment effects (black bars). Transcript levels were assessed by q-PCR at each time and compared to its matched GFP-dsRNA control. For each dsRNA tested, data are represented as mean fold-difference (+/−S.E.) relative to the GFP control (1.00). However, statistical analyses were based on raw q-PCR values using the ΔΔCt method followed by statistical analysis using the Mann-Whitney U-test, N = 4, *P≤0.05. Two-day comparisons (stippled bars): dsRNA-EF1a/GFP, P = 0.028; -calcineurin B/GFP, P = 0.021; -SOD/GFP, P = 0.015; -lactate dehydrogenase/GFP, P = 0.041; - RHO2/GFP, P = 0.021; –Smad4/GFP, P = 0.028; -ring box/GFP, P = 0.3; -Smad2/GFP, P = 1.0; and -myosin/GFP, P = 0.059. Four-day comparisons (gray bars): dsRNA-EF1a/GFP, P = 0.0319; -calcineurin B/GFP, P = 0.03; -SOD/GFP, P = 0.028; -lactate dehydrogenase/GFP, P = 0.029; - RHO2/GFP, P = 0.021; –Smad4/GFP, P = 0.0286; -ring box/GFP, P = 0.884; -Smad2/GFP, P = 0.98; and -myosin/GFP, P = 0.9. Data for 7 day dsRNA treatments (black bars) were taken from identically performed experiments (data shown previously in Fig. 5), and are reproduced in Fig. 6 for graphic comparisons only. Statistics for this group of genes are provided in the Fig. 5 legend.
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pntd-0000502-g006: Transcript levels in S. mansoni sporocysts at different times post-dsRNA treatment.Time-course of steady-state transcript levels was assessed in sporocysts treated with dsRNAs under culture conditions. Sporocysts were treated with dsRNA-EF1α, -calcineurin B, -SOD, -lactate dehydrogenase, -RHO2, Smad2, -Smad4, -myosin and -ring box for 2 days (stippled bars) or 4 days (gray bars), and compared to 7 day dsRNA treatment effects (black bars). Transcript levels were assessed by q-PCR at each time and compared to its matched GFP-dsRNA control. For each dsRNA tested, data are represented as mean fold-difference (+/−S.E.) relative to the GFP control (1.00). However, statistical analyses were based on raw q-PCR values using the ΔΔCt method followed by statistical analysis using the Mann-Whitney U-test, N = 4, *P≤0.05. Two-day comparisons (stippled bars): dsRNA-EF1a/GFP, P = 0.028; -calcineurin B/GFP, P = 0.021; -SOD/GFP, P = 0.015; -lactate dehydrogenase/GFP, P = 0.041; - RHO2/GFP, P = 0.021; –Smad4/GFP, P = 0.028; -ring box/GFP, P = 0.3; -Smad2/GFP, P = 1.0; and -myosin/GFP, P = 0.059. Four-day comparisons (gray bars): dsRNA-EF1a/GFP, P = 0.0319; -calcineurin B/GFP, P = 0.03; -SOD/GFP, P = 0.028; -lactate dehydrogenase/GFP, P = 0.029; - RHO2/GFP, P = 0.021; –Smad4/GFP, P = 0.0286; -ring box/GFP, P = 0.884; -Smad2/GFP, P = 0.98; and -myosin/GFP, P = 0.9. Data for 7 day dsRNA treatments (black bars) were taken from identically performed experiments (data shown previously in Fig. 5), and are reproduced in Fig. 6 for graphic comparisons only. Statistics for this group of genes are provided in the Fig. 5 legend.

Mentions: A comparison was made between dsRNA species that produced a detectable phenotype and those generating a significant transcript knockdown (or induced expression) in an attempt to directly correlate phenotype and gene expression. Notably, only 7 of the 11 target dsRNA-treatments that produced a “shortened” larval phenotype presented a significant alteration in transcript levels (Smad4, lactate dehydrogenase, Cav2A, EF1α, calcineurin B, GST26 and SOD) when compared to dsRNA-GFP treated sporocysts (Fig. 5). Smad4 and LDH dsRNA treatments showed a small, but consistently significant 15% decrease, while Cav2A, calcineurin B, EF1α and GST26 exhibited knockdowns of 42%, 65%, 70% and 85%, respectively. SOD, whose transcript levels were dramatically increased in dsRNA-treated sporocysts, also was phenotype-associated. In addition, as noted in Figure 5, 6 dsRNA treatment groups that did not exhibit significant or consistent changes in larval length expressed significantly lower transcript levels than controls ranging from an approximately 30% (SmZF1) to 75% (fibrillarin, GST28, GPx, TPx1, and TPx2) after 7 days of exposure. No changes in transcript levels were observed for phenotype-associated Smad1, Smad2, RHO2 and ring box dsRNA treatments and phenotype-non-associated myosin, PKCB, Pepck, calpain, 14.3.3 protein, glycoprotein K5, and SPO1 dsRNAs.


