High throughput functional analysis of HIV-1 env genes without cloning
Functional human immunodeficiency virus type 1 (HIV-1) env genes have been widely used for vaccine design, neutralization assays, and pathogenesis studies. However, obtaining bona fide functional env clones is a time consuming and labor intensive process. A new high throughput method has been develo...
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| Veröffentlicht in: | Journal of virological methods 2007-07, Vol.143 (1), p.104-111 |
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| creator | Kirchherr, Jennifer L. Lu, Xiaozhi Kasongo, Webster Chalwe, Victor Mwananyanda, Lawrence Musonda, Rosemary M. Xia, Shi-Mao Scearce, Richard M. Liao, Hua-Xin Montefiori, David C. Haynes, Barton F. Gao, Feng |
| description | Functional human immunodeficiency virus type 1 (HIV-1)
env genes have been widely used for vaccine design, neutralization assays, and pathogenesis studies. However, obtaining bona fide functional
env clones is a time consuming and labor intensive process. A new high throughput method has been developed to characterize HIV-1
env genes. Multiple
rev/
env gene cassettes were obtained from each of seven HIV-1 strains using single genome amplification (SGA) PCR. The cytomegalovirus (CMV) promoter was amplified separately by PCR. A promoter PCR (pPCR) method was developed to link both PCR products using an overlapping PCR method. Pseudovirions were generated by cotransfection of pPCR products and pSG3Δ
env backbone into 293T cells. After infecting TZM-bl cells, 75 out of 87 (86%) of the
rev/
env gene cassettes were functional. Pseudoviruses generated with pPCR products or corresponding plasmid DNA showed similar sensitivity to six HIV-1 positive sera and three monoclonal antibodies, suggesting neutralization properties are not altered in pPCR pseudovirions. Furthermore, sufficient amounts of pseudovirions can be obtained for a large number of neutralization assays. The new pPCR method eliminates cloning, transformation, and plasmid DNA preparation steps in the generation of HIV-1 pseudovirions. This allows for quick analysis of multiple
env genes from HIV-1 infected individuals. |
| doi_str_mv | 10.1016/j.jviromet.2007.02.015 |
| format | Article |
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env genes have been widely used for vaccine design, neutralization assays, and pathogenesis studies. However, obtaining bona fide functional
env clones is a time consuming and labor intensive process. A new high throughput method has been developed to characterize HIV-1
env genes. Multiple
rev/
env gene cassettes were obtained from each of seven HIV-1 strains using single genome amplification (SGA) PCR. The cytomegalovirus (CMV) promoter was amplified separately by PCR. A promoter PCR (pPCR) method was developed to link both PCR products using an overlapping PCR method. Pseudovirions were generated by cotransfection of pPCR products and pSG3Δ
env backbone into 293T cells. After infecting TZM-bl cells, 75 out of 87 (86%) of the
rev/
env gene cassettes were functional. Pseudoviruses generated with pPCR products or corresponding plasmid DNA showed similar sensitivity to six HIV-1 positive sera and three monoclonal antibodies, suggesting neutralization properties are not altered in pPCR pseudovirions. Furthermore, sufficient amounts of pseudovirions can be obtained for a large number of neutralization assays. The new pPCR method eliminates cloning, transformation, and plasmid DNA preparation steps in the generation of HIV-1 pseudovirions. This allows for quick analysis of multiple
env genes from HIV-1 infected individuals.</description><identifier>ISSN: 0166-0934</identifier><identifier>EISSN: 1879-0984</identifier><identifier>DOI: 10.1016/j.jviromet.2007.02.015</identifier><identifier>PMID: 17416428</identifier><identifier>CODEN: JVMEDH</identifier><language>eng</language><publisher>London: Elsevier B.V</publisher><subject>Acquired Immunodeficiency Syndrome ; Biological and medical sciences ; Cell Line ; Cytomegalovirus ; Cytomegalovirus - genetics ; Envelope ; Fundamental and applied biological sciences. Psychology ; Gene Products, env - isolation & purification ; Genes, env ; HIV-1 ; HIV-1 - genetics ; HIV-1 - isolation & purification ; Human immunodeficiency virus 1 ; Humans ; Microbiology ; Neutralization ; PCR ; Polymerase Chain Reaction - methods ; Promoter Regions, Genetic ; Pseudovirion ; Techniques used in virology ; Virology ; Zambia</subject><ispartof>Journal of virological methods, 2007-07, Vol.143 (1), p.104-111</ispartof><rights>2007 Elsevier B.V.