CORD-19-Sentences  

CORD-19:a01a6cdf83b4f97c924da009bc034e8d01ad6f10 JSONTXT 11 Projects

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Id Subject Object Predicate Lexical cue
TextSentencer_T1 0-75 Sentence denotes Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells
TextSentencer_T1 0-75 Sentence denotes Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells
TextSentencer_T1 0-75 Sentence denotes Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells
TextSentencer_T2 77-85 Sentence denotes Abstract
TextSentencer_T2 77-85 Sentence denotes Abstract
TextSentencer_T2 77-85 Sentence denotes Abstract
TextSentencer_T3 86-323 Sentence denotes 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.
TextSentencer_T3 86-323 Sentence denotes 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.
TextSentencer_T3 86-323 Sentence denotes 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.
TextSentencer_T4 324-541 Sentence denotes 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.
TextSentencer_T4 324-541 Sentence denotes 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.
TextSentencer_T4 324-541 Sentence denotes 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.
TextSentencer_T5 542-772 Sentence denotes 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.
TextSentencer_T5 542-772 Sentence denotes 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.
TextSentencer_T5 542-772 Sentence denotes 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.
TextSentencer_T6 773-984 Sentence denotes 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.
TextSentencer_T6 773-984 Sentence denotes 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.
TextSentencer_T6 773-984 Sentence denotes 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.
TextSentencer_T7 985-1240 Sentence denotes 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.
TextSentencer_T7 985-1240 Sentence denotes 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.
TextSentencer_T7 985-1240 Sentence denotes 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.
TextSentencer_T8 1241-1612 Sentence denotes 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.
TextSentencer_T8 1241-1612 Sentence denotes 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.
TextSentencer_T8 1241-1612 Sentence denotes 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.
TextSentencer_T9 1614-1776 Sentence denotes The coronaviruses are a diverse group of enveloped positive-strand RNA viruses that can cause respiratory, enteric, and neurologic diseases in their host species.
TextSentencer_T9 1614-1776 Sentence denotes The coronaviruses are a diverse group of enveloped positive-strand RNA viruses that can cause respiratory, enteric, and neurologic diseases in their host species.
TextSentencer_T9 1614-1776 Sentence denotes The coronaviruses are a diverse group of enveloped positive-strand RNA viruses that can cause respiratory, enteric, and neurologic diseases in their host species.
TextSentencer_T10 1777-2041 Sentence denotes The recently identified severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been proved as a new group of coronaviruses [1] [2] [3] , which consists of four major structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N).
TextSentencer_T10 1777-2041 Sentence denotes The recently identified severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been proved as a new group of coronaviruses [1] [2] [3] , which consists of four major structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N).
TextSentencer_T10 1777-2041 Sentence denotes The recently identified severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been proved as a new group of coronaviruses [1] [2] [3] , which consists of four major structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N).
TextSentencer_T11 2042-2320 Sentence denotes The S protein, with 1255 amino acids in size, is a surface glycoprotein and contains signal peptide at the N-terminus (residues 1-13), a large amino-terminal ectodomain (residues , and a short carboxy-terminal endodomain bridged with a transmembrane domain (residues 1196-1255).
TextSentencer_T11 2042-2320 Sentence denotes The S protein, with 1255 amino acids in size, is a surface glycoprotein and contains signal peptide at the N-terminus (residues 1-13), a large amino-terminal ectodomain (residues , and a short carboxy-terminal endodomain bridged with a transmembrane domain (residues 1196-1255).
TextSentencer_T11 2042-2320 Sentence denotes The S protein, with 1255 amino acids in size, is a surface glycoprotein and contains signal peptide at the N-terminus (residues 1-13), a large amino-terminal ectodomain (residues , and a short carboxy-terminal endodomain bridged with a transmembrane domain (residues 1196-1255).
TextSentencer_T12 2321-2434 Sentence denotes There is only 20-27% amino acid identity between SARS-CoV S protein and other coronavirus family members [3, 4] .
TextSentencer_T12 2321-2434 Sentence denotes There is only 20-27% amino acid identity between SARS-CoV S protein and other coronavirus family members [3, 4] .
TextSentencer_T12 2321-2434 Sentence denotes There is only 20-27% amino acid identity between SARS-CoV S protein and other coronavirus family members [3, 4] .
TextSentencer_T13 2435-2564 Sentence denotes Similar to other coronaviruses, SARS-CoV S glycoprotein contains two non-covalently associated subunits (S1 and S2) functionally.
TextSentencer_T13 2435-2564 Sentence denotes Similar to other coronaviruses, SARS-CoV S glycoprotein contains two non-covalently associated subunits (S1 and S2) functionally.
TextSentencer_T13 2435-2564 Sentence denotes Similar to other coronaviruses, SARS-CoV S glycoprotein contains two non-covalently associated subunits (S1 and S2) functionally.
TextSentencer_T14 2565-2751 Sentence denotes It is believed that binding of S1 subunit to its cellular receptor induces conformational changes in S2, leading to the fusion between viral and cellular membranes, and viral entry [5] .
TextSentencer_T14 2565-2751 Sentence denotes It is believed that binding of S1 subunit to its cellular receptor induces conformational changes in S2, leading to the fusion between viral and cellular membranes, and viral entry [5] .
TextSentencer_T14 2565-2751 Sentence denotes It is believed that binding of S1 subunit to its cellular receptor induces conformational changes in S2, leading to the fusion between viral and cellular membranes, and viral entry [5] .
TextSentencer_T15 2752-2893 Sentence denotes In addition, SARS-CoV S glycoprotein has been proved an important antigen and is able to induce the generation of neutralizing antibody [6] .
TextSentencer_T15 2752-2893 Sentence denotes In addition, SARS-CoV S glycoprotein has been proved an important antigen and is able to induce the generation of neutralizing antibody [6] .
TextSentencer_T15 2752-2893 Sentence denotes In addition, SARS-CoV S glycoprotein has been proved an important antigen and is able to induce the generation of neutralizing antibody [6] .
TextSentencer_T16 2894-3104 Sentence denotes RNA interference (RNAi) is a cellular process in that double-stranded RNA (dsRNA) molecules of 19-23 nucleotides (nt) can silence targeted genes through sequence-specific cleavage of the cognate RNA transcript.
TextSentencer_T16 2894-3104 Sentence denotes RNA interference (RNAi) is a cellular process in that double-stranded RNA (dsRNA) molecules of 19-23 nucleotides (nt) can silence targeted genes through sequence-specific cleavage of the cognate RNA transcript.
TextSentencer_T16 2894-3104 Sentence denotes RNA interference (RNAi) is a cellular process in that double-stranded RNA (dsRNA) molecules of 19-23 nucleotides (nt) can silence targeted genes through sequence-specific cleavage of the cognate RNA transcript.
TextSentencer_T17 3105-3331 Sentence denotes The phenomenon was originally discovered in Caenorhabditis elegans in 1998 [7] but has since been observed in numerous organisms including plant, fungus, Drosophila, seaweed, protozoan, and mammal [8] [9] [10] [11] [12] [13] .
TextSentencer_T17 3105-3331 Sentence denotes The phenomenon was originally discovered in Caenorhabditis elegans in 1998 [7] but has since been observed in numerous organisms including plant, fungus, Drosophila, seaweed, protozoan, and mammal [8] [9] [10] [11] [12] [13] .
TextSentencer_T17 3105-3331 Sentence denotes The phenomenon was originally discovered in Caenorhabditis elegans in 1998 [7] but has since been observed in numerous organisms including plant, fungus, Drosophila, seaweed, protozoan, and mammal [8] [9] [10] [11] [12] [13] .
TextSentencer_T18 3332-3403 Sentence denotes The RNAi mechanism appears to be mediated by small dsRNA intermediates.
TextSentencer_T18 3332-3403 Sentence denotes The RNAi mechanism appears to be mediated by small dsRNA intermediates.
TextSentencer_T18 3332-3403 Sentence denotes The RNAi mechanism appears to be mediated by small dsRNA intermediates.
TextSentencer_T19 3404-3640 Sentence denotes The parent, larger dsRNA is processed by ribonuclease III-like enzymes into smaller fragments in vivo, and the resulting small interfering RNAs (siRNAs) direct posttranscriptional, but pretranslational, degradation of the targeted mRNA.
TextSentencer_T19 3404-3640 Sentence denotes The parent, larger dsRNA is processed by ribonuclease III-like enzymes into smaller fragments in vivo, and the resulting small interfering RNAs (siRNAs) direct posttranscriptional, but pretranslational, degradation of the targeted mRNA.
TextSentencer_T19 3404-3640 Sentence denotes The parent, larger dsRNA is processed by ribonuclease III-like enzymes into smaller fragments in vivo, and the resulting small interfering RNAs (siRNAs) direct posttranscriptional, but pretranslational, degradation of the targeted mRNA.
TextSentencer_T20 3641-3900 Sentence denotes RNAi is becoming a powerful tool to investigate gene function through specific suppression of a particular mRNA and has been employed in therapeutic studies of human diseases including cancer, neurogenerative diseases, and viral infectious diseases [14, 15] .
TextSentencer_T20 3641-3900 Sentence denotes RNAi is becoming a powerful tool to investigate gene function through specific suppression of a particular mRNA and has been employed in therapeutic studies of human diseases including cancer, neurogenerative diseases, and viral infectious diseases [14, 15] .
TextSentencer_T20 3641-3900 Sentence denotes RNAi is becoming a powerful tool to investigate gene function through specific suppression of a particular mRNA and has been employed in therapeutic studies of human diseases including cancer, neurogenerative diseases, and viral infectious diseases [14, 15] .
TextSentencer_T21 3901-3964 Sentence denotes Recently, it has also been used in the study of SARS-CoV [16] .
TextSentencer_T21 3901-3964 Sentence denotes Recently, it has also been used in the study of SARS-CoV [16] .
TextSentencer_T21 3901-3964 Sentence denotes Recently, it has also been used in the study of SARS-CoV [16] .
TextSentencer_T22 3965-4194 Sentence denotes To explore the possibility of silencing SARS-CoV S gene by RNAi technique, we cloned and expressed SARS-CoV spike gene, which encodes N-terminal amino acid residues 1-690 and contains the identified receptor-binding domain (RBD).
TextSentencer_T22 3965-4194 Sentence denotes To explore the possibility of silencing SARS-CoV S gene by RNAi technique, we cloned and expressed SARS-CoV spike gene, which encodes N-terminal amino acid residues 1-690 and contains the identified receptor-binding domain (RBD).
TextSentencer_T22 3965-4194 Sentence denotes To explore the possibility of silencing SARS-CoV S gene by RNAi technique, we cloned and expressed SARS-CoV spike gene, which encodes N-terminal amino acid residues 1-690 and contains the identified receptor-binding domain (RBD).
TextSentencer_T23 4195-4303 Sentence denotes The in vitro transcribed siRNAs were then introduced into HEK 293T cells expressing SARS-CoV S glycoprotein.
TextSentencer_T23 4195-4303 Sentence denotes The in vitro transcribed siRNAs were then introduced into HEK 293T cells expressing SARS-CoV S glycoprotein.
TextSentencer_T23 4195-4303 Sentence denotes The in vitro transcribed siRNAs were then introduced into HEK 293T cells expressing SARS-CoV S glycoprotein.
