Efficient <it>in vitro </it>RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, <it>Brugia malayi</it>
<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode <it>Caenorhabditis elegans&l...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2012-01-01
|
| Series: | Parasites & Vectors |
| Subjects: | |
| Online Access: | http://www.parasitesandvectors.com/content/5/1/16 |
| _version_ | 1818519818886709248 |
|---|---|
| author | Landmann Frédéric Foster Jeremy M Slatko Barton E Sullivan William |
| author_facet | Landmann Frédéric Foster Jeremy M Slatko Barton E Sullivan William |
| author_sort | Landmann Frédéric |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode <it>Caenorhabditis elegans</it>, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from <it>C. elegans</it>.</p> <p>Results</p> <p>We report improved and effective <it>in vitro </it>RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, <it>Brugia malayi</it>. The cellular disorganization observed in <it>B. malayi </it>embryos following RNAi targeting the genes encoding γ-tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their <it>C. elegans </it>orthologs. Targeting the <it>B. malayi </it>cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in <it>C. elegans</it>. Cellular phenotypes induced by our <it>in vitro </it>RNAi procedure can be observed by immunofluorescence in as little as one week.</p> <p>Conclusions</p> <p>We observed cytological defects following RNAi targeting all seven <it>B. malayi </it>transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism <it>C. elegans</it>. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis.</p> |
| first_indexed | 2024-12-11T01:29:09Z |
| format | Article |
| id | doaj.art-5fea525c12b44bc99d386d8cafde6e94 |
| institution | Directory Open Access Journal |
| issn | 1756-3305 |
| language | English |
| last_indexed | 2024-12-11T01:29:09Z |
| publishDate | 2012-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Parasites & Vectors |
| spelling | doaj.art-5fea525c12b44bc99d386d8cafde6e942022-12-22T01:25:26ZengBMCParasites & Vectors1756-33052012-01-01511610.1186/1756-3305-5-16Efficient <it>in vitro </it>RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, <it>Brugia malayi</it>Landmann FrédéricFoster Jeremy MSlatko Barton ESullivan William<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode <it>Caenorhabditis elegans</it>, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from <it>C. elegans</it>.</p> <p>Results</p> <p>We report improved and effective <it>in vitro </it>RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, <it>Brugia malayi</it>. The cellular disorganization observed in <it>B. malayi </it>embryos following RNAi targeting the genes encoding γ-tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their <it>C. elegans </it>orthologs. Targeting the <it>B. malayi </it>cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in <it>C. elegans</it>. Cellular phenotypes induced by our <it>in vitro </it>RNAi procedure can be observed by immunofluorescence in as little as one week.</p> <p>Conclusions</p> <p>We observed cytological defects following RNAi targeting all seven <it>B. malayi </it>transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism <it>C. elegans</it>. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis.</p>http://www.parasitesandvectors.com/content/5/1/16RNAinematodeimmunostaining<it>Brugia</it>filaria |
| spellingShingle | Landmann Frédéric Foster Jeremy M Slatko Barton E Sullivan William Efficient <it>in vitro </it>RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, <it>Brugia malayi</it> Parasites & Vectors RNAi nematode immunostaining <it>Brugia</it> filaria |
| title | Efficient <it>in vitro </it>RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, <it>Brugia malayi</it> |
| title_full | Efficient <it>in vitro </it>RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, <it>Brugia malayi</it> |
| title_fullStr | Efficient <it>in vitro </it>RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, <it>Brugia malayi</it> |
| title_full_unstemmed | Efficient <it>in vitro </it>RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, <it>Brugia malayi</it> |
| title_short | Efficient <it>in vitro </it>RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, <it>Brugia malayi</it> |
| title_sort | efficient it in vitro it rna interference and immunofluorescence based phenotype analysis in a human parasitic nematode it brugia malayi it |
| topic | RNAi nematode immunostaining <it>Brugia</it> filaria |
| url | http://www.parasitesandvectors.com/content/5/1/16 |
| work_keys_str_mv | AT landmannfrederic efficientitinvitroitrnainterferenceandimmunofluorescencebasedphenotypeanalysisinahumanparasiticnematodeitbrugiamalayiit AT fosterjeremym efficientitinvitroitrnainterferenceandimmunofluorescencebasedphenotypeanalysisinahumanparasiticnematodeitbrugiamalayiit AT slatkobartone efficientitinvitroitrnainterferenceandimmunofluorescencebasedphenotypeanalysisinahumanparasiticnematodeitbrugiamalayiit AT sullivanwilliam efficientitinvitroitrnainterferenceandimmunofluorescencebasedphenotypeanalysisinahumanparasiticnematodeitbrugiamalayiit |