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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pubmed:15504339Le document en format XML
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<author><name sortKey="Zhang, Xiao Lian" sort="Zhang, Xiao Lian" uniqKey="Zhang X" first="Xiao-Lian" last="Zhang">Xiao-Lian Zhang</name>
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<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>
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<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>
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