TextSentencer_T24 4304-4428 Sentence denotes Our results demonstrated that the codon-optimized spike gene obviously improved S glycoprotein expression in cultured cells.
TextSentencer_T24 4304-4428 Sentence denotes Our results demonstrated that the codon-optimized spike gene obviously improved S glycoprotein expression in cultured cells.
TextSentencer_T24 4304-4428 Sentence denotes Our results demonstrated that the codon-optimized spike gene obviously improved S glycoprotein expression in cultured cells.
TextSentencer_T25 4429-4569 Sentence denotes The specific siRNAs corresponding to SARS-CoV spike gene specifically degraded S mRNA and significantly inhibited S glycoprotein expression.
TextSentencer_T25 4429-4569 Sentence denotes The specific siRNAs corresponding to SARS-CoV spike gene specifically degraded S mRNA and significantly inhibited S glycoprotein expression.
TextSentencer_T25 4429-4569 Sentence denotes The specific siRNAs corresponding to SARS-CoV spike gene specifically degraded S mRNA and significantly inhibited S glycoprotein expression.
TextSentencer_T26 4570-4595 Sentence denotes Construction of plasmids.
TextSentencer_T26 4570-4595 Sentence denotes Construction of plasmids.
TextSentencer_T26 4570-4595 Sentence denotes Construction of plasmids.
TextSentencer_T27 4596-4710 Sentence denotes Two DNA fragments, corresponding to nt 1-1720 and 1626-2934 of SARS-CoV spike gene, were kindly pro-vided by Prof.
TextSentencer_T27 4596-4710 Sentence denotes Two DNA fragments, corresponding to nt 1-1720 and 1626-2934 of SARS-CoV spike gene, were kindly pro-vided by Prof.
TextSentencer_T27 4596-4710 Sentence denotes Two DNA fragments, corresponding to nt 1-1720 and 1626-2934 of SARS-CoV spike gene, were kindly pro-vided by Prof.
TextSentencer_T28 4711-4715 Sentence denotes Z.H.
TextSentencer_T28 4711-4715 Sentence denotes Z.H.
TextSentencer_T28 4711-4715 Sentence denotes Z.H.
TextSentencer_T29 4716-4781 Sentence denotes Yuan (Molecular Virology Laboratory, Fudan University, Shanghai).
TextSentencer_T29 4716-4781 Sentence denotes Yuan (Molecular Virology Laboratory, Fudan University, Shanghai).
TextSentencer_T29 4716-4781 Sentence denotes Yuan (Molecular Virology Laboratory, Fudan University, Shanghai).
TextSentencer_T30 4782-5159 Sentence denotes The DNA fragment encoding N-terminal amino acid residues 1-690 of S glycoprotein, flanked by the ÔKozakÕ consensus sequence, was amplified from the two DNA fragments with the approach of splice overlap extension-polymerase chain reaction (SOE-PCR), and inserted into the multi-cloning site of a eukaryotic expression vector pEGFP-N1 (Clonetech) between its SalI and PstI sites.
TextSentencer_T30 4782-5159 Sentence denotes The DNA fragment encoding N-terminal amino acid residues 1-690 of S glycoprotein, flanked by the ÔKozakÕ consensus sequence, was amplified from the two DNA fragments with the approach of splice overlap extension-polymerase chain reaction (SOE-PCR), and inserted into the multi-cloning site of a eukaryotic expression vector pEGFP-N1 (Clonetech) between its SalI and PstI sites.
TextSentencer_T30 4782-5159 Sentence denotes The DNA fragment encoding N-terminal amino acid residues 1-690 of S glycoprotein, flanked by the ÔKozakÕ consensus sequence, was amplified from the two DNA fragments with the approach of splice overlap extension-polymerase chain reaction (SOE-PCR), and inserted into the multi-cloning site of a eukaryotic expression vector pEGFP-N1 (Clonetech) between its SalI and PstI sites.
TextSentencer_T31 5160-5291 Sentence denotes The resulting plasmid was named pEGFP-S, in which enhanced green fluorescence protein (EGFP) gene was located downstream of S gene.
TextSentencer_T31 5160-5291 Sentence denotes The resulting plasmid was named pEGFP-S, in which enhanced green fluorescence protein (EGFP) gene was located downstream of S gene.
TextSentencer_T31 5160-5291 Sentence denotes The resulting plasmid was named pEGFP-S, in which enhanced green fluorescence protein (EGFP) gene was located downstream of S gene.
TextSentencer_T32 5292-5359 Sentence denotes Identity of S gene to the published sequence (GenBank Accession No.
TextSentencer_T32 5292-5359 Sentence denotes Identity of S gene to the published sequence (GenBank Accession No.
TextSentencer_T32 5292-5359 Sentence denotes Identity of S gene to the published sequence (GenBank Accession No.
TextSentencer_T33 5360-5418 Sentence denotes AY278554) was confirmed by sequencing (Bioasia, Shanghai).
TextSentencer_T33 5360-5418 Sentence denotes AY278554) was confirmed by sequencing (Bioasia, Shanghai).
TextSentencer_T33 5360-5418 Sentence denotes AY278554) was confirmed by sequencing (Bioasia, Shanghai).
TextSentencer_T34 5419-5608 Sentence denotes Some of the nucleotides encoding the amino acid residues 1-110 of S glycoprotein in the plasmid pEGFP-S were optimized utilizing mammalian preferred codons, creating the plasmid pEGFP-optS.
TextSentencer_T34 5419-5608 Sentence denotes Some of the nucleotides encoding the amino acid residues 1-110 of S glycoprotein in the plasmid pEGFP-S were optimized utilizing mammalian preferred codons, creating the plasmid pEGFP-optS.
TextSentencer_T34 5419-5608 Sentence denotes Some of the nucleotides encoding the amino acid residues 1-110 of S glycoprotein in the plasmid pEGFP-S were optimized utilizing mammalian preferred codons, creating the plasmid pEGFP-optS.
TextSentencer_T35 5609-5644 Sentence denotes Design and transcription of siRNAs.
TextSentencer_T35 5609-5644 Sentence denotes Design and transcription of siRNAs.
TextSentencer_T35 5609-5644 Sentence denotes Design and transcription of siRNAs.
TextSentencer_T36 5645-5753 Sentence denotes The siRNAs corresponding to SARS-CoV spike gene were designed according to AmbionÕs siRNA design guidelines.
TextSentencer_T36 5645-5753 Sentence denotes The siRNAs corresponding to SARS-CoV spike gene were designed according to AmbionÕs siRNA design guidelines.
TextSentencer_T36 5645-5753 Sentence denotes The siRNAs corresponding to SARS-CoV spike gene were designed according to AmbionÕs siRNA design guidelines.
TextSentencer_T37 5754-5866 Sentence denotes Scramble siRNA [17] and EGFP siRNA [18] were used as negative and positive controls for silencing, respectively.
TextSentencer_T37 5754-5866 Sentence denotes Scramble siRNA [17] and EGFP siRNA [18] were used as negative and positive controls for silencing, respectively.
TextSentencer_T37 5754-5866 Sentence denotes Scramble siRNA [17] and EGFP siRNA [18] were used as negative and positive controls for silencing, respectively.
TextSentencer_T38 5867-6179 Sentence denotes All sequences of the siRNAs were BLAST searched in the National Center for Biotechnology InformationÕs (NCBI) ''search for short nearly exact matches'' mode against all human sequences deposited in the GenBank and RefSeq databases, and were not found to have significant homology to genes other than the targets.
TextSentencer_T38 5867-6179 Sentence denotes All sequences of the siRNAs were BLAST searched in the National Center for Biotechnology InformationÕs (NCBI) ''search for short nearly exact matches'' mode against all human sequences deposited in the GenBank and RefSeq databases, and were not found to have significant homology to genes other than the targets.
TextSentencer_T38 5867-6179 Sentence denotes All sequences of the siRNAs were BLAST searched in the National Center for Biotechnology InformationÕs (NCBI) ''search for short nearly exact matches'' mode against all human sequences deposited in the GenBank and RefSeq databases, and were not found to have significant homology to genes other than the targets.
TextSentencer_T39 6180-6288 Sentence denotes The template deoxynucleotides (Table 1 ) used for siRNA transcription were synthesized by Bioasia, Shanghai.
TextSentencer_T39 6180-6288 Sentence denotes The template deoxynucleotides (Table 1 ) used for siRNA transcription were synthesized by Bioasia, Shanghai.
TextSentencer_T39 6180-6288 Sentence denotes The template deoxynucleotides (Table 1 ) used for siRNA transcription were synthesized by Bioasia, Shanghai.
TextSentencer_T40 6289-6398 Sentence denotes The oligonucleotide directed production of siRNAs with T7 RNA polymerase has been described previously [18] .
TextSentencer_T40 6289-6398 Sentence denotes The oligonucleotide directed production of siRNAs with T7 RNA polymerase has been described previously [18] .
TextSentencer_T40 6289-6398 Sentence denotes The oligonucleotide directed production of siRNAs with T7 RNA polymerase has been described previously [18] .
TextSentencer_T41 6399-6553 Sentence denotes For each transcription reaction, 300 lM of each oligonucleotide template and T7 promoter primer were mixed and denaturalized by heating at 95°C for 2 min.
TextSentencer_T41 6399-6553 Sentence denotes For each transcription reaction, 300 lM of each oligonucleotide template and T7 promoter primer were mixed and denaturalized by heating at 95°C for 2 min.
TextSentencer_T41 6399-6553 Sentence denotes For each transcription reaction, 300 lM of each oligonucleotide template and T7 promoter primer were mixed and denaturalized by heating at 95°C for 2 min.
TextSentencer_T42 6554-6726 Sentence denotes The mixture was then added with 10· Klenow reaction buffer, dNTP mix (Promega, USA), Exo-Klenow (TaKaRa, Dalian), and nuclease-free water, and incubated at 37°C for 30 min.
TextSentencer_T42 6554-6726 Sentence denotes The mixture was then added with 10· Klenow reaction buffer, dNTP mix (Promega, USA), Exo-Klenow (TaKaRa, Dalian), and nuclease-free water, and incubated at 37°C for 30 min.
TextSentencer_T42 6554-6726 Sentence denotes The mixture was then added with 10· Klenow reaction buffer, dNTP mix (Promega, USA), Exo-Klenow (TaKaRa, Dalian), and nuclease-free water, and incubated at 37°C for 30 min.
TextSentencer_T43 6727-6795 Sentence denotes The in vitro transcription was performed in 20 ll transcription mix:
TextSentencer_T43 6727-6795 Sentence denotes The in vitro transcription was performed in 20 ll transcription mix:
TextSentencer_T43 6727-6795 Sentence denotes The in vitro transcription was performed in 20 ll transcription mix:
TextSentencer_T44 6796-6924 Sentence denotes 6 ll hybridization solution, 4 ll of 5· T7 reaction buffer, 6 ll rNTP mix, 2 ll T7 RNA polymerase, and 2 ll nuclease-free water.
TextSentencer_T44 6796-6924 Sentence denotes 6 ll hybridization solution, 4 ll of 5· T7 reaction buffer, 6 ll rNTP mix, 2 ll T7 RNA polymerase, and 2 ll nuclease-free water.
TextSentencer_T44 6796-6924 Sentence denotes 6 ll hybridization solution, 4 ll of 5· T7 reaction buffer, 6 ll rNTP mix, 2 ll T7 RNA polymerase, and 2 ll nuclease-free water.
