Potato Virus Y mRNA Expression Knockdown Mediated by siRNAs in Cultured Mammalian Cell Line
RNA interference （RNAi） is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes. The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to b...
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| Veröffentlicht in: | Virologica Sinica 2011-04, Vol.26 (2), p.105-113 |
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| creator | Tabassum, Bushra Nasir, Idrees Ahmad Husnain, Tayyab |
| description | RNA interference （RNAi） is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes. The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion. In our study, a 480bp fragment of the capsid protein gene of potato virus Y （CP-PVY） was used as a target to downregulate PVY mRNA expression in-vitro, as the CP gene interferes with viral uncoating, translation and replication. A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells. CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs. Six biological replicates were performed in this study. In our findings, one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%. |
| doi_str_mv | 10.1007/s12250-011-3161-x |
| format | Article |
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The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion. In our study, a 480bp fragment of the capsid protein gene of potato virus Y （CP-PVY） was used as a target to downregulate PVY mRNA expression in-vitro, as the CP gene interferes with viral uncoating, translation and replication. A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells. CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs. Six biological replicates were performed in this study. In our findings, one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.</description><identifier>ISSN: 1674-0769</identifier><identifier>EISSN: 1995-820X</identifier><identifier>DOI: 10.1007/s12250-011-3161-x</identifier><identifier>PMID: 21468933</identifier><language>eng</language><publisher>Heidelberg: Springer-Verlag</publisher><subject>Animals ; Biochemistry ; Biomedical and Life Sciences ; Biomedicine ; capsid ; Capsid Proteins - genetics ; Capsid Proteins - metabolism ; CHO Cells ; CP基因 ; Cricetinae ; Cricetulus ; Down-Regulation ; gene expression ; Gene Expression Regulation, Viral ; Gene Knockdown Techniques ; genes ; mammals ; Medical Microbiology ; messenger RNA ; Microbial Genetics and Genomics ; Microbiology ; Oncology ; PCR技术 ; Plant Diseases - prevention & control ; Plant Diseases - virology ; Potato virus Y ; potatoes ; Potyvirus - genetics ; Potyvirus - physiology ; quantitative polymerase chain reaction ; RNA Interference ; RNA, Messenger - genetics ; RNA, Messenger - metabolism ; RNA, Small Interfering - genetics ; RNA, Small Interfering - metabolism ; RNA干扰 ; screening ; siRNAs ; small interfering RNA ; Solanum tuberosum ; Solanum tuberosum - virology ; Transfection ; translation (genetics) ; Virology ; 哺乳动物 ; 基因表达 ; 细胞株 ; 马铃薯Y病毒</subject><ispartof>Virologica Sinica, 2011-04, Vol.26 (2), p.105-113</ispartof><rights>Wuhan Institute of Virology, CAS and Springer-Verlag Berlin Heidelberg 2011</rights><rights>Copyright © Wanfang Data Co. 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All Rights Reserved.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4704-367645c3d00cd866beb2e6162d624c3ad9673351055cb2bb7b4e443beee082073</citedby><cites>FETCH-LOGICAL-c4704-367645c3d00cd866beb2e6162d624c3ad9673351055cb2bb7b4e443beee082073</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://image.cqvip.com/vip1000/qk/92590B/92590B.jpg</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8222446/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8222446/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,27901,27902,41464,42533,51294,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21468933$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Tabassum, Bushra</creatorcontrib><creatorcontrib>Nasir, Idrees Ahmad</creatorcontrib><creatorcontrib>Husnain, Tayyab</creatorcontrib><title>Potato Virus Y mRNA Expression Knockdown Mediated by siRNAs in Cultured Mammalian Cell Line</title><title>Virologica Sinica</title><addtitle>Virol. Sin</addtitle><addtitle>Virologica Sinica</addtitle><description>RNA interference （RNAi） is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes. The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion. In our study, a 480bp fragment of the capsid protein gene of potato virus Y （CP-PVY） was used as a target to downregulate PVY mRNA expression in-vitro, as the CP gene interferes with viral uncoating, translation and replication. A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells. CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs. Six biological replicates were performed in this study. In our findings, one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.