Identification of effective siRNA blocking the expression of SARS viral envelope E and RDRP genes.
Identifieur interne : 003E64 ( Main/Exploration ); précédent : 003E63; suivant : 003E65Identification of effective siRNA blocking the expression of SARS viral envelope E and RDRP genes.
Auteurs : Bo Meng ; Yue-Woon Lui ; Shi Meng ; Changxiu Cao ; Yinghe HuSource :
- Molecular biotechnology [ 1559-0305 ] ; 2006.
Descripteurs français
- KwdFr :
- MESH :
English descriptors
- KwdEn :
- Animals, Gene Expression Regulation, Viral, Mice, Mutagenesis, Insertional, NIH 3T3 Cells, RNA Interference, RNA Replicase (genetics), RNA, Small Interfering (genetics), Reverse Transcriptase Polymerase Chain Reaction, SARS Virus (enzymology), SARS Virus (genetics), Viral Envelope Proteins (genetics).
- MESH :
- chemical , genetics : RNA Replicase, RNA, Small Interfering, Viral Envelope Proteins.
- enzymology : SARS Virus.
- genetics : SARS Virus.
- Animals, Gene Expression Regulation, Viral, Mice, Mutagenesis, Insertional, NIH 3T3 Cells, RNA Interference, Reverse Transcriptase Polymerase Chain Reaction.
Abstract
A cell-based assay was developed to screen small interference RNA (siRNA) to block the expression of two genes of the severe acute respiratory syndrome (SARS) virus. These two genes encode RNA-dependent RNA polymerase (RDRP) and envelope E protein. The RDRP plays an essential role in viral RNA replication where envelope E protein is involved in envelope formation and virus assembly. The RDRP and envelope E genes, based on published sequences, have been synthesized and cloned into mammalian expression vectors. In addition, four siRNA sites for the RDRP gene and two siRNA sites for envelope E gene were designed and tested. The siRNA or short hairpin RNA (shRNA) expression cassettes were co-transfected with the SARS viral RDRP or envelope E expression vectors into NIH 3T3 cells. The expression levels of RDRP and envelope E genes were examined by reverse transcription followed by quantitative real-time polymerase chain reaction (PCR). Two of the siRNA expression cassettes for RDRP successfully inhibited the expression of the gene, whereas both of the siRNA expression cassettes for envelope E decreased approx 90% of the envelope E gene expression. The siRNA and shRNA for one of the siRNA sites of the RDRP gene were also tested, and it was found that both inhibited exogenous RDRP expression in a dose-dependent manner. These siRNA molecules could be used to examine the function of these genes in SARS virus replication and assembly. Furthermore, these molecules could potentially be developed into therapeutic agents for the treatment of patients with SARS.
DOI: 10.1385/MB:33:2:141
PubMed: 16757801
Affiliations:
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Identification of effective siRNA blocking the expression of SARS viral envelope E and RDRP genes.</title><author><name sortKey="Meng, Bo" sort="Meng, Bo" uniqKey="Meng B" first="Bo" last="Meng">Bo Meng</name><affiliation><nlm:affiliation>Key Lab of Brain Functional Genomics, MOE & STCSM, Shanghai Institute of Brain Functional Genomics, East China Normal University, Shanghai, China 200062.</nlm:affiliation><wicri:noCountry code="subField">China 200062</wicri:noCountry></affiliation></author><author><name sortKey="Lui, Yue Woon" sort="Lui, Yue Woon" uniqKey="Lui Y" first="Yue-Woon" last="Lui">Yue-Woon Lui</name></author><author><name sortKey="Meng, Shi" sort="Meng, Shi" uniqKey="Meng S" first="Shi" last="Meng">Shi Meng</name></author><author><name sortKey="Cao, Changxiu" sort="Cao, Changxiu" uniqKey="Cao C" first="Changxiu" last="Cao">Changxiu Cao</name></author><author><name sortKey="Hu, Yinghe" sort="Hu, Yinghe" uniqKey="Hu Y" first="Yinghe" last="Hu">Yinghe Hu</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2006">2006</date><idno type="RBID">pubmed:16757801</idno><idno type="pmid">16757801</idno><idno type="doi">10.1385/MB:33:2:141</idno><idno type="wicri:Area/PubMed/Corpus">002216</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002216</idno><idno type="wicri:Area/PubMed/Curation">002216</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">002216</idno><idno type="wicri:Area/PubMed/Checkpoint">002157</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">002157</idno><idno type="wicri:Area/Ncbi/Merge">001522</idno><idno type="wicri:Area/Ncbi/Curation">001522</idno><idno type="wicri:Area/Ncbi/Checkpoint">001522</idno><idno type="wicri:Area/Main/Merge">004053</idno><idno type="wicri:Area/Main/Curation">003E64</idno><idno type="wicri:Area/Main/Exploration">003E64</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Identification of effective siRNA blocking the expression of SARS viral envelope E and RDRP genes.</title><author><name sortKey="Meng, Bo" sort="Meng, Bo" uniqKey="Meng B" first="Bo" last="Meng">Bo Meng</name><affiliation><nlm:affiliation>Key Lab of Brain Functional Genomics, MOE & STCSM, Shanghai Institute of Brain Functional Genomics, East China Normal University, Shanghai, China 200062.