TextSentencer_T45 6925-7108 Sentence denotes After incubation at 37°C for 2 h, sense and antisense RNAs generated in separate reactions were annealed by mixing both crude transcription reactions and incubating at 37°C overnight.
TextSentencer_T45 6925-7108 Sentence denotes After incubation at 37°C for 2 h, sense and antisense RNAs generated in separate reactions were annealed by mixing both crude transcription reactions and incubating at 37°C overnight.
TextSentencer_T45 6925-7108 Sentence denotes After incubation at 37°C for 2 h, sense and antisense RNAs generated in separate reactions were annealed by mixing both crude transcription reactions and incubating at 37°C overnight.
TextSentencer_T46 7109-7231 Sentence denotes The concentration of the generated dsRNA was measured by the absorbance at 260 nm in a BioPhotometer (Eppendorf, Germany).
TextSentencer_T46 7109-7231 Sentence denotes The concentration of the generated dsRNA was measured by the absorbance at 260 nm in a BioPhotometer (Eppendorf, Germany).
TextSentencer_T46 7109-7231 Sentence denotes The concentration of the generated dsRNA was measured by the absorbance at 260 nm in a BioPhotometer (Eppendorf, Germany).
TextSentencer_T47 7232-7394 Sentence denotes S1 nuclease and RNase-free DNase I (TaKaRa, Dalian) were added to final concentrations of 10 and 1 U/lg siRNA, respectively, for the digestion of ssRNA and dsDNA.
TextSentencer_T47 7232-7394 Sentence denotes S1 nuclease and RNase-free DNase I (TaKaRa, Dalian) were added to final concentrations of 10 and 1 U/lg siRNA, respectively, for the digestion of ssRNA and dsDNA.
TextSentencer_T47 7232-7394 Sentence denotes S1 nuclease and RNase-free DNase I (TaKaRa, Dalian) were added to final concentrations of 10 and 1 U/lg siRNA, respectively, for the digestion of ssRNA and dsDNA.
TextSentencer_T48 7395-7457 Sentence denotes SiRNAs were assessed by RNA gel electrophoresis on 2% agarose.
TextSentencer_T48 7395-7457 Sentence denotes SiRNAs were assessed by RNA gel electrophoresis on 2% agarose.
TextSentencer_T48 7395-7457 Sentence denotes SiRNAs were assessed by RNA gel electrophoresis on 2% agarose.
TextSentencer_T49 7458-7481 Sentence denotes Purification of siRNAs.
TextSentencer_T49 7458-7481 Sentence denotes Purification of siRNAs.
TextSentencer_T49 7458-7481 Sentence denotes Purification of siRNAs.
TextSentencer_T50 7482-7664 Sentence denotes The in vitro transcribed siRNA was added with one volume of TE-saturated (pH 4.5) phenol:chloroform:isoamyl alcohol (25:24:1), and spun at 13,000 rpm in a microcentrifuge for 10 min.
TextSentencer_T50 7482-7664 Sentence denotes The in vitro transcribed siRNA was added with one volume of TE-saturated (pH 4.5) phenol:chloroform:isoamyl alcohol (25:24:1), and spun at 13,000 rpm in a microcentrifuge for 10 min.
TextSentencer_T50 7482-7664 Sentence denotes The in vitro transcribed siRNA was added with one volume of TE-saturated (pH 4.5) phenol:chloroform:isoamyl alcohol (25:24:1), and spun at 13,000 rpm in a microcentrifuge for 10 min.
TextSentencer_T51 7665-7822 Sentence denotes The upper, aqueous phase was collected to a fresh tube, mixed with one volume of chloroform:isoamyl alcohol (24:1), and centrifuged at 13,000 rpm for 10 min.
TextSentencer_T51 7665-7822 Sentence denotes The upper, aqueous phase was collected to a fresh tube, mixed with one volume of chloroform:isoamyl alcohol (24:1), and centrifuged at 13,000 rpm for 10 min.
TextSentencer_T51 7665-7822 Sentence denotes The upper, aqueous phase was collected to a fresh tube, mixed with one volume of chloroform:isoamyl alcohol (24:1), and centrifuged at 13,000 rpm for 10 min.
TextSentencer_T52 7823-8104 Sentence denotes The upper, aqueous phase was then mixed with two volumes of ethanol and 0.1 volume of 3 mmol/L sodium acetate (pH 5.2) and placed on ice for 30 min, followed by centrifugation at Table 1 Sequences of template deoxynucleotides for siRNAs used for target genes 13,000 rpm for 10 min.
TextSentencer_T52 7823-8104 Sentence denotes The upper, aqueous phase was then mixed with two volumes of ethanol and 0.1 volume of 3 mmol/L sodium acetate (pH 5.2) and placed on ice for 30 min, followed by centrifugation at Table 1 Sequences of template deoxynucleotides for siRNAs used for target genes 13,000 rpm for 10 min.
TextSentencer_T52 7823-8104 Sentence denotes The upper, aqueous phase was then mixed with two volumes of ethanol and 0.1 volume of 3 mmol/L sodium acetate (pH 5.2) and placed on ice for 30 min, followed by centrifugation at Table 1 Sequences of template deoxynucleotides for siRNAs used for target genes 13,000 rpm for 10 min.
TextSentencer_T53 8105-8205 Sentence denotes The pellet was washed with 1 ml of 70% ethanol and suspended in nuclease-free water for further use.
TextSentencer_T53 8105-8205 Sentence denotes The pellet was washed with 1 ml of 70% ethanol and suspended in nuclease-free water for further use.
TextSentencer_T53 8105-8205 Sentence denotes The pellet was washed with 1 ml of 70% ethanol and suspended in nuclease-free water for further use.
TextSentencer_T54 8206-8272 Sentence denotes The purity and integrity of siRNAs were checked on an agarose gel.
TextSentencer_T54 8206-8272 Sentence denotes The purity and integrity of siRNAs were checked on an agarose gel.
TextSentencer_T54 8206-8272 Sentence denotes The purity and integrity of siRNAs were checked on an agarose gel.
TextSentencer_T55 8273-8303 Sentence denotes Cell culture and transfection.
TextSentencer_T55 8273-8303 Sentence denotes Cell culture and transfection.
TextSentencer_T55 8273-8303 Sentence denotes Cell culture and transfection.
TextSentencer_T56 8304-8586 Sentence denotes Human embryonic kidney cell line (HEK) 293T cells were grown at 37°C in DulbeccoÕs modified EagleÕs medium (DMEM) (Sigma) containing 10% heat-inactivated fetal bovine serum (Gibco-BRL, USA) supplemented with L L-glutamine (1 mM), streptomycin (100 lg/ml), and penicillin (100 U/ml).
TextSentencer_T56 8304-8586 Sentence denotes Human embryonic kidney cell line (HEK) 293T cells were grown at 37°C in DulbeccoÕs modified EagleÕs medium (DMEM) (Sigma) containing 10% heat-inactivated fetal bovine serum (Gibco-BRL, USA) supplemented with L L-glutamine (1 mM), streptomycin (100 lg/ml), and penicillin (100 U/ml).
TextSentencer_T56 8304-8586 Sentence denotes Human embryonic kidney cell line (HEK) 293T cells were grown at 37°C in DulbeccoÕs modified EagleÕs medium (DMEM) (Sigma) containing 10% heat-inactivated fetal bovine serum (Gibco-BRL, USA) supplemented with L L-glutamine (1 mM), streptomycin (100 lg/ml), and penicillin (100 U/ml).
TextSentencer_T57 8587-8762 Sentence denotes Twentyfour hours prior to transfection, the cells were seeded into 24-well plates at a density of 0.5-2 · 10 5 cells/well with fresh medium (500 ll/ well) without antibiotics.
TextSentencer_T57 8587-8762 Sentence denotes Twentyfour hours prior to transfection, the cells were seeded into 24-well plates at a density of 0.5-2 · 10 5 cells/well with fresh medium (500 ll/ well) without antibiotics.
TextSentencer_T57 8587-8762 Sentence denotes Twentyfour hours prior to transfection, the cells were seeded into 24-well plates at a density of 0.5-2 · 10 5 cells/well with fresh medium (500 ll/ well) without antibiotics.
TextSentencer_T58 8763-8973 Sentence denotes For the transfection of adherent HEK 293T cells, a total of 0.8 lg of plasmid DNA or/and 2 ll of siRNA mixed with Lipofectamine 2000 (Invitrogen, CA, USA) were used according to the manufacturerÕs instructions.
TextSentencer_T58 8763-8973 Sentence denotes For the transfection of adherent HEK 293T cells, a total of 0.8 lg of plasmid DNA or/and 2 ll of siRNA mixed with Lipofectamine 2000 (Invitrogen, CA, USA) were used according to the manufacturerÕs instructions.
TextSentencer_T58 8763-8973 Sentence denotes For the transfection of adherent HEK 293T cells, a total of 0.8 lg of plasmid DNA or/and 2 ll of siRNA mixed with Lipofectamine 2000 (Invitrogen, CA, USA) were used according to the manufacturerÕs instructions.
TextSentencer_T59 8974-9070 Sentence denotes The cells were incubated at 37°C for various time lengths for gene transcription and expression.
TextSentencer_T59 8974-9070 Sentence denotes The cells were incubated at 37°C for various time lengths for gene transcription and expression.
TextSentencer_T59 8974-9070 Sentence denotes The cells were incubated at 37°C for various time lengths for gene transcription and expression.
TextSentencer_T60 9071-9175 Sentence denotes The expression of S-EGFP fusion protein was observed directly under an inverted fluorescence microscope.
TextSentencer_T60 9071-9175 Sentence denotes The expression of S-EGFP fusion protein was observed directly under an inverted fluorescence microscope.
TextSentencer_T60 9071-9175 Sentence denotes The expression of S-EGFP fusion protein was observed directly under an inverted fluorescence microscope.
TextSentencer_T61 9176-9217 Sentence denotes Fluorescence and flow cytometry analysis.
TextSentencer_T61 9176-9217 Sentence denotes Fluorescence and flow cytometry analysis.
TextSentencer_T61 9176-9217 Sentence denotes Fluorescence and flow cytometry analysis.
TextSentencer_T62 9218-9367 Sentence denotes Expression of S-EGFP in transfected cells was examined with fluorescence microscope (Olympus CK40, Japan) at 24, 48, 72, and 96 h after transfection.
TextSentencer_T62 9218-9367 Sentence denotes Expression of S-EGFP in transfected cells was examined with fluorescence microscope (Olympus CK40, Japan) at 24, 48, 72, and 96 h after transfection.
TextSentencer_T62 9218-9367 Sentence denotes Expression of S-EGFP in transfected cells was examined with fluorescence microscope (Olympus CK40, Japan) at 24, 48, 72, and 96 h after transfection.
TextSentencer_T63 9368-9680 Sentence denotes For flow cytometry analysis of S-EGFP expression, the cells were harvested at 48 h posttransfection and digested with 0.25% trypsin, washed with PBS twice, and then resuspended in PBS to measure the fluorescence using a Becton-Dickinson FACScan flow cytometer with filters (emission, 507 nm; excitation, 488 nm).
