Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells.
Identifieur interne : 002A81 ( PubMed/Corpus ); précédent : 002A80; suivant : 002A82Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells.
Auteurs : Zhao-Ling Qin ; Ping Zhao ; Xiao-Lian Zhang ; Jian-Guo Yu ; Ming-Mei Cao ; Lan-Juan Zhao ; Jie Luan ; Zhong-Tian QiSource :
- Biochemical and biophysical research communications [ 0006-291X ] ; 2004.
English descriptors
- KwdEn :
- Cell Line, Humans, Membrane Glycoproteins (biosynthesis), Membrane Glycoproteins (genetics), RNA Interference, RNA, Small Interfering (biosynthesis), RNA, Small Interfering (genetics), RNA, Small Interfering (pharmacology), SARS Virus (genetics), Spike Glycoprotein, Coronavirus, Viral Envelope Proteins (biosynthesis), Viral Envelope Proteins (genetics).
- MESH :
- chemical , biosynthesis : Membrane Glycoproteins, RNA, Small Interfering, Viral Envelope Proteins.
- chemical , genetics : Membrane Glycoproteins, RNA, Small Interfering, Viral Envelope Proteins.
- chemical , pharmacology : RNA, Small Interfering.
- genetics : SARS Virus.
- Cell Line, Humans, RNA Interference, Spike Glycoprotein, Coronavirus.
Abstract
Two candidate small interfering RNAs (siRNAs) corresponding to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike gene were designed and in vitro transcribed to explore the possibility of silencing SARS-CoV S gene. The plasmid pEGFP-optS, which contains the codon-optimized SARS-CoV S gene and expresses spike-EGFP fusion protein (S-EGFP) as silencing target and expressing reporter, was transfected with siRNAs into HEK 293T cells. At various time points of posttransfection, the levels of S-EGFP expression and amounts of spike mRNA transcript were detected by fluorescence microscopy, flow cytometry, Western blot, and real-time quantitative PCR, respectively. The results showed that the cells transfected with pEGFP-optS expressed S-EGFP fusion protein at a higher level compared with those transfected with pEGFP-S, which contains wildtype SARS-CoV spike gene sequence. The green fluorescence, mean fluorescence intensity, and SARS-CoV S RNA transcripts were found significantly reduced, and the expression of SARS-CoV S glycoprotein was strongly inhibited in those cells co-transfected with either EGFP- or S-specific siRNAs. Our findings demonstrated that the S-specific siRNAs used in this study were able to specifically and effectively inhibit SARS-CoV S glycoprotein expression in cultured cells through blocking the accumulation of S mRNA, which may provide an approach for studies on the functions of SARS-CoV S gene and development of novel prophylactic or therapeutic agents for SARS-CoV.
DOI: 10.1016/j.bbrc.2004.09.180
PubMed: 15504339
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells.</title><author><name sortKey="Qin, Zhao Ling" sort="Qin, Zhao Ling" uniqKey="Qin Z" first="Zhao-Ling" last="Qin">Zhao-Ling Qin</name><affiliation><nlm:affiliation>Department of Microbiology, Second Military Medical University, Shanghai 200433, China.</nlm:affiliation></affiliation></author><author><name sortKey="Zhao, Ping" sort="Zhao, Ping" uniqKey="Zhao P" first="Ping" last="Zhao">Ping Zhao</name></author><author><name sortKey="Zhang, Xiao Lian" sort="Zhang, Xiao Lian" uniqKey="Zhang X" first="Xiao-Lian" last="Zhang">Xiao-Lian Zhang</name></author><author><name sortKey="Yu, Jian Guo" sort="Yu, Jian Guo" uniqKey="Yu J" first="Jian-Guo" last="Yu">Jian-Guo Yu</name></author><author><name sortKey="Cao, Ming Mei" sort="Cao, Ming Mei" uniqKey="Cao M" first="Ming-Mei" last="Cao">Ming-Mei Cao</name></author><author><name sortKey="Zhao, Lan Juan" sort="Zhao, Lan Juan" uniqKey="Zhao L" first="Lan-Juan" last="Zhao">Lan-Juan Zhao</name></author><author><name sortKey="Luan, Jie" sort="Luan, Jie" uniqKey="Luan J" first="Jie" last="Luan">Jie Luan</name></author><author><name sortKey="Qi, Zhong Tian" sort="Qi, Zhong Tian" uniqKey="Qi Z" first="Zhong-Tian" last="Qi">Zhong-Tian Qi</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2004">2004</date><idno type="RBID">pubmed:15504339</idno><idno type="pmid">15504339</idno><idno type="doi">10.1016/j.bbrc.2004.09.180</idno><idno type="wicri:Area/PubMed/Corpus">002A81</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002A81</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells.</title><author><name sortKey="Qin, Zhao Ling" sort="Qin, Zhao Ling" uniqKey="Qin Z" first="Zhao-Ling" last="Qin">Zhao-Ling Qin</name><affiliation><nlm:affiliation>Department of Microbiology, Second Military Medical University, Shanghai 200433, China.</nlm:affiliation></affiliation></author><author><name sortKey="Zhao, Ping" sort="Zhao, Ping" uniqKey="Zhao P" first="Ping" last="Zhao">Ping Zhao</name></author><author><name sortKey="Zhang, Xiao Lian" sort="Zhang, Xiao Lian" uniqKey="Zhang X" first="Xiao-Lian" last="Zhang">Xiao-Lian Zhang</name></author><author><name sortKey="Yu, Jian Guo" sort="Yu, Jian Guo" uniqKey="Yu J" first="Jian-Guo" last="Yu">Jian-Guo Yu</name></author><author><name sortKey="Cao, Ming Mei" sort="Cao, Ming Mei" uniqKey="Cao M" first="Ming-Mei" last="Cao">Ming-Mei Cao</name></author><author><name sortKey="Zhao, Lan Juan" sort="Zhao, Lan Juan" uniqKey="Zhao L" first="Lan-Juan" last="Zhao">Lan-Juan Zhao</name></author><author><name sortKey="Luan, Jie" sort="Luan, Jie" uniqKey="Luan J" first="Jie" last="Luan">Jie Luan</name></author><author><name sortKey="Qi, Zhong Tian" sort="Qi, Zhong Tian" uniqKey="Qi Z" first="Zhong-Tian" last="Qi">Zhong-Tian Qi</name></author></analytic><series><title level="j">Biochemical and biophysical research communications</title><idno type="ISSN">0006-291X</idno><imprint><date when="2004" type="published">2004</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Cell Line</term><term>Humans</term><term>Membrane Glycoproteins (biosynthesis)</term><term>Membrane Glycoproteins (genetics)</term><term>RNA Interference</term><term>RNA, Small Interfering (biosynthesis)</term><term>RNA, Small Interfering (genetics)</term><term>RNA, Small Interfering (pharmacology)</term><term>SARS Virus (genetics)</term><term>Spike Glycoprotein, Coronavirus</term><term>Viral Envelope Proteins (biosynthesis)</term><term>Viral Envelope Proteins (genetics)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>Membrane Glycoproteins</term><term>RNA, Small Interfering</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Membrane Glycoproteins</term><term>RNA, Small Interfering</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>RNA, Small Interfering</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Cell Line</term><term>Humans</term><term>RNA Interference</term><term>Spike Glycoprotein, Coronavirus</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Two candidate small interfering RNAs (siRNAs) corresponding to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike gene were designed and in vitro transcribed to explore the possibility of silencing SARS-CoV S gene. The plasmid pEGFP-optS, which contains the codon-optimized SARS-CoV S gene and expresses spike-EGFP fusion protein (S-EGFP) as silencing target and expressing reporter, was transfected with siRNAs into HEK 293T cells. At various time points of posttransfection, the levels of S-EGFP expression and amounts of spike mRNA transcript were detected by fluorescence microscopy, flow cytometry, Western blot, and real-time quantitative PCR, respectively. The results showed that the cells transfected with pEGFP-optS expressed S-EGFP fusion protein at a higher level compared with those transfected with pEGFP-S, which contains wildtype SARS-CoV spike gene sequence. The green fluorescence, mean fluorescence intensity, and SARS-CoV S RNA transcripts were found significantly reduced, and the expression of SARS-CoV S glycoprotein was strongly inhibited in those cells co-transfected with either EGFP- or S-specific siRNAs. Our findings demonstrated that the S-specific siRNAs used in this study were able to specifically and effectively inhibit SARS-CoV S glycoprotein expression in cultured cells through blocking the accumulation of S mRNA, which may provide an approach for studies on the functions of SARS-CoV S gene and development of novel prophylactic or therapeutic agents for SARS-CoV.