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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

Observations of differential expression of EF1α protein levels in S. mansoni sporocysts after dsRNA treatments.Immunfluorescence photomicrographs of GFP dsRNA (control; A) and EF1α dsRNA (B) treated sporocysts showing EF1α protein-knockdown post RNAi treatments. Larvae were cultured with dsRNAs for 7 days and fixed prior to treatment with anti-EF1α antibody and Alexa 488-conjugated secondary antibody. Strong immunoreactivity (green fluorescence) is distributed among various cells and tissues within interior of control sporocysts (A), compared to only weak reactivity in EF1α dsRNA-treated sporocysts (B). Confocal images; 400×. N = 2.
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pntd-0000502-g008: Observations of differential expression of EF1α protein levels in S. mansoni sporocysts after dsRNA treatments.Immunfluorescence photomicrographs of GFP dsRNA (control; A) and EF1α dsRNA (B) treated sporocysts showing EF1α protein-knockdown post RNAi treatments. Larvae were cultured with dsRNAs for 7 days and fixed prior to treatment with anti-EF1α antibody and Alexa 488-conjugated secondary antibody. Strong immunoreactivity (green fluorescence) is distributed among various cells and tissues within interior of control sporocysts (A), compared to only weak reactivity in EF1α dsRNA-treated sporocysts (B). Confocal images; 400×. N = 2.

Mentions: Finally, because we had available an antibody that was specifically reactive to the S. mansoni EF1α protein, we assessed the effect of EF1α dsRNA treatment on EF1α protein levels using Western blot and immunofluorescence imaging. Western blot analysis clearly showed that EF1α dsRNA-treated 7-day sporocyst extracts were significantly reduced in EF1α protein (50 kDa band) compared to the GFP dsRNA-treated control group (Fig. 7). The presence of a 25 kDa GST26 band (used as an antibody specificity and loading control) in both the EF1α and GFP dsRNA-treated samples suggested both a specific EF1α transcript silencing and associated protein knockout (Fig. 7). This result was quantitatively confirmed by densitometry showing that, following normalization of transcripts to the loading control, EF1α protein was highly reduced by >80% in the EF1α dsRNA-treated sample compared to the GFP dsRNA control. Confocal immunolocalization of EF1α in intact dsRNA-treated sporocysts was consistent with the Western blot analysis: EF1α dsRNA-treated larvae displayed little immunoreactivity, while abundant anti-EF1α-reactivity was evident within cells and parenchymal tissues of GFP dsRNA-treated sporocysts (Fig. 8).


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)

Observations of differential expression of EF1α protein levels in S. mansoni sporocysts after dsRNA treatments.Immunfluorescence photomicrographs of GFP dsRNA (control; A) and EF1α dsRNA (B) treated sporocysts showing EF1α protein-knockdown post RNAi treatments. Larvae were cultured with dsRNAs for 7 days and fixed prior to treatment with anti-EF1α antibody and Alexa 488-conjugated secondary antibody. Strong immunoreactivity (green fluorescence) is distributed among various cells and tissues within interior of control sporocysts (A), compared to only weak reactivity in EF1α dsRNA-treated sporocysts (B). Confocal images; 400×. N = 2.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000502-g008: Observations of differential expression of EF1α protein levels in S. mansoni sporocysts after dsRNA treatments.Immunfluorescence photomicrographs of GFP dsRNA (control; A) and EF1α dsRNA (B) treated sporocysts showing EF1α protein-knockdown post RNAi treatments. Larvae were cultured with dsRNAs for 7 days and fixed prior to treatment with anti-EF1α antibody and Alexa 488-conjugated secondary antibody. Strong immunoreactivity (green fluorescence) is distributed among various cells and tissues within interior of control sporocysts (A), compared to only weak reactivity in EF1α dsRNA-treated sporocysts (B). Confocal images; 400×. N = 2.
Mentions: Finally, because we had available an antibody that was specifically reactive to the S. mansoni EF1α protein, we assessed the effect of EF1α dsRNA treatment on EF1α protein levels using Western blot and immunofluorescence imaging. Western blot analysis clearly showed that EF1α dsRNA-treated 7-day sporocyst extracts were significantly reduced in EF1α protein (50 kDa band) compared to the GFP dsRNA-treated control group (Fig. 7). The presence of a 25 kDa GST26 band (used as an antibody specificity and loading control) in both the EF1α and GFP dsRNA-treated samples suggested both a specific EF1α transcript silencing and associated protein knockout (Fig. 7). This result was quantitatively confirmed by densitometry showing that, following normalization of transcripts to the loading control, EF1α protein was highly reduced by >80% in the EF1α dsRNA-treated sample compared to the GFP dsRNA control. Confocal immunolocalization of EF1α in intact dsRNA-treated sporocysts was consistent with the Western blot analysis: EF1α dsRNA-treated larvae displayed little immunoreactivity, while abundant anti-EF1α-reactivity was evident within cells and parenchymal tissues of GFP dsRNA-treated sporocysts (Fig. 8).

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