TextSentencer_T63 9368-9680 Sentence denotes For flow cytometry analysis of S-EGFP expression, the cells were harvested at 48 h posttransfection and digested with 0.25% trypsin, washed with PBS twice, and then resuspended in PBS to measure the fluorescence using a Becton-Dickinson FACScan flow cytometer with filters (emission, 507 nm; excitation, 488 nm).
TextSentencer_T63 9368-9680 Sentence denotes For flow cytometry analysis of S-EGFP expression, the cells were harvested at 48 h posttransfection and digested with 0.25% trypsin, washed with PBS twice, and then resuspended in PBS to measure the fluorescence using a Becton-Dickinson FACScan flow cytometer with filters (emission, 507 nm; excitation, 488 nm).
TextSentencer_T64 9681-9811 Sentence denotes Samples (about 10 6 cells each) were counted and analyzed with CellQuest software, using nontransfected HEK 293T cells as control.
TextSentencer_T64 9681-9811 Sentence denotes Samples (about 10 6 cells each) were counted and analyzed with CellQuest software, using nontransfected HEK 293T cells as control.
TextSentencer_T64 9681-9811 Sentence denotes Samples (about 10 6 cells each) were counted and analyzed with CellQuest software, using nontransfected HEK 293T cells as control.
TextSentencer_T65 9812-9997 Sentence denotes The values were calculated as the percentage of the cell population that exceeded the fluorescence intensity of the control cells and the mean fluorescence intensity of this population.
TextSentencer_T65 9812-9997 Sentence denotes The values were calculated as the percentage of the cell population that exceeded the fluorescence intensity of the control cells and the mean fluorescence intensity of this population.
TextSentencer_T65 9812-9997 Sentence denotes The values were calculated as the percentage of the cell population that exceeded the fluorescence intensity of the control cells and the mean fluorescence intensity of this population.
TextSentencer_T66 9998-10055 Sentence denotes Reverse transcription-PCR and real-time quantitative PCR.
TextSentencer_T66 9998-10055 Sentence denotes Reverse transcription-PCR and real-time quantitative PCR.
TextSentencer_T66 9998-10055 Sentence denotes Reverse transcription-PCR and real-time quantitative PCR.
TextSentencer_T67 10056-10244 Sentence denotes Total RNA from the transfected or non-transfected cells was extracted at 48 h posttransfection using RNAex Reagent (Watson, Shanghai) and digested with RNase-free DNase I (TaKaRa, Dalian).
TextSentencer_T67 10056-10244 Sentence denotes Total RNA from the transfected or non-transfected cells was extracted at 48 h posttransfection using RNAex Reagent (Watson, Shanghai) and digested with RNase-free DNase I (TaKaRa, Dalian).
TextSentencer_T67 10056-10244 Sentence denotes Total RNA from the transfected or non-transfected cells was extracted at 48 h posttransfection using RNAex Reagent (Watson, Shanghai) and digested with RNase-free DNase I (TaKaRa, Dalian).
TextSentencer_T68 10245-10454 Sentence denotes One microgram of the RNA was then reverse transcribed into cDNA with oligo(dT) 15 and the avian myeloblastosis virus (AMV) reverse transcriptase XL (TaKaRa, Dalian) according to manufacturerÕs recommendations.
TextSentencer_T68 10245-10454 Sentence denotes One microgram of the RNA was then reverse transcribed into cDNA with oligo(dT) 15 and the avian myeloblastosis virus (AMV) reverse transcriptase XL (TaKaRa, Dalian) according to manufacturerÕs recommendations.
TextSentencer_T68 10245-10454 Sentence denotes One microgram of the RNA was then reverse transcribed into cDNA with oligo(dT) 15 and the avian myeloblastosis virus (AMV) reverse transcriptase XL (TaKaRa, Dalian) according to manufacturerÕs recommendations.
TextSentencer_T69 10455-10578 Sentence denotes Reactions with no reverse transcriptase added were performed in parallel with most experiments and yielded no PCR products.
TextSentencer_T69 10455-10578 Sentence denotes Reactions with no reverse transcriptase added were performed in parallel with most experiments and yielded no PCR products.
TextSentencer_T69 10455-10578 Sentence denotes Reactions with no reverse transcriptase added were performed in parallel with most experiments and yielded no PCR products.
TextSentencer_T70 10579-10870 Sentence denotes 1 lM of forward primer (5 0 -CCCATGGGTACACAGACACA-3 0 ) and reverse primer (5 0 -AATAGGCTGCAGCTGACGTG-3 0 ) for SARS-CoV spike gene was used for semi-quantitative reverse transcription (RT)-PCR with 20 cycles of denaturation (94°C, 55 s), annealing (56°C, 55 s), and extension (72°C, 1 min).
TextSentencer_T70 10579-10870 Sentence denotes 1 lM of forward primer (5 0 -CCCATGGGTACACAGACACA-3 0 ) and reverse primer (5 0 -AATAGGCTGCAGCTGACGTG-3 0 ) for SARS-CoV spike gene was used for semi-quantitative reverse transcription (RT)-PCR with 20 cycles of denaturation (94°C, 55 s), annealing (56°C, 55 s), and extension (72°C, 1 min).
TextSentencer_T70 10579-10870 Sentence denotes 1 lM of forward primer (5 0 -CCCATGGGTACACAGACACA-3 0 ) and reverse primer (5 0 -AATAGGCTGCAGCTGACGTG-3 0 ) for SARS-CoV spike gene was used for semi-quantitative reverse transcription (RT)-PCR with 20 cycles of denaturation (94°C, 55 s), annealing (56°C, 55 s), and extension (72°C, 1 min).
TextSentencer_T71 10871-11140 Sentence denotes Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, the primers (forward primer, 5 0 -TGGGCTACACTGAGCACCAG-3 0 ; reverse primer, 5 0 -AAGTGGTCGTTGAGGGCAAT-3 0 ) were synthesized based on the human GAPDH mRNA sequence (GenBank Accession No.
TextSentencer_T71 10871-11140 Sentence denotes Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, the primers (forward primer, 5 0 -TGGGCTACACTGAGCACCAG-3 0 ; reverse primer, 5 0 -AAGTGGTCGTTGAGGGCAAT-3 0 ) were synthesized based on the human GAPDH mRNA sequence (GenBank Accession No.
TextSentencer_T71 10871-11140 Sentence denotes Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, the primers (forward primer, 5 0 -TGGGCTACACTGAGCACCAG-3 0 ; reverse primer, 5 0 -AAGTGGTCGTTGAGGGCAAT-3 0 ) were synthesized based on the human GAPDH mRNA sequence (GenBank Accession No.
TextSentencer_T72 11141-11151 Sentence denotes BC013310).
TextSentencer_T72 11141-11151 Sentence denotes BC013310).
TextSentencer_T72 11141-11151 Sentence denotes BC013310).
TextSentencer_T73 11152-11272 Sentence denotes Its reaction conditions were 25 cycles of denaturation (94°C, 50 s), annealing (60°C, 30 s), and extension (72°C, 30 s).
TextSentencer_T73 11152-11272 Sentence denotes Its reaction conditions were 25 cycles of denaturation (94°C, 50 s), annealing (60°C, 30 s), and extension (72°C, 30 s).
TextSentencer_T73 11152-11272 Sentence denotes Its reaction conditions were 25 cycles of denaturation (94°C, 50 s), annealing (60°C, 30 s), and extension (72°C, 30 s).
TextSentencer_T74 11273-11430 Sentence denotes Then real-time quantitative PCR (Lightcycler, Roche) was performed as described [19] using SYBR Premix Ex Taq (TaKaRa, Dalian) with the following exceptions.
TextSentencer_T74 11273-11430 Sentence denotes Then real-time quantitative PCR (Lightcycler, Roche) was performed as described [19] using SYBR Premix Ex Taq (TaKaRa, Dalian) with the following exceptions.
TextSentencer_T74 11273-11430 Sentence denotes Then real-time quantitative PCR (Lightcycler, Roche) was performed as described [19] using SYBR Premix Ex Taq (TaKaRa, Dalian) with the following exceptions.
TextSentencer_T75 11431-11530 Sentence denotes Briefly, reactions were carried out in 20 ll volumes containing 2 ll reverse transcription product.
TextSentencer_T75 11431-11530 Sentence denotes Briefly, reactions were carried out in 20 ll volumes containing 2 ll reverse transcription product.
TextSentencer_T75 11431-11530 Sentence denotes Briefly, reactions were carried out in 20 ll volumes containing 2 ll reverse transcription product.
TextSentencer_T76 11531-11812 Sentence denotes To quantitate SARS-CoV spike gene transcript levels, dilutions of cDNA from the HEK 293T cells transfected with pEGFP-optS were always run in parallel with cDNA from the transfected HEK 293T cells for use as standard curve (dilutions ranged from 10 6 -10 0 copies of each plasmid).
TextSentencer_T76 11531-11812 Sentence denotes To quantitate SARS-CoV spike gene transcript levels, dilutions of cDNA from the HEK 293T cells transfected with pEGFP-optS were always run in parallel with cDNA from the transfected HEK 293T cells for use as standard curve (dilutions ranged from 10 6 -10 0 copies of each plasmid).
TextSentencer_T76 11531-11812 Sentence denotes To quantitate SARS-CoV spike gene transcript levels, dilutions of cDNA from the HEK 293T cells transfected with pEGFP-optS were always run in parallel with cDNA from the transfected HEK 293T cells for use as standard curve (dilutions ranged from 10 6 -10 0 copies of each plasmid).
TextSentencer_T77 11813-11993 Sentence denotes To perform analysis of relative expression of SARS-CoV spike gene using real-time PCR, we adopted the relative quantitative method [20] , by normalizing to GAPDH expression levels.
TextSentencer_T77 11813-11993 Sentence denotes To perform analysis of relative expression of SARS-CoV spike gene using real-time PCR, we adopted the relative quantitative method [20] , by normalizing to GAPDH expression levels.
TextSentencer_T77 11813-11993 Sentence denotes To perform analysis of relative expression of SARS-CoV spike gene using real-time PCR, we adopted the relative quantitative method [20] , by normalizing to GAPDH expression levels.
TextSentencer_T78 11994-12128 Sentence denotes Then the percentages of SARS-CoV spike mRNA in siRNAs co-transfected cells relative to that in mock-transfected cells were calculated.
TextSentencer_T78 11994-12128 Sentence denotes Then the percentages of SARS-CoV spike mRNA in siRNAs co-transfected cells relative to that in mock-transfected cells were calculated.
TextSentencer_T78 11994-12128 Sentence denotes Then the percentages of SARS-CoV spike mRNA in siRNAs co-transfected cells relative to that in mock-transfected cells were calculated.
TextSentencer_T79 12129-12184 Sentence denotes The primers for GAPDH were the same as described above.
TextSentencer_T79 12129-12184 Sentence denotes The primers for GAPDH were the same as described above.
TextSentencer_T79 12129-12184 Sentence denotes The primers for GAPDH were the same as described above.
TextSentencer_T80 12185-12352 Sentence denotes The primers used for the amplification of SARS-CoV spike gene were 5 0 -TCTGATGCCTTT TCGCTTGA-3 0 (forward primer) and 5 0 -GTGCCCCAAATGTCTT GAGC-3 0 (reverse primer).
TextSentencer_T80 12185-12352 Sentence denotes The primers used for the amplification of SARS-CoV spike gene were 5 0 -TCTGATGCCTTT TCGCTTGA-3 0 (forward primer) and 5 0 -GTGCCCCAAATGTCTT GAGC-3 0 (reverse primer).