</rights><rights>2007 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c530t-b734a3b094bdeb072af35280ef9b0714ad5e817414ac7bb7f58ccd24c6ccc7633</citedby><cites>FETCH-LOGICAL-c530t-b734a3b094bdeb072af35280ef9b0714ad5e817414ac7bb7f58ccd24c6ccc7633</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0166093407000924$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>230,314,776,780,881,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=18808421$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17416428$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kirchherr, Jennifer L.</creatorcontrib><creatorcontrib>Lu, Xiaozhi</creatorcontrib><creatorcontrib>Kasongo, Webster</creatorcontrib><creatorcontrib>Chalwe, Victor</creatorcontrib><creatorcontrib>Mwananyanda, Lawrence</creatorcontrib><creatorcontrib>Musonda, Rosemary M.</creatorcontrib><creatorcontrib>Xia, Shi-Mao</creatorcontrib><creatorcontrib>Scearce, Richard M.</creatorcontrib><creatorcontrib>Liao, Hua-Xin</creatorcontrib><creatorcontrib>Montefiori, David C.</creatorcontrib><creatorcontrib>Haynes, Barton F.</creatorcontrib><creatorcontrib>Gao, Feng</creatorcontrib><title>High throughput functional analysis of HIV-1 env genes without cloning</title><title>Journal of virological methods</title><addtitle>J Virol Methods</addtitle><description>Functional human immunodeficiency virus type 1 (HIV-1)
env genes have been widely used for vaccine design, neutralization assays, and pathogenesis studies. However, obtaining bona fide functional
env clones is a time consuming and labor intensive process. A new high throughput method has been developed to characterize HIV-1
env genes. Multiple
rev/
env gene cassettes were obtained from each of seven HIV-1 strains using single genome amplification (SGA) PCR. The cytomegalovirus (CMV) promoter was amplified separately by PCR. A promoter PCR (pPCR) method was developed to link both PCR products using an overlapping PCR method. Pseudovirions were generated by cotransfection of pPCR products and pSG3Δ
env backbone into 293T cells. After infecting TZM-bl cells, 75 out of 87 (86%) of the
rev/
env gene cassettes were functional. Pseudoviruses generated with pPCR products or corresponding plasmid DNA showed similar sensitivity to six HIV-1 positive sera and three monoclonal antibodies, suggesting neutralization properties are not altered in pPCR pseudovirions. Furthermore, sufficient amounts of pseudovirions can be obtained for a large number of neutralization assays. The new pPCR method eliminates cloning, transformation, and plasmid DNA preparation steps in the generation of HIV-1 pseudovirions. This allows for quick analysis of multiple
env genes from HIV-1 infected individuals.</description><subject>Acquired Immunodeficiency Syndrome</subject><subject>Biological and medical sciences</subject><subject>Cell Line</subject><subject>Cytomegalovirus</subject><subject>Cytomegalovirus - genetics</subject><subject>Envelope</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Products, env - isolation & purification</subject><subject>Genes, env</subject><subject>HIV-1</subject><subject>HIV-1 - genetics</subject><subject>HIV-1 - isolation & purification</subject><subject>Human immunodeficiency virus 1</subject><subject>Humans</subject><subject>Microbiology</subject><subject>Neutralization</subject><subject>PCR</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Promoter Regions, Genetic</subject><subject>Pseudovirion</subject><subject>Techniques used in virology</subject><subject>Virology</subject><subject>Zambia</subject><issn>0166-0934</issn><issn>1879-0984</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUtv1DAUhS0EotPCX6iygV3C9SOxZ4NAFWUqVWIDbC3HuUk8ytiDnQzqv8ejGVpYdeOH_J2j43sIuaZQUaDNh221PbgYdjhXDEBWwCqg9QuyokquS1gr8ZKsMtjkMxcX5DKlLQDUkvPX5IJKQRvB1IrcbtwwFvMYwzKM-2Uu-sXb2QVvpsLk5SG5VIS-2Nz9LGmB_lAM6DEVv908hozbKXjnhzfkVW-mhG_P-xX5cfvl-82mvP_29e7m831paw5z2UouDG9hLdoOW5DM9LxmCrBf5xsVpqtRHcMJY2Xbyr5W1nZM2MZaKxvOr8jHk-9-aXfYWfRzNJPeR7cz8UEH4_T_L96NeggHTddCATsavD8bxPBrwTTrnUsWp8l4DEvSEmrRUCaeBfPUOWVQZ7A5gTaGlCL2j2ko6GNXeqv_dnVUSQ1M566y8PrfvzzJzuVk4N0ZMMmaqY_GW5eeOKVACUYz9-nEYZ78wWHUyTr0FjsX0c66C-65LH8AJwG3Ww</recordid><startdate>20070701</startdate><enddate>20070701</enddate><creator>Kirchherr, Jennifer L.</creator><creator>Lu, Xiaozhi</creator><creator>Kasongo, Webster</creator><creator>Chalwe, Victor</creator><creator>Mwananyanda, Lawrence</creator><creator>Musonda, Rosemary M.</creator><creator>Xia, Shi-Mao</creator><creator>Scearce, Richard M.</creator><creator>Liao, Hua-Xin</creator><creator>Montefiori, David C.</creator><creator>Haynes, Barton F.