</nlm:affiliation><wicri:noCountry code="subField">China 200062</wicri:noCountry></affiliation></author><author><name sortKey="Lui, Yue Woon" sort="Lui, Yue Woon" uniqKey="Lui Y" first="Yue-Woon" last="Lui">Yue-Woon Lui</name></author><author><name sortKey="Meng, Shi" sort="Meng, Shi" uniqKey="Meng S" first="Shi" last="Meng">Shi Meng</name></author><author><name sortKey="Cao, Changxiu" sort="Cao, Changxiu" uniqKey="Cao C" first="Changxiu" last="Cao">Changxiu Cao</name></author><author><name sortKey="Hu, Yinghe" sort="Hu, Yinghe" uniqKey="Hu Y" first="Yinghe" last="Hu">Yinghe Hu</name></author></analytic><series><title level="j">Molecular biotechnology</title><idno type="eISSN">1559-0305</idno><imprint><date when="2006" type="published">2006</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Gene Expression Regulation, Viral</term><term>Mice</term><term>Mutagenesis, Insertional</term><term>NIH 3T3 Cells</term><term>RNA Interference</term><term>RNA Replicase (genetics)</term><term>RNA, Small Interfering (genetics)</term><term>Reverse Transcriptase Polymerase Chain Reaction</term><term>SARS Virus (enzymology)</term><term>SARS Virus (genetics)</term><term>Viral Envelope Proteins (genetics)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Animaux</term><term>Cellules NIH 3T3</term><term>Interférence par ARN</term><term>Mutagenèse par insertion</term><term>Petit ARN interférent (génétique)</term><term>Protéines de l'enveloppe virale (génétique)</term><term>RNA replicase (génétique)</term><term>RT-PCR</term><term>Régulation de l'expression des gènes viraux</term><term>Souris</term><term>Virus du SRAS (enzymologie)</term><term>Virus du SRAS (génétique)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>RNA Replicase</term><term>RNA, Small Interfering</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Petit ARN interférent</term><term>Protéines de l'enveloppe virale</term><term>RNA replicase</term><term>Virus du SRAS</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Gene Expression Regulation, Viral</term><term>Mice</term><term>Mutagenesis, Insertional</term><term>NIH 3T3 Cells</term><term>RNA Interference</term><term>Reverse Transcriptase Polymerase Chain Reaction</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Cellules NIH 3T3</term><term>Interférence par ARN</term><term>Mutagenèse par insertion</term><term>RT-PCR</term><term>Régulation de l'expression des gènes viraux</term><term>Souris</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">A cell-based assay was developed to screen small interference RNA (siRNA) to block the expression of two genes of the severe acute respiratory syndrome (SARS) virus. These two genes encode RNA-dependent RNA polymerase (RDRP) and envelope E protein. The RDRP plays an essential role in viral RNA replication where envelope E protein is involved in envelope formation and virus assembly. The RDRP and envelope E genes, based on published sequences, have been synthesized and cloned into mammalian expression vectors. In addition, four siRNA sites for the RDRP gene and two siRNA sites for envelope E gene were designed and tested. The siRNA or short hairpin RNA (shRNA) expression cassettes were co-transfected with the SARS viral RDRP or envelope E expression vectors into NIH 3T3 cells. The expression levels of RDRP and envelope E genes were examined by reverse transcription followed by quantitative real-time polymerase chain reaction (PCR). Two of the siRNA expression cassettes for RDRP successfully inhibited the expression of the gene, whereas both of the siRNA expression cassettes for envelope E decreased approx 90% of the envelope E gene expression. The siRNA and shRNA for one of the siRNA sites of the RDRP gene were also tested, and it was found that both inhibited exogenous RDRP expression in a dose-dependent manner. These siRNA molecules could be used to examine the function of these genes in SARS virus replication and assembly. Furthermore, these molecules could potentially be developed into therapeutic agents for the treatment of patients with SARS.</div></front></TEI><affiliations><list></list><tree><noCountry><name sortKey="Cao, Changxiu" sort="Cao, Changxiu" uniqKey="Cao C" first="Changxiu" last="Cao">Changxiu Cao</name><name sortKey="Hu, Yinghe" sort="Hu, Yinghe" uniqKey="Hu Y" first="Yinghe" last="Hu">Yinghe Hu</name><name sortKey="Lui, Yue Woon" sort="Lui, Yue Woon" uniqKey="Lui Y" first="Yue-Woon" last="Lui">Yue-Woon Lui</name><name sortKey="Meng, Bo" sort="Meng, Bo" uniqKey="Meng B" first="Bo" last="Meng">Bo Meng</name><name sortKey="Meng, Shi" sort="Meng, Shi" uniqKey="Meng S" first="Shi" last="Meng">Shi Meng</name></noCountry></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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