TextSentencer_T80 12185-12352 Sentence denotes The primers used for the amplification of SARS-CoV spike gene were 5 0 -TCTGATGCCTTT TCGCTTGA-3 0 (forward primer) and 5 0 -GTGCCCCAAATGTCTT GAGC-3 0 (reverse primer).
TextSentencer_T81 12353-12370 Sentence denotes Western blotting.
TextSentencer_T81 12353-12370 Sentence denotes Western blotting.
TextSentencer_T81 12353-12370 Sentence denotes Western blotting.
TextSentencer_T82 12371-12568 Sentence denotes The transfected or non-transfected cells were harvested and lysed with lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 1% Triton X-100, 1 lg/ml aprotinin, and 100 lg/ml PMSF).
TextSentencer_T82 12371-12568 Sentence denotes The transfected or non-transfected cells were harvested and lysed with lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 1% Triton X-100, 1 lg/ml aprotinin, and 100 lg/ml PMSF).
TextSentencer_T82 12371-12568 Sentence denotes The transfected or non-transfected cells were harvested and lysed with lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 1% Triton X-100, 1 lg/ml aprotinin, and 100 lg/ml PMSF).
TextSentencer_T83 12569-12822 Sentence denotes Equal amounts of total proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to nitrocellulose membrane following the protocol suggested by the manufacturer (Bio-Rad, CA).
TextSentencer_T83 12569-12822 Sentence denotes Equal amounts of total proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to nitrocellulose membrane following the protocol suggested by the manufacturer (Bio-Rad, CA).
TextSentencer_T83 12569-12822 Sentence denotes Equal amounts of total proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to nitrocellulose membrane following the protocol suggested by the manufacturer (Bio-Rad, CA).
TextSentencer_T84 12823-12955 Sentence denotes After blocking non-specific-binding sites with 5% non-fat milk, the membrane was incubated with primary antibodies at 4°C overnight.
TextSentencer_T84 12823-12955 Sentence denotes After blocking non-specific-binding sites with 5% non-fat milk, the membrane was incubated with primary antibodies at 4°C overnight.
TextSentencer_T84 12823-12955 Sentence denotes After blocking non-specific-binding sites with 5% non-fat milk, the membrane was incubated with primary antibodies at 4°C overnight.
TextSentencer_T85 12956-13144 Sentence denotes The primary antibodies used in this experiment were: anti-GFP mouse monoclonal antibody (Santa Cruz, 1:500 dilution) and anti-b-actin goat monoclonal antibody (Santa Cruz, 1:500 dilution).
TextSentencer_T85 12956-13144 Sentence denotes The primary antibodies used in this experiment were: anti-GFP mouse monoclonal antibody (Santa Cruz, 1:500 dilution) and anti-b-actin goat monoclonal antibody (Santa Cruz, 1:500 dilution).
TextSentencer_T85 12956-13144 Sentence denotes The primary antibodies used in this experiment were: anti-GFP mouse monoclonal antibody (Santa Cruz, 1:500 dilution) and anti-b-actin goat monoclonal antibody (Santa Cruz, 1:500 dilution).
TextSentencer_T86 13145-13306 Sentence denotes After washing, the blot was incubated with alkaline phosphatase-conjugated goat anti-mouse IgG and horseradish peroxidase-conjugated anti-goat IgG, respectively.
TextSentencer_T86 13145-13306 Sentence denotes After washing, the blot was incubated with alkaline phosphatase-conjugated goat anti-mouse IgG and horseradish peroxidase-conjugated anti-goat IgG, respectively.
TextSentencer_T86 13145-13306 Sentence denotes After washing, the blot was incubated with alkaline phosphatase-conjugated goat anti-mouse IgG and horseradish peroxidase-conjugated anti-goat IgG, respectively.
TextSentencer_T87 13307-13553 Sentence denotes Immunoreactive bands were visualized with the 5-bromo-4-chioro-3-indolylphosphate/nitroblue tetrazolium (BCIP/ NBT) substrate (Sino-American, Shanghai) and the luminol/enhancer chemiluminescent substrate (ECL) kit (PFBIO, Shanghai), respectively.
TextSentencer_T87 13307-13553 Sentence denotes Immunoreactive bands were visualized with the 5-bromo-4-chioro-3-indolylphosphate/nitroblue tetrazolium (BCIP/ NBT) substrate (Sino-American, Shanghai) and the luminol/enhancer chemiluminescent substrate (ECL) kit (PFBIO, Shanghai), respectively.
TextSentencer_T87 13307-13553 Sentence denotes Immunoreactive bands were visualized with the 5-bromo-4-chioro-3-indolylphosphate/nitroblue tetrazolium (BCIP/ NBT) substrate (Sino-American, Shanghai) and the luminol/enhancer chemiluminescent substrate (ECL) kit (PFBIO, Shanghai), respectively.
TextSentencer_T88 13554-13731 Sentence denotes Expression of SARS-CoV S glycoprotein in cultured cells SARS-CoV spike gene was cloned into pEGFP-N1 with a C-terminal EGFP tag that could be used to monitor protein expression.
TextSentencer_T88 13554-13731 Sentence denotes Expression of SARS-CoV S glycoprotein in cultured cells SARS-CoV spike gene was cloned into pEGFP-N1 with a C-terminal EGFP tag that could be used to monitor protein expression.
TextSentencer_T88 13554-13731 Sentence denotes Expression of SARS-CoV S glycoprotein in cultured cells SARS-CoV spike gene was cloned into pEGFP-N1 with a C-terminal EGFP tag that could be used to monitor protein expression.
TextSentencer_T89 13732-14004 Sentence denotes At 48 h after the transfection, very mild fluorescence was detected in the pEGFP-S transfected HEK 293T cells, while strong fluorescence was observed in the pEGFP-optS transfected cells (Fig. 1A) , which was repeated at least three times but similar results were obtained.
TextSentencer_T89 13732-14004 Sentence denotes At 48 h after the transfection, very mild fluorescence was detected in the pEGFP-S transfected HEK 293T cells, while strong fluorescence was observed in the pEGFP-optS transfected cells (Fig. 1A) , which was repeated at least three times but similar results were obtained.
TextSentencer_T89 13732-14004 Sentence denotes At 48 h after the transfection, very mild fluorescence was detected in the pEGFP-S transfected HEK 293T cells, while strong fluorescence was observed in the pEGFP-optS transfected cells (Fig. 1A) , which was repeated at least three times but similar results were obtained.
TextSentencer_T90 14005-14201 Sentence denotes To determine the levels of S-EGFP fusion protein expressed in transfected cells, we used convalescent SARS patient sera to detect the fusion protein but obtained very faint bands (data not shown).
TextSentencer_T90 14005-14201 Sentence denotes To determine the levels of S-EGFP fusion protein expressed in transfected cells, we used convalescent SARS patient sera to detect the fusion protein but obtained very faint bands (data not shown).
TextSentencer_T90 14005-14201 Sentence denotes To determine the levels of S-EGFP fusion protein expressed in transfected cells, we used convalescent SARS patient sera to detect the fusion protein but obtained very faint bands (data not shown).
TextSentencer_T91 14202-14281 Sentence denotes Therefore, we alternatively employed anti-GFP monoclonal antibody to detect it.
TextSentencer_T91 14202-14281 Sentence denotes Therefore, we alternatively employed anti-GFP monoclonal antibody to detect it.
TextSentencer_T91 14202-14281 Sentence denotes Therefore, we alternatively employed anti-GFP monoclonal antibody to detect it.
TextSentencer_T92 14282-14479 Sentence denotes Western blot analysis of the cell lysates showed that the S-EGFP fusion protein was expressed at high level in pEGFP-optS transfected cells but at low level in pEGFP-S transfected cells (Fig. 1B) .
TextSentencer_T92 14282-14479 Sentence denotes Western blot analysis of the cell lysates showed that the S-EGFP fusion protein was expressed at high level in pEGFP-optS transfected cells but at low level in pEGFP-S transfected cells (Fig. 1B) .
TextSentencer_T92 14282-14479 Sentence denotes Western blot analysis of the cell lysates showed that the S-EGFP fusion protein was expressed at high level in pEGFP-optS transfected cells but at low level in pEGFP-S transfected cells (Fig. 1B) .
TextSentencer_T93 14480-14610 Sentence denotes However, the molecular weight of expressed S-EGFP is larger than the predicted one (about 82 kDa) based on amino acid composition.
TextSentencer_T93 14480-14610 Sentence denotes However, the molecular weight of expressed S-EGFP is larger than the predicted one (about 82 kDa) based on amino acid composition.
TextSentencer_T93 14480-14610 Sentence denotes However, the molecular weight of expressed S-EGFP is larger than the predicted one (about 82 kDa) based on amino acid composition.
TextSentencer_T94 14611-14738 Sentence denotes This may represent glycosylated protein which resulted from posttranslational modification by the eukaryotic expression system.
TextSentencer_T94 14611-14738 Sentence denotes This may represent glycosylated protein which resulted from posttranslational modification by the eukaryotic expression system.
TextSentencer_T94 14611-14738 Sentence denotes This may represent glycosylated protein which resulted from posttranslational modification by the eukaryotic expression system.
TextSentencer_T95 14739-14890 Sentence denotes The only difference between the two plasmids was that the plasmid pEGFP-optS contained codon-optimized spike gene utilizing mammalian preferred codons.
TextSentencer_T95 14739-14890 Sentence denotes The only difference between the two plasmids was that the plasmid pEGFP-optS contained codon-optimized spike gene utilizing mammalian preferred codons.
TextSentencer_T95 14739-14890 Sentence denotes The only difference between the two plasmids was that the plasmid pEGFP-optS contained codon-optimized spike gene utilizing mammalian preferred codons.
TextSentencer_T96 14891-15260 Sentence denotes The data indicated that replacement of codons in the spike gene with those frequently used in mammalian increased S-EGFP fusion protein expression in cultured cells, supporting the hypothesis that the observed differences in efficiency of expression of modified and unmodified spike genes were related to codon usage and GC content, as shown in other studies [21, 22] .
TextSentencer_T96 14891-15260 Sentence denotes The data indicated that replacement of codons in the spike gene with those frequently used in mammalian increased S-EGFP fusion protein expression in cultured cells, supporting the hypothesis that the observed differences in efficiency of expression of modified and unmodified spike genes were related to codon usage and GC content, as shown in other studies [21, 22] .
TextSentencer_T96 14891-15260 Sentence denotes The data indicated that replacement of codons in the spike gene with those frequently used in mammalian increased S-EGFP fusion protein expression in cultured cells, supporting the hypothesis that the observed differences in efficiency of expression of modified and unmodified spike genes were related to codon usage and GC content, as shown in other studies [21, 22] .
TextSentencer_T97 15261-15486 Sentence denotes The underlying mechanisms remain to be elucidated but it is possible that partial optimized codon contained in the spike gene could much more efficiently initiate gene transcription and improve efficiency of gene translation.
TextSentencer_T97 15261-15486 Sentence denotes The underlying mechanisms remain to be elucidated but it is possible that partial optimized codon contained in the spike gene could much more efficiently initiate gene transcription and improve efficiency of gene translation.