</creator><creator>Gao, Feng</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>IQODW</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>7U9</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20070701</creationdate><title>High throughput functional analysis of HIV-1 env genes without cloning</title><author>Kirchherr, Jennifer L. ; Lu, Xiaozhi ; Kasongo, Webster ; Chalwe, Victor ; Mwananyanda, Lawrence ; Musonda, Rosemary M. ; Xia, Shi-Mao ; Scearce, Richard M. ; Liao, Hua-Xin ; Montefiori, David C. ; Haynes, Barton F. ; Gao, Feng</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c530t-b734a3b094bdeb072af35280ef9b0714ad5e817414ac7bb7f58ccd24c6ccc7633</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2007</creationdate><topic>Acquired Immunodeficiency Syndrome</topic><topic>Biological and medical sciences</topic><topic>Cell Line</topic><topic>Cytomegalovirus</topic><topic>Cytomegalovirus - genetics</topic><topic>Envelope</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Products, env - isolation & purification</topic><topic>Genes, env</topic><topic>HIV-1</topic><topic>HIV-1 - genetics</topic><topic>HIV-1 - isolation & purification</topic><topic>Human immunodeficiency virus 1</topic><topic>Humans</topic><topic>Microbiology</topic><topic>Neutralization</topic><topic>PCR</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Promoter Regions, Genetic</topic><topic>Pseudovirion</topic><topic>Techniques used in virology</topic><topic>Virology</topic><topic>Zambia</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kirchherr, Jennifer L.</creatorcontrib><creatorcontrib>Lu, Xiaozhi</creatorcontrib><creatorcontrib>Kasongo, Webster</creatorcontrib><creatorcontrib>Chalwe, Victor</creatorcontrib><creatorcontrib>Mwananyanda, Lawrence</creatorcontrib><creatorcontrib>Musonda, Rosemary M.</creatorcontrib><creatorcontrib>Xia, Shi-Mao</creatorcontrib><creatorcontrib>Scearce, Richard M.</creatorcontrib><creatorcontrib>Liao, Hua-Xin</creatorcontrib><creatorcontrib>Montefiori, David C.</creatorcontrib><creatorcontrib>Haynes, Barton F.</creatorcontrib><creatorcontrib>Gao, Feng</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of virological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kirchherr, Jennifer L.</au><au>Lu, Xiaozhi</au><au>Kasongo, Webster</au><au>Chalwe, Victor</au><au>Mwananyanda, Lawrence</au><au>Musonda, Rosemary M.</au><au>Xia, Shi-Mao</au><au>Scearce, Richard M.</au><au>Liao, Hua-Xin</au><au>Montefiori, David C.</au><au>Haynes, Barton F.</au><au>Gao, Feng</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>High throughput functional analysis of HIV-1 env genes without cloning</atitle><jtitle>Journal of virological methods</jtitle><addtitle>J Virol Methods</addtitle><date>2007-07-01</date><risdate>2007</risdate><volume>143</volume><issue>1</issue><spage>104</spage><epage>111</epage><pages>104-111</pages><issn>0166-0934</issn><eissn>1879-0984</eissn><coden>JVMEDH</coden><abstract>Functional human immunodeficiency virus type 1 (HIV-1)
env genes have been widely used for vaccine design, neutralization assays, and pathogenesis studies. However, obtaining bona fide functional
env clones is a time consuming and labor intensive process. A new high throughput method has been developed to characterize HIV-1
env genes. Multiple
rev/
env gene cassettes were obtained from each of seven HIV-1 strains using single genome amplification (SGA) PCR. The cytomegalovirus (CMV) promoter was amplified separately by PCR. A promoter PCR (pPCR) method was developed to link both PCR products using an overlapping PCR method. Pseudovirions were generated by cotransfection of pPCR products and pSG3Δ
env backbone into 293T cells. After infecting TZM-bl cells, 75 out of 87 (86%) of the
rev/
env gene cassettes were functional. Pseudoviruses generated with pPCR products or corresponding plasmid DNA showed similar sensitivity to six HIV-1 positive sera and three monoclonal antibodies, suggesting neutralization properties are not altered in pPCR pseudovirions. Furthermore, sufficient amounts of pseudovirions can be obtained for a large number of neutralization assays. The new pPCR method eliminates cloning, transformation, and plasmid DNA preparation steps in the generation of HIV-1 pseudovirions. This allows for quick analysis of multiple
env genes from HIV-1 infected individuals.</abstract><cop>London</cop><cop>Amsterdam</cop><cop>New York, NY</cop><pub>Elsevier B.V</pub><pmid>17416428</pmid><doi>10.1016/j.jviromet.2007.02.015</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Acquired Immunodeficiency Syndrome Biological and medical sciences Cell Line Cytomegalovirus Cytomegalovirus - genetics Envelope Fundamental and applied biological sciences. Psychology Gene Products, env - isolation & purification Genes, env HIV-1 HIV-1 - genetics HIV-1 - isolation & purification Human immunodeficiency virus 1 Humans Microbiology Neutralization PCR Polymerase Chain Reaction - methods Promoter Regions, Genetic Pseudovirion Techniques used in virology Virology Zambia |
| title | High throughput functional analysis of HIV-1 env genes without cloning |
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