</description><subject>Animals</subject><subject>Biochemistry</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedicine</subject><subject>capsid</subject><subject>Capsid Proteins - genetics</subject><subject>Capsid Proteins - metabolism</subject><subject>CHO Cells</subject><subject>CP基因</subject><subject>Cricetinae</subject><subject>Cricetulus</subject><subject>Down-Regulation</subject><subject>gene expression</subject><subject>Gene Expression Regulation, Viral</subject><subject>Gene Knockdown Techniques</subject><subject>genes</subject><subject>mammals</subject><subject>Medical Microbiology</subject><subject>messenger RNA</subject><subject>Microbial Genetics and Genomics</subject><subject>Microbiology</subject><subject>Oncology</subject><subject>PCR技术</subject><subject>Plant Diseases - prevention & control</subject><subject>Plant Diseases - virology</subject><subject>Potato virus Y</subject><subject>potatoes</subject><subject>Potyvirus - genetics</subject><subject>Potyvirus - physiology</subject><subject>quantitative polymerase chain reaction</subject><subject>RNA Interference</subject><subject>RNA, Messenger - genetics</subject><subject>RNA, Messenger - metabolism</subject><subject>RNA, Small Interfering - genetics</subject><subject>RNA, Small Interfering - metabolism</subject><subject>RNA干扰</subject><subject>screening</subject><subject>siRNAs</subject><subject>small interfering RNA</subject><subject>Solanum tuberosum</subject><subject>Solanum tuberosum - virology</subject><subject>Transfection</subject><subject>translation (genetics)</subject><subject>Virology</subject><subject>哺乳动物</subject><subject>基因表达</subject><subject>细胞株</subject><subject>马铃薯Y病毒</subject><issn>1674-0769</issn><issn>1995-820X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkktv1DAUhSMEoqXwA9iAxQJWgetH7GSDVI3KQ0wBAUUgFpbteFK3iT21Ezrl1-NRhgIbxMqW73eP7_FxUdzH8BQDiGcJE1JBCRiXFHNcbm4U-7hpqrIm8OVm3nPBShC82SvupHQGwElN6e1ij2DG64bS_eLb-zCqMaDPLk4JfUXDh7eH6GizjjYlFzx644M5b8OlR8e2dWq0LdJXKLmMJeQ8Wkz9OMV8eqyGQfVO5SPb92jpvL1b3FqpPtl7u_WgOHlx9Gnxqly-e_l6cbgsDRPASsoFZ5WhLYBpa8611cRyzEnLCTNUtQ0XlFYYqspoorXQzDJGtbUWslFBD4rns-560oNtjfVjVL1cRzeoeCWDcvLvinensgvfZU0IYYxngcezwKXyK-U7eRam6PPI8ken2w3JDwwEgGXwye6mGC4mm0Y5uGSyX-VtmJKsOWuquuHVf5BQi4Y3WxLPpIkhpWhX14NjkNuY5RyzzFPIbcxyk3se_On4uuNXrhkgM5ByyXc2_rb0L9WHc9NKBam66JI8-ZjNM8g_h2MBmXi0m_U0-O4iK0utzPnK9VZSgUUtCKE_Ad2txpo</recordid><startdate>201104</startdate><enddate>201104</enddate><creator>Tabassum, Bushra</creator><creator>Nasir, Idrees Ahmad</creator><creator>Husnain, Tayyab</creator><general>Springer-Verlag</general><general>SP Wuhan Institute of Virology, CAS</general><general>National Centre of Excellence in Molecular Biology(CEMB), University of the Punjab, 87-West Canal Bank Road Thokar Niaz Baig Lahore, 53700, Pakistan</general><scope>2RA</scope><scope>92L</scope><scope>CQIGP</scope><scope>W94</scope><scope>WU4</scope><scope>~WA</scope><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>7TM</scope><scope>7U9</scope><scope>H94</scope><scope>2B.</scope><scope>4A8</scope><scope>92I</scope><scope>93N</scope><scope>PSX</scope><scope>TCJ</scope><scope>5PM</scope></search><sort><creationdate>201104</creationdate><title>Potato Virus Y mRNA Expression Knockdown Mediated by siRNAs in Cultured Mammalian Cell Line</title><author>Tabassum, Bushra ; Nasir, Idrees Ahmad ; Husnain, Tayyab</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4704-367645c3d00cd866beb2e6162d624c3ad9673351055cb2bb7b4e443beee082073</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Animals</topic><topic>Biochemistry</topic><topic>Biomedical and Life Sciences</topic><topic>Biomedicine</topic><topic>capsid</topic><topic>Capsid Proteins - genetics</topic><topic>Capsid Proteins - metabolism</topic><topic>CHO Cells</topic><topic>CP基因</topic><topic>Cricetinae</topic><topic>Cricetulus</topic><topic>Down-Regulation</topic><topic>gene expression</topic><topic>Gene Expression Regulation, Viral</topic><topic>Gene Knockdown Techniques</topic><topic>genes</topic><topic>mammals</topic><topic>Medical