TextSentencer_T97 15261-15486 Sentence denotes The underlying mechanisms remain to be elucidated but it is possible that partial optimized codon contained in the spike gene could much more efficiently initiate gene transcription and improve efficiency of gene translation.
TextSentencer_T98 15487-15716 Sentence denotes After all, this transient expression system, transfected with plasmid pEGFP-optS, effectively expresses SARS-CoV S glycoprotein and is suitable for use in the silencing of SARS-CoV S gene expression by siRNA transcribed in vitro.
TextSentencer_T98 15487-15716 Sentence denotes After all, this transient expression system, transfected with plasmid pEGFP-optS, effectively expresses SARS-CoV S glycoprotein and is suitable for use in the silencing of SARS-CoV S gene expression by siRNA transcribed in vitro.
TextSentencer_T98 15487-15716 Sentence denotes After all, this transient expression system, transfected with plasmid pEGFP-optS, effectively expresses SARS-CoV S glycoprotein and is suitable for use in the silencing of SARS-CoV S gene expression by siRNA transcribed in vitro.
TextSentencer_T99 15717-15868 Sentence denotes To silence the expression of SARS-CoV S glycoprotein using RNAi technology, we adopted transcription in vitro with T7 RNA polymerase to acquire siRNAs.
TextSentencer_T99 15717-15868 Sentence denotes To silence the expression of SARS-CoV S glycoprotein using RNAi technology, we adopted transcription in vitro with T7 RNA polymerase to acquire siRNAs.
TextSentencer_T99 15717-15868 Sentence denotes To silence the expression of SARS-CoV S glycoprotein using RNAi technology, we adopted transcription in vitro with T7 RNA polymerase to acquire siRNAs.
TextSentencer_T100 15869-16053 Sentence denotes The sense and antisense of siRNA templates were separately transcribed in vitro with T7 RNA polymerase and annealed to form double-stranded siRNA as described in Materials and methods.
TextSentencer_T100 15869-16053 Sentence denotes The sense and antisense of siRNA templates were separately transcribed in vitro with T7 RNA polymerase and annealed to form double-stranded siRNA as described in Materials and methods.
TextSentencer_T100 15869-16053 Sentence denotes The sense and antisense of siRNA templates were separately transcribed in vitro with T7 RNA polymerase and annealed to form double-stranded siRNA as described in Materials and methods.
TextSentencer_T101 16054-16243 Sentence denotes The 5 0 overhanging leader sequence of the generated dsRNA and DNA template remainders were digested with single-strand specific nuclease (S1 nuclease) and RNase-free DNase I, respectively.
TextSentencer_T101 16054-16243 Sentence denotes The 5 0 overhanging leader sequence of the generated dsRNA and DNA template remainders were digested with single-strand specific nuclease (S1 nuclease) and RNase-free DNase I, respectively.
TextSentencer_T101 16054-16243 Sentence denotes The 5 0 overhanging leader sequence of the generated dsRNA and DNA template remainders were digested with single-strand specific nuclease (S1 nuclease) and RNase-free DNase I, respectively.
TextSentencer_T102 16244-16341 Sentence denotes The double-stranded siRNAs of 21 nt in length were obtained and found intact as shown in Fig. 2 .
TextSentencer_T102 16244-16341 Sentence denotes The double-stranded siRNAs of 21 nt in length were obtained and found intact as shown in Fig. 2 .
TextSentencer_T102 16244-16341 Sentence denotes The double-stranded siRNAs of 21 nt in length were obtained and found intact as shown in Fig. 2 .
TextSentencer_T103 16342-16611 Sentence denotes In mammalian cells, dsRNA duplexes >30 nt in size lead to the induction of interferon response and global RNA degradation through the activation of RNase L, while duplexes of 21 nt synthesized in vitro induce RNAi after being introduced into human and mouse cell lines.
TextSentencer_T103 16342-16611 Sentence denotes In mammalian cells, dsRNA duplexes >30 nt in size lead to the induction of interferon response and global RNA degradation through the activation of RNase L, while duplexes of 21 nt synthesized in vitro induce RNAi after being introduced into human and mouse cell lines.
TextSentencer_T103 16342-16611 Sentence denotes In mammalian cells, dsRNA duplexes >30 nt in size lead to the induction of interferon response and global RNA degradation through the activation of RNase L, while duplexes of 21 nt synthesized in vitro induce RNAi after being introduced into human and mouse cell lines.
TextSentencer_T104 16612-16734 Sentence denotes This effect of gene silencing induced by siRNA (<30 nt) was sequence specific without the activation of RNase L [23, 24] .
TextSentencer_T104 16612-16734 Sentence denotes This effect of gene silencing induced by siRNA (<30 nt) was sequence specific without the activation of RNase L [23, 24] .
TextSentencer_T104 16612-16734 Sentence denotes This effect of gene silencing induced by siRNA (<30 nt) was sequence specific without the activation of RNase L [23, 24] .
TextSentencer_T105 16735-16925 Sentence denotes Although siRNAs can be acquired through several methods [25] [26] [27] [28] [29] [30] [31] , transcription in vitro by T7 polymerase adopted here is more convenient and reliable than others.
TextSentencer_T105 16735-16925 Sentence denotes Although siRNAs can be acquired through several methods [25] [26] [27] [28] [29] [30] [31] , transcription in vitro by T7 polymerase adopted here is more convenient and reliable than others.
TextSentencer_T105 16735-16925 Sentence denotes Although siRNAs can be acquired through several methods [25] [26] [27] [28] [29] [30] [31] , transcription in vitro by T7 polymerase adopted here is more convenient and reliable than others.
TextSentencer_T106 16926-17042 Sentence denotes To examine RNA levels of SARS-CoV spike gene in siRNAs co-transfected cells, semi-quantitative RT-PCR was performed.
TextSentencer_T106 16926-17042 Sentence denotes To examine RNA levels of SARS-CoV spike gene in siRNAs co-transfected cells, semi-quantitative RT-PCR was performed.
TextSentencer_T106 16926-17042 Sentence denotes To examine RNA levels of SARS-CoV spike gene in siRNAs co-transfected cells, semi-quantitative RT-PCR was performed.
TextSentencer_T107 17043-17374 Sentence denotes The data showed that EGFP-siRNA, S-siRNA1, and S-siRNA2 reduced the accumulation of S mRNA (Fig. 3A, upper panel, lane 3 , 5, and 6 compared to lane 2), and scramble siRNA almost had no effect on S mRNA (Fig. 3A , upper panel, lane 4 compared to lane 2) while GAPDH mRNA was not effected by the four siRNAs (Fig. 3A, lower panel) .
TextSentencer_T107 17043-17374 Sentence denotes The data showed that EGFP-siRNA, S-siRNA1, and S-siRNA2 reduced the accumulation of S mRNA (Fig. 3A, upper panel, lane 3 , 5, and 6 compared to lane 2), and scramble siRNA almost had no effect on S mRNA (Fig. 3A , upper panel, lane 4 compared to lane 2) while GAPDH mRNA was not effected by the four siRNAs (Fig. 3A, lower panel) .
TextSentencer_T107 17043-17374 Sentence denotes The data showed that EGFP-siRNA, S-siRNA1, and S-siRNA2 reduced the accumulation of S mRNA (Fig. 3A, upper panel, lane 3 , 5, and 6 compared to lane 2), and scramble siRNA almost had no effect on S mRNA (Fig. 3A , upper panel, lane 4 compared to lane 2) while GAPDH mRNA was not effected by the four siRNAs (Fig. 3A, lower panel) .
TextSentencer_T108 17375-17527 Sentence denotes To more accurately quantitate RNA levels of SARS-CoV spike gene, we performed real-time quantitative PCR using primers specific to spike gene and GAPDH.
TextSentencer_T108 17375-17527 Sentence denotes To more accurately quantitate RNA levels of SARS-CoV spike gene, we performed real-time quantitative PCR using primers specific to spike gene and GAPDH.
TextSentencer_T108 17375-17527 Sentence denotes To more accurately quantitate RNA levels of SARS-CoV spike gene, we performed real-time quantitative PCR using primers specific to spike gene and GAPDH.
TextSentencer_T109 17528-17619 Sentence denotes Dilutions of cDNA from the pEGFP-optS transfected cells were run for use as standard curve.
TextSentencer_T109 17528-17619 Sentence denotes Dilutions of cDNA from the pEGFP-optS transfected cells were run for use as standard curve.
TextSentencer_T109 17528-17619 Sentence denotes Dilutions of cDNA from the pEGFP-optS transfected cells were run for use as standard curve.
TextSentencer_T110 17620-17924 Sentence denotes Based on the standard curve generated, the relative expression of SARS-CoV spike gene in each RNA transcript was obtained by normalizing to GAPDH expression levels, and then the percentages of SARS-CoV spike mRNA in siR-NAs co-transfected cells relative to that in mock-transfected cells were calculated.
TextSentencer_T110 17620-17924 Sentence denotes Based on the standard curve generated, the relative expression of SARS-CoV spike gene in each RNA transcript was obtained by normalizing to GAPDH expression levels, and then the percentages of SARS-CoV spike mRNA in siR-NAs co-transfected cells relative to that in mock-transfected cells were calculated.
TextSentencer_T110 17620-17924 Sentence denotes Based on the standard curve generated, the relative expression of SARS-CoV spike gene in each RNA transcript was obtained by normalizing to GAPDH expression levels, and then the percentages of SARS-CoV spike mRNA in siR-NAs co-transfected cells relative to that in mock-transfected cells were calculated.
TextSentencer_T111 17925-18161 Sentence denotes The results of relative quantitative analysis revealed that the transcript levels of SARS-CoV spike gene at 48 h posttransfection were decreased about 10-and 9-fold in cells transfected with SARS-CoV S-siRNA1 and S-siRNA2, respectively.
TextSentencer_T111 17925-18161 Sentence denotes The results of relative quantitative analysis revealed that the transcript levels of SARS-CoV spike gene at 48 h posttransfection were decreased about 10-and 9-fold in cells transfected with SARS-CoV S-siRNA1 and S-siRNA2, respectively.
TextSentencer_T111 17925-18161 Sentence denotes The results of relative quantitative analysis revealed that the transcript levels of SARS-CoV spike gene at 48 h posttransfection were decreased about 10-and 9-fold in cells transfected with SARS-CoV S-siRNA1 and S-siRNA2, respectively.
TextSentencer_T112 18162-18351 Sentence denotes Correspondingly, spike transcript levels were decreased about 16-fold in cells transfected with EGFP-siRNA but not significantly changed in cells transfected with scramble siRNA (Fig. 3B) .
TextSentencer_T112 18162-18351 Sentence denotes Correspondingly, spike transcript levels were decreased about 16-fold in cells transfected with EGFP-siRNA but not significantly changed in cells transfected with scramble siRNA (Fig. 3B) .
TextSentencer_T112 18162-18351 Sentence denotes Correspondingly, spike transcript levels were decreased about 16-fold in cells transfected with EGFP-siRNA but not significantly changed in cells transfected with scramble siRNA (Fig. 3B) .
TextSentencer_T113 18352-18655 Sentence denotes RT-PCR and real-time quantitative PCR analysis showed that SARS-CoV spike transcript level in S-siRNA1, S-siRNA2 or EGFP-siRNA transfected HEK 293T cells decreased obviously, whereas no significant inhibition of SARS-CoV spike transcript level was observed in scramble siRNA transfected cells (Fig. 3) .
