Flow Cytometric Application of Helper Adenovirus (HAd) Containing GFP Gene Flanked by Two Parallel loxP Sites to Evaluation of 293 Cre-Complementing Cell Line and Monitoring of HAd in Gutless Ad Production
Gutless adenoviruses (GAds), namely, all gene‐deleted adenoviruses, were developed to minimize their immune responses and toxic effects for a successful gene delivery tool in gene therapy. The Cre/ loxP system has been widely used for GAd production. To produce GAd with a low amount of helper adenov...
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| Veröffentlicht in: | Biotechnology progress 2004-05, Vol.20 (3), p.913-920 |
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| creator | Park, Min Tae Hwang, Su-Jeong Lee, Gyun Min |
| description | Gutless adenoviruses (GAds), namely, all gene‐deleted adenoviruses, were developed to minimize their immune responses and toxic effects for a successful gene delivery tool in gene therapy. The Cre/ loxP system has been widely used for GAd production. To produce GAd with a low amount of helper adenovirus (HAd) as byproduct, it is indispensable to use 293Cre cells expressing a high level