Microbiology</topic><topic>messenger RNA</topic><topic>Microbial Genetics and Genomics</topic><topic>Microbiology</topic><topic>Oncology</topic><topic>PCR技术</topic><topic>Plant Diseases - prevention & control</topic><topic>Plant Diseases - virology</topic><topic>Potato virus Y</topic><topic>potatoes</topic><topic>Potyvirus - genetics</topic><topic>Potyvirus - physiology</topic><topic>quantitative polymerase chain reaction</topic><topic>RNA Interference</topic><topic>RNA, Messenger - genetics</topic><topic>RNA, Messenger - metabolism</topic><topic>RNA, Small Interfering - genetics</topic><topic>RNA, Small Interfering - metabolism</topic><topic>RNA干扰</topic><topic>screening</topic><topic>siRNAs</topic><topic>small interfering RNA</topic><topic>Solanum tuberosum</topic><topic>Solanum tuberosum - virology</topic><topic>Transfection</topic><topic>translation (genetics)</topic><topic>Virology</topic><topic>哺乳动物</topic><topic>基因表达</topic><topic>细胞株</topic><topic>马铃薯Y病毒</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Tabassum, Bushra</creatorcontrib><creatorcontrib>Nasir, Idrees Ahmad</creatorcontrib><creatorcontrib>Husnain, Tayyab</creatorcontrib><collection>中文科技期刊数据库</collection><collection>中文科技期刊数据库-CALIS站点</collection><collection>中文科技期刊数据库-7.0平台</collection><collection>中文科技期刊数据库-自然科学</collection><collection>中文科技期刊数据库-自然科学-生物科学</collection><collection>中文科技期刊数据库- 镜像站点</collection><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Wanfang Data Journals - Hong Kong</collection><collection>WANFANG Data Centre</collection><collection>Wanfang Data Journals</collection><collection>万方数据期刊 - 香港版</collection><collection>China Online Journals (COJ)</collection><collection>China Online Journals (COJ)</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Virologica Sinica</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Tabassum, Bushra</au><au>Nasir, Idrees Ahmad</au><au>Husnain, Tayyab</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Potato Virus Y mRNA Expression Knockdown Mediated by siRNAs in Cultured Mammalian Cell Line</atitle><jtitle>Virologica Sinica</jtitle><stitle>Virol. Sin</stitle><addtitle>Virologica Sinica</addtitle><date>2011-04</date><risdate>2011</risdate><volume>26</volume><issue>2</issue><spage>105</spage><epage>113</epage><pages>105-113</pages><issn>1674-0769</issn><eissn>1995-820X</eissn><abstract>RNA interference （RNAi） is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes. The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion. In our study, a 480bp fragment of the capsid protein gene of potato virus Y （CP-PVY） was used as a target to downregulate PVY mRNA expression in-vitro, as the CP gene interferes with viral uncoating, translation and replication. A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells. CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs. Six biological replicates were performed in this study. In our findings, one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.</abstract><cop>Heidelberg</cop><pub>Springer-Verlag</pub><pmid>21468933</pmid><doi>10.1007/s12250-011-3161-x</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1674-0769 |
| ispartof | Virologica Sinica, 2011-04, Vol.26 (2), p.105-113 |
| issn | 1674-0769 1995-820X |
| language | eng |
| recordid | cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_8222446 |
| source | MEDLINE; Springer Nature - Complete Springer Journals; PubMed Central; Alma/SFX Local Collection |
| subjects | Animals Biochemistry Biomedical and Life Sciences Biomedicine capsid Capsid Proteins - genetics Capsid Proteins - metabolism CHO Cells CP基因 Cricetinae Cricetulus Down-Regulation gene expression Gene Expression Regulation, Viral Gene Knockdown Techniques genes mammals Medical Microbiology messenger RNA Microbial Genetics and Genomics Microbiology Oncology PCR技术 Plant Diseases - prevention & control Plant Diseases - virology Potato virus Y potatoes Potyvirus - genetics Potyvirus - physiology quantitative polymerase chain reaction RNA Interference RNA, Messenger - genetics RNA, Messenger - metabolism RNA, Small Interfering - genetics RNA, Small Interfering - metabolism RNA干扰 screening siRNAs small interfering RNA Solanum tuberosum Solanum tuberosum - virology Transfection translation (genetics) Virology 哺乳动物 基因表达 细胞株 马铃薯Y病毒 |
| title | Potato Virus Y mRNA Expression Knockdown Mediated by siRNAs in Cultured Mammalian Cell Line |
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