TextSentencer_T113 18352-18655 Sentence denotes RT-PCR and real-time quantitative PCR analysis showed that SARS-CoV spike transcript level in S-siRNA1, S-siRNA2 or EGFP-siRNA transfected HEK 293T cells decreased obviously, whereas no significant inhibition of SARS-CoV spike transcript level was observed in scramble siRNA transfected cells (Fig. 3) .
TextSentencer_T113 18352-18655 Sentence denotes RT-PCR and real-time quantitative PCR analysis showed that SARS-CoV spike transcript level in S-siRNA1, S-siRNA2 or EGFP-siRNA transfected HEK 293T cells decreased obviously, whereas no significant inhibition of SARS-CoV spike transcript level was observed in scramble siRNA transfected cells (Fig. 3) .
TextSentencer_T114 18656-18804 Sentence denotes Whether RNAi spreading, as demonstrated in plants and C. elegans [32, 33] , has occurred in this experiment required further experiments to address.
TextSentencer_T114 18656-18804 Sentence denotes Whether RNAi spreading, as demonstrated in plants and C. elegans [32, 33] , has occurred in this experiment required further experiments to address.
TextSentencer_T114 18656-18804 Sentence denotes Whether RNAi spreading, as demonstrated in plants and C. elegans [32, 33] , has occurred in this experiment required further experiments to address.
TextSentencer_T115 18805-18951 Sentence denotes The data above suggested that the effect of gene silencing induced by siRNA should be sequence specific and entire open reading frame (ORF) based.
TextSentencer_T115 18805-18951 Sentence denotes The data above suggested that the effect of gene silencing induced by siRNA should be sequence specific and entire open reading frame (ORF) based.
TextSentencer_T115 18805-18951 Sentence denotes The data above suggested that the effect of gene silencing induced by siRNA should be sequence specific and entire open reading frame (ORF) based.
TextSentencer_T116 18952-19209 Sentence denotes The siRNAs transcribed in vitro can effectively down-regulate SARS-CoV S RNA levels in either EGFP-or S-specific siRNA transfected HEK 293T cells, corresponding to the reported mechanism that siRNAs degraded target mRNA at RNA level instead of at DNA level.
TextSentencer_T116 18952-19209 Sentence denotes The siRNAs transcribed in vitro can effectively down-regulate SARS-CoV S RNA levels in either EGFP-or S-specific siRNA transfected HEK 293T cells, corresponding to the reported mechanism that siRNAs degraded target mRNA at RNA level instead of at DNA level.
TextSentencer_T116 18952-19209 Sentence denotes The siRNAs transcribed in vitro can effectively down-regulate SARS-CoV S RNA levels in either EGFP-or S-specific siRNA transfected HEK 293T cells, corresponding to the reported mechanism that siRNAs degraded target mRNA at RNA level instead of at DNA level.
TextSentencer_T117 19210-19413 Sentence denotes To determine whether the synthesized siRNA could effectively silence SARS-CoV S glycoprotein expression in cultured cells, we co-transfected HEK 293T cells with plasmid pEGFP-optS and the various siRNAs.
TextSentencer_T117 19210-19413 Sentence denotes To determine whether the synthesized siRNA could effectively silence SARS-CoV S glycoprotein expression in cultured cells, we co-transfected HEK 293T cells with plasmid pEGFP-optS and the various siRNAs.
TextSentencer_T117 19210-19413 Sentence denotes To determine whether the synthesized siRNA could effectively silence SARS-CoV S glycoprotein expression in cultured cells, we co-transfected HEK 293T cells with plasmid pEGFP-optS and the various siRNAs.
TextSentencer_T118 19414-19502 Sentence denotes The cells were examined microscopically at 48 h posttransfection for green fluorescence.
TextSentencer_T118 19414-19502 Sentence denotes The cells were examined microscopically at 48 h posttransfection for green fluorescence.
TextSentencer_T118 19414-19502 Sentence denotes The cells were examined microscopically at 48 h posttransfection for green fluorescence.
TextSentencer_T119 19503-19689 Sentence denotes As can be seen in Fig. 4 , the fluorescence imaging was much stronger in the HEK 293T cells transfected with scramble siRNA than in those transfected with SARS-CoV S-siRNA1 and S-siRNA2.
TextSentencer_T119 19503-19689 Sentence denotes As can be seen in Fig. 4 , the fluorescence imaging was much stronger in the HEK 293T cells transfected with scramble siRNA than in those transfected with SARS-CoV S-siRNA1 and S-siRNA2.
TextSentencer_T119 19503-19689 Sentence denotes As can be seen in Fig. 4 , the fluorescence imaging was much stronger in the HEK 293T cells transfected with scramble siRNA than in those transfected with SARS-CoV S-siRNA1 and S-siRNA2.
TextSentencer_T120 19690-19764 Sentence denotes Very mild green fluorescence was observed in EGFP-siRNA transfected cells.
TextSentencer_T120 19690-19764 Sentence denotes Very mild green fluorescence was observed in EGFP-siRNA transfected cells.
TextSentencer_T120 19690-19764 Sentence denotes Very mild green fluorescence was observed in EGFP-siRNA transfected cells.
TextSentencer_T121 19765-19927 Sentence denotes The fluorescence intensity of HEK 293T cells co-transfected with scramble siRNA showed no significant difference compared with pEGFP-optS transfected cells (Figs.
TextSentencer_T121 19765-19927 Sentence denotes The fluorescence intensity of HEK 293T cells co-transfected with scramble siRNA showed no significant difference compared with pEGFP-optS transfected cells (Figs.
TextSentencer_T121 19765-19927 Sentence denotes The fluorescence intensity of HEK 293T cells co-transfected with scramble siRNA showed no significant difference compared with pEGFP-optS transfected cells (Figs.
TextSentencer_T122 19928-19952 Sentence denotes 4, upper panel A and C).
TextSentencer_T122 19928-19952 Sentence denotes 4, upper panel A and C).
TextSentencer_T122 19928-19952 Sentence denotes 4, upper panel A and C).
TextSentencer_T123 19953-20033 Sentence denotes The lower panels represent the corresponding image observed by light microscope.
TextSentencer_T123 19953-20033 Sentence denotes The lower panels represent the corresponding image observed by light microscope.
TextSentencer_T123 19953-20033 Sentence denotes The lower panels represent the corresponding image observed by light microscope.
TextSentencer_T124 20034-20135 Sentence denotes Specific silencing of the green fluorescence was confirmed at least in three independent experiments.
TextSentencer_T124 20034-20135 Sentence denotes Specific silencing of the green fluorescence was confirmed at least in three independent experiments.
TextSentencer_T124 20034-20135 Sentence denotes Specific silencing of the green fluorescence was confirmed at least in three independent experiments.
TextSentencer_T125 20136-20395 Sentence denotes To further examine the silencing effect of SARS-CoV S-siRNAs, we collected cells and analyzed with fluorescence-activated cell sorting (FACS) 48 h after transfection for EGFP expression with CellQuest software, using non-transfected HEK 293T cells as control.
TextSentencer_T125 20136-20395 Sentence denotes To further examine the silencing effect of SARS-CoV S-siRNAs, we collected cells and analyzed with fluorescence-activated cell sorting (FACS) 48 h after transfection for EGFP expression with CellQuest software, using non-transfected HEK 293T cells as control.
TextSentencer_T125 20136-20395 Sentence denotes To further examine the silencing effect of SARS-CoV S-siRNAs, we collected cells and analyzed with fluorescence-activated cell sorting (FACS) 48 h after transfection for EGFP expression with CellQuest software, using non-transfected HEK 293T cells as control.
TextSentencer_T126 20396-20750 Sentence denotes As shown in Fig. 5 , compared with the cells transfected with plasmid pEGFP-optS alone, the cells co-transfected with pEGFP-optS and scramble siRNA gave no significant reduction of EGFP expression, whereas the EGFP-siRNA gave an about 3.7-and 3.8-fold reduction in percentage of fluorescence cell population and mean fluorescence intensity, respectively.
TextSentencer_T126 20396-20750 Sentence denotes As shown in Fig. 5 , compared with the cells transfected with plasmid pEGFP-optS alone, the cells co-transfected with pEGFP-optS and scramble siRNA gave no significant reduction of EGFP expression, whereas the EGFP-siRNA gave an about 3.7-and 3.8-fold reduction in percentage of fluorescence cell population and mean fluorescence intensity, respectively.
TextSentencer_T126 20396-20750 Sentence denotes As shown in Fig. 5 , compared with the cells transfected with plasmid pEGFP-optS alone, the cells co-transfected with pEGFP-optS and scramble siRNA gave no significant reduction of EGFP expression, whereas the EGFP-siRNA gave an about 3.7-and 3.8-fold reduction in percentage of fluorescence cell population and mean fluorescence intensity, respectively.
TextSentencer_T127 20751-20904 Sentence denotes Percentage of fluorescence cell population and mean fluorescence intensity reduced about 1.6-and 3.3-fold for S-siRNA1 and 5.8-and 6.1-fold for S-siRNA2.
TextSentencer_T127 20751-20904 Sentence denotes Percentage of fluorescence cell population and mean fluorescence intensity reduced about 1.6-and 3.3-fold for S-siRNA1 and 5.8-and 6.1-fold for S-siRNA2.
TextSentencer_T127 20751-20904 Sentence denotes Percentage of fluorescence cell population and mean fluorescence intensity reduced about 1.6-and 3.3-fold for S-siRNA1 and 5.8-and 6.1-fold for S-siRNA2.
TextSentencer_T128 20905-20969 Sentence denotes This result was in keeping with fluorescence imaging in Fig. 4 .
TextSentencer_T128 20905-20969 Sentence denotes This result was in keeping with fluorescence imaging in Fig. 4 .
TextSentencer_T128 20905-20969 Sentence denotes This result was in keeping with fluorescence imaging in Fig. 4 .
TextSentencer_T129 20970-21126 Sentence denotes To explore the mechanism of siRNA in transfected cells, we examined transfected cells for S-EGFP fusion protein levels in the presence or absence of siRNAs.
TextSentencer_T129 20970-21126 Sentence denotes To explore the mechanism of siRNA in transfected cells, we examined transfected cells for S-EGFP fusion protein levels in the presence or absence of siRNAs.
TextSentencer_T129 20970-21126 Sentence denotes To explore the mechanism of siRNA in transfected cells, we examined transfected cells for S-EGFP fusion protein levels in the presence or absence of siRNAs.
TextSentencer_T130 21127-21435 Sentence denotes Western blot analysis showed that compared with the cells transfected with plasmid pEGFP-optS alone, S-EGFP fusion protein expression was reduced in cells co-transfected with EGFP-siRNA, S-siRNA1 or S-siR-NA2, but not reduced in cells co-transfected with pEGFP-optS and scramble siRNA (Fig. 6, upper panel) .
TextSentencer_T130 21127-21435 Sentence denotes Western blot analysis showed that compared with the cells transfected with plasmid pEGFP-optS alone, S-EGFP fusion protein expression was reduced in cells co-transfected with EGFP-siRNA, S-siRNA1 or S-siR-NA2, but not reduced in cells co-transfected with pEGFP-optS and scramble siRNA (Fig. 6, upper panel) .