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)

Transcript levels in S. mansoni sporocysts at different times post-dsRNA treatment.Time-course of steady-state transcript levels was assessed in sporocysts treated with dsRNAs under culture conditions. Sporocysts were treated with dsRNA-EF1α, -calcineurin B, -SOD, -lactate dehydrogenase, -RHO2, Smad2, -Smad4, -myosin and -ring box for 2 days (stippled bars) or 4 days (gray bars), and compared to 7 day dsRNA treatment effects (black bars). Transcript levels were assessed by q-PCR at each time and compared to its matched GFP-dsRNA control. For each dsRNA tested, data are represented as mean fold-difference (+/−S.E.) relative to the GFP control (1.00). However, statistical analyses were based on raw q-PCR values using the ΔΔCt method followed by statistical analysis using the Mann-Whitney U-test, N = 4, *P≤0.05. Two-day comparisons (stippled bars): dsRNA-EF1a/GFP, P = 0.028; -calcineurin B/GFP, P = 0.021; -SOD/GFP, P = 0.015; -lactate dehydrogenase/GFP, P = 0.041; - RHO2/GFP, P = 0.021; –Smad4/GFP, P = 0.028; -ring box/GFP, P = 0.3; -Smad2/GFP, P = 1.0; and -myosin/GFP, P = 0.059. Four-day comparisons (gray bars): dsRNA-EF1a/GFP, P = 0.0319; -calcineurin B/GFP, P = 0.03; -SOD/GFP, P = 0.028; -lactate dehydrogenase/GFP, P = 0.029; - RHO2/GFP, P = 0.021; –Smad4/GFP, P = 0.0286; -ring box/GFP, P = 0.884; -Smad2/GFP, P = 0.98; and -myosin/GFP, P = 0.9. Data for 7 day dsRNA treatments (black bars) were taken from identically performed experiments (data shown previously in Fig. 5), and are reproduced in Fig. 6 for graphic comparisons only. Statistics for this group of genes are provided in the Fig. 5 legend.
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Related In: Results  -  Collection

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

pntd-0000502-g006: Transcript levels in S. mansoni sporocysts at different times post-dsRNA treatment.Time-course of steady-state transcript levels was assessed in sporocysts treated with dsRNAs under culture conditions. Sporocysts were treated with dsRNA-EF1α, -calcineurin B, -SOD, -lactate dehydrogenase, -RHO2, Smad2, -Smad4, -myosin and -ring box for 2 days (stippled bars) or 4 days (gray bars), and compared to 7 day dsRNA treatment effects (black bars). Transcript levels were assessed by q-PCR at each time and compared to its matched GFP-dsRNA control. For each dsRNA tested, data are represented as mean fold-difference (+/−S.E.) relative to the GFP control (1.00). However, statistical analyses were based on raw q-PCR values using the ΔΔCt method followed by statistical analysis using the Mann-Whitney U-test, N = 4, *P≤0.05. Two-day comparisons (stippled bars): dsRNA-EF1a/GFP, P = 0.028; -calcineurin B/GFP, P = 0.021; -SOD/GFP, P = 0.015; -lactate dehydrogenase/GFP, P = 0.041; - RHO2/GFP, P = 0.021; –Smad4/GFP, P = 0.028; -ring box/GFP, P = 0.3; -Smad2/GFP, P = 1.0; and -myosin/GFP, P = 0.059. Four-day comparisons (gray bars): dsRNA-EF1a/GFP, P = 0.0319; -calcineurin B/GFP, P = 0.03; -SOD/GFP, P = 0.028; -lactate dehydrogenase/GFP, P = 0.029; - RHO2/GFP, P = 0.021; –Smad4/GFP, P = 0.0286; -ring box/GFP, P = 0.884; -Smad2/GFP, P = 0.98; and -myosin/GFP, P = 0.9. Data for 7 day dsRNA treatments (black bars) were taken from identically performed experiments (data shown previously in Fig. 5), and are reproduced in Fig. 6 for graphic comparisons only. Statistics for this group of genes are provided in the Fig. 5 legend.
Mentions: A comparison was made between dsRNA species that produced a detectable phenotype and those generating a significant transcript knockdown (or induced expression) in an attempt to directly correlate phenotype and gene expression. Notably, only 7 of the 11 target dsRNA-treatments that produced a “shortened” larval phenotype presented a significant alteration in transcript levels (Smad4, lactate dehydrogenase, Cav2A, EF1α, calcineurin B, GST26 and SOD) when compared to dsRNA-GFP treated sporocysts (Fig. 5). Smad4 and LDH dsRNA treatments showed a small, but consistently significant 15% decrease, while Cav2A, calcineurin B, EF1α and GST26 exhibited knockdowns of 42%, 65%, 70% and 85%, respectively. SOD, whose transcript levels were dramatically increased in dsRNA-treated sporocysts, also was phenotype-associated. In addition, as noted in Figure 5, 6 dsRNA treatment groups that did not exhibit significant or consistent changes in larval length expressed significantly lower transcript levels than controls ranging from an approximately 30% (SmZF1) to 75% (fibrillarin, GST28, GPx, TPx1, and TPx2) after 7 days of exposure. No changes in transcript levels were observed for phenotype-associated Smad1, Smad2, RHO2 and ring box dsRNA treatments and phenotype-non-associated myosin, PKCB, Pepck, calpain, 14.3.3 protein, glycoprotein K5, and SPO1 dsRNAs.

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