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">15504339</PMID><DateCompleted><Year>2005</Year><Month>02</Month><Day>07</Day></DateCompleted><DateRevised><Year>2020</Year><Month>03</Month><Day>31</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0006-291X</ISSN><JournalIssue CitedMedium="Print"><Volume>324</Volume><Issue>4</Issue><PubDate><Year>2004</Year><Month>Nov</Month><Day>26</Day></PubDate></JournalIssue><Title>Biochemical and biophysical research communications</Title><ISOAbbreviation>Biochem. Biophys. Res. Commun.</ISOAbbreviation></Journal><ArticleTitle>Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells.</ArticleTitle><Pagination><MedlinePgn>1186-93</MedlinePgn></Pagination><Abstract><AbstractText>Two candidate small interfering RNAs (siRNAs) corresponding to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike gene were designed and in vitro transcribed to explore the possibility of silencing SARS-CoV S gene. The plasmid pEGFP-optS, which contains the codon-optimized SARS-CoV S gene and expresses spike-EGFP fusion protein (S-EGFP) as silencing target and expressing reporter, was transfected with siRNAs into HEK 293T cells. At various time points of posttransfection, the levels of S-EGFP expression and amounts of spike mRNA transcript were detected by fluorescence microscopy, flow cytometry, Western blot, and real-time quantitative PCR, respectively. The results showed that the cells transfected with pEGFP-optS expressed S-EGFP fusion protein at a higher level compared with those transfected with pEGFP-S, which contains wildtype SARS-CoV spike gene sequence. The green fluorescence, mean fluorescence intensity, and SARS-CoV S RNA transcripts were found significantly reduced, and the expression of SARS-CoV S glycoprotein was strongly inhibited in those cells co-transfected with either EGFP- or S-specific siRNAs. Our findings demonstrated that the S-specific siRNAs used in this study were able to specifically and effectively inhibit SARS-CoV S glycoprotein expression in cultured cells through blocking the accumulation of S mRNA, which may provide an approach for studies on the functions of SARS-CoV S gene and development of novel prophylactic or therapeutic agents for SARS-CoV.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Qin</LastName><ForeName>Zhao-ling</ForeName><Initials>ZL</Initials><AffiliationInfo><Affiliation>Department of Microbiology, Second Military Medical University, Shanghai 200433, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Zhao</LastName><ForeName>Ping</ForeName><Initials>P</Initials></Author><Author ValidYN="Y"><LastName>Zhang</LastName><ForeName>Xiao-lian</ForeName><Initials>XL</Initials></Author><Author ValidYN="Y"><LastName>Yu</LastName><ForeName>Jian-guo</ForeName><Initials>JG</Initials></Author><Author ValidYN="Y"><LastName>Cao</LastName><ForeName>Ming-mei</ForeName><Initials>MM</Initials></Author><Author ValidYN="Y"><LastName>Zhao</LastName><ForeName>Lan-juan</ForeName><Initials>LJ</Initials></Author><Author ValidYN="Y"><LastName>Luan</LastName><ForeName>Jie</ForeName><Initials>J</Initials></Author><Author ValidYN="Y"><LastName>Qi</LastName><ForeName>Zhong-tian</ForeName><Initials>ZT</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Biochem Biophys Res Commun</MedlineTA><NlmUniqueID>0372516</NlmUniqueID><ISSNLinking>0006-291X</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D008562">Membrane 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