TextSentencer_T130 21127-21435 Sentence denotes Western blot analysis showed that compared with the cells transfected with plasmid pEGFP-optS alone, S-EGFP fusion protein expression was reduced in cells co-transfected with EGFP-siRNA, S-siRNA1 or S-siR-NA2, but not reduced in cells co-transfected with pEGFP-optS and scramble siRNA (Fig. 6, upper panel) .
TextSentencer_T131 21436-21740 Sentence denotes Interestingly, a little difference in the level of S-EGFP fusion protein expression in S-siRNA1 and S-siRNA2 transfected cells was detected, indicating that the effect of silencing S-EGFP fusion protein expression by S-siRNA2 was better than S-siRNA1, which was consistent with the previous data in Figs.
TextSentencer_T131 21436-21740 Sentence denotes Interestingly, a little difference in the level of S-EGFP fusion protein expression in S-siRNA1 and S-siRNA2 transfected cells was detected, indicating that the effect of silencing S-EGFP fusion protein expression by S-siRNA2 was better than S-siRNA1, which was consistent with the previous data in Figs.
TextSentencer_T131 21436-21740 Sentence denotes Interestingly, a little difference in the level of S-EGFP fusion protein expression in S-siRNA1 and S-siRNA2 transfected cells was detected, indicating that the effect of silencing S-EGFP fusion protein expression by S-siRNA2 was better than S-siRNA1, which was consistent with the previous data in Figs.
TextSentencer_T132 21741-21749 Sentence denotes 4 and 5.
TextSentencer_T132 21741-21749 Sentence denotes 4 and 5.
TextSentencer_T132 21741-21749 Sentence denotes 4 and 5.
TextSentencer_T133 21750-21853 Sentence denotes As a loading control, b-actin was not affected by any of the four siRNAs tested (Fig. 6, lower panel) .
TextSentencer_T133 21750-21853 Sentence denotes As a loading control, b-actin was not affected by any of the four siRNAs tested (Fig. 6, lower panel) .
TextSentencer_T133 21750-21853 Sentence denotes As a loading control, b-actin was not affected by any of the four siRNAs tested (Fig. 6, lower panel) .
TextSentencer_T134 21854-21975 Sentence denotes These data indicated that siRNAs silenced SARS-CoV S glycoprotein expression through blocking the accumulation of S mRNA.
TextSentencer_T134 21854-21975 Sentence denotes These data indicated that siRNAs silenced SARS-CoV S glycoprotein expression through blocking the accumulation of S mRNA.
TextSentencer_T134 21854-21975 Sentence denotes These data indicated that siRNAs silenced SARS-CoV S glycoprotein expression through blocking the accumulation of S mRNA.
TextSentencer_T135 21976-22169 Sentence denotes SARS-CoV S glycoprotein initiates entry of SARS-CoV into susceptible cells mediated by combining with cell surface receptors followed by conformational changes leading to membrane fusion [34] .
TextSentencer_T135 21976-22169 Sentence denotes SARS-CoV S glycoprotein initiates entry of SARS-CoV into susceptible cells mediated by combining with cell surface receptors followed by conformational changes leading to membrane fusion [34] .
TextSentencer_T135 21976-22169 Sentence denotes SARS-CoV S glycoprotein initiates entry of SARS-CoV into susceptible cells mediated by combining with cell surface receptors followed by conformational changes leading to membrane fusion [34] .
TextSentencer_T136 22170-22393 Sentence denotes Recently, the angiotensin-converting enzyme 2 (ACE2) was identified as a functional receptor for SARS-CoV [35] and its binding site on the SARS-CoV S glycoprotein was localized between amino acid residues 303 and 537 [36] .
TextSentencer_T136 22170-22393 Sentence denotes Recently, the angiotensin-converting enzyme 2 (ACE2) was identified as a functional receptor for SARS-CoV [35] and its binding site on the SARS-CoV S glycoprotein was localized between amino acid residues 303 and 537 [36] .
TextSentencer_T136 22170-22393 Sentence denotes Recently, the angiotensin-converting enzyme 2 (ACE2) was identified as a functional receptor for SARS-CoV [35] and its binding site on the SARS-CoV S glycoprotein was localized between amino acid residues 303 and 537 [36] .
TextSentencer_T137 22394-22516 Sentence denotes Therefore, to block the combination of S glycoprotein with receptor efficiently is crucial to prevent the virus infection.
TextSentencer_T137 22394-22516 Sentence denotes Therefore, to block the combination of S glycoprotein with receptor efficiently is crucial to prevent the virus infection.
TextSentencer_T137 22394-22516 Sentence denotes Therefore, to block the combination of S glycoprotein with receptor efficiently is crucial to prevent the virus infection.
TextSentencer_T138 22517-22732 Sentence denotes As a cellular defense mechanism, RNAi has been employed in anti-virus infections in viruses such as human immunodeficiency virus and hepatitis C/B virus [37] and inhi-bition of viral replication in host cells [38] .
TextSentencer_T138 22517-22732 Sentence denotes As a cellular defense mechanism, RNAi has been employed in anti-virus infections in viruses such as human immunodeficiency virus and hepatitis C/B virus [37] and inhi-bition of viral replication in host cells [38] .
TextSentencer_T138 22517-22732 Sentence denotes As a cellular defense mechanism, RNAi has been employed in anti-virus infections in viruses such as human immunodeficiency virus and hepatitis C/B virus [37] and inhi-bition of viral replication in host cells [38] .
TextSentencer_T139 22733-22994 Sentence denotes Zhang et al. [16] introduced vector-mediated RNAi technology into the study of SARS-CoV infection, and found that the siRNAs generated from the DNA vector blocked the expression of SARS-CoV S gene and inhibited the replication of SARS-CoV in the infected cells.
TextSentencer_T139 22733-22994 Sentence denotes Zhang et al. [16] introduced vector-mediated RNAi technology into the study of SARS-CoV infection, and found that the siRNAs generated from the DNA vector blocked the expression of SARS-CoV S gene and inhibited the replication of SARS-CoV in the infected cells.
TextSentencer_T139 22733-22994 Sentence denotes Zhang et al. [16] introduced vector-mediated RNAi technology into the study of SARS-CoV infection, and found that the siRNAs generated from the DNA vector blocked the expression of SARS-CoV S gene and inhibited the replication of SARS-CoV in the infected cells.
TextSentencer_T140 22995-23203 Sentence denotes Their work enlightened us to explore the possibility of silencing SARS-CoV S glycoprotein expression by siRNA transcribed in vitro by T7 RNA polymerase and blocking its combination with cell surface receptor.
TextSentencer_T140 22995-23203 Sentence denotes Their work enlightened us to explore the possibility of silencing SARS-CoV S glycoprotein expression by siRNA transcribed in vitro by T7 RNA polymerase and blocking its combination with cell surface receptor.
TextSentencer_T140 22995-23203 Sentence denotes Their work enlightened us to explore the possibility of silencing SARS-CoV S glycoprotein expression by siRNA transcribed in vitro by T7 RNA polymerase and blocking its combination with cell surface receptor.
TextSentencer_T141 23204-23497 Sentence denotes In the present work, we cloned wild and codon-optimized SARS-CoV spike gene encoding N-terminal amino acid residues 1-690, which contained the identified receptor-binding domain (RBD), and found that the codonoptimized spike gene can improve the expression of S glycoprotein in cultured cells.
TextSentencer_T141 23204-23497 Sentence denotes In the present work, we cloned wild and codon-optimized SARS-CoV spike gene encoding N-terminal amino acid residues 1-690, which contained the identified receptor-binding domain (RBD), and found that the codonoptimized spike gene can improve the expression of S glycoprotein in cultured cells.
TextSentencer_T141 23204-23497 Sentence denotes In the present work, we cloned wild and codon-optimized SARS-CoV spike gene encoding N-terminal amino acid residues 1-690, which contained the identified receptor-binding domain (RBD), and found that the codonoptimized spike gene can improve the expression of S glycoprotein in cultured cells.
TextSentencer_T142 23498-23759 Sentence denotes Our results demonstrated that the siRNAs transcribed in vitro were effective, and S-siRNA1 and S-siRNA2 can both silence SARS-CoV S glycoprotein expression, which provided evidence for the further study of SARS-CoV infection of cells mediated by S glycoprotein.
TextSentencer_T142 23498-23759 Sentence denotes Our results demonstrated that the siRNAs transcribed in vitro were effective, and S-siRNA1 and S-siRNA2 can both silence SARS-CoV S glycoprotein expression, which provided evidence for the further study of SARS-CoV infection of cells mediated by S glycoprotein.
TextSentencer_T142 23498-23759 Sentence denotes Our results demonstrated that the siRNAs transcribed in vitro were effective, and S-siRNA1 and S-siRNA2 can both silence SARS-CoV S glycoprotein expression, which provided evidence for the further study of SARS-CoV infection of cells mediated by S glycoprotein.
TextSentencer_T143 23760-23869 Sentence denotes Studies on the interaction of SARS-CoV S glycoprotein and its receptor using RNAi technology are in progress.
TextSentencer_T143 23760-23869 Sentence denotes Studies on the interaction of SARS-CoV S glycoprotein and its receptor using RNAi technology are in progress.
TextSentencer_T143 23760-23869 Sentence denotes Studies on the interaction of SARS-CoV S glycoprotein and its receptor using RNAi technology are in progress.
TextSentencer_T144 23870-24054 Sentence denotes Our findings in this work could help in the development of novel SARS-CoV therapeutic agent and provide a new strategy for the prevention and treatment of SARS-CoV infection in humans.
TextSentencer_T144 23870-24054 Sentence denotes Our findings in this work could help in the development of novel SARS-CoV therapeutic agent and provide a new strategy for the prevention and treatment of SARS-CoV infection in humans.
TextSentencer_T144 23870-24054 Sentence denotes Our findings in this work could help in the development of novel SARS-CoV therapeutic agent and provide a new strategy for the prevention and treatment of SARS-CoV infection in humans.
TextSentencer_T145 24055-24063 Sentence denotes Fig. 6 .
TextSentencer_T145 24055-24063 Sentence denotes Fig. 6 .
TextSentencer_T145 24055-24063 Sentence denotes Fig. 6 .
TextSentencer_T146 24064-24127 Sentence denotes Effect of siRNA on S-EGFP protein expression in HEK 293T cells.
TextSentencer_T146 24064-24127 Sentence denotes Effect of siRNA on S-EGFP protein expression in HEK 293T cells.
TextSentencer_T146 24064-24127 Sentence denotes Effect of siRNA on S-EGFP protein expression in HEK 293T cells.
TextSentencer_T147 24128-24394 Sentence denotes Western blot analysis was performed on equal amounts of proteins harvested from mock-or siRNA co-transfected HEK 293T cells at 48 h posttransfection by using GFP-and GAPDH-specific antibodies described in Materials and methods. b-Actin was used as a loading control.
TextSentencer_T147 24128-24394 Sentence denotes Western blot analysis was performed on equal amounts of proteins harvested from mock-or siRNA co-transfected HEK 293T cells at 48 h posttransfection by using GFP-and GAPDH-specific antibodies described in Materials and methods. b-Actin was used as a loading control.
TextSentencer_T147 24128-24394 Sentence denotes Western blot analysis was performed on equal amounts of proteins harvested from mock-or siRNA co-transfected HEK 293T cells at 48 h posttransfection by using GFP-and GAPDH-specific antibodies described in Materials and methods. b-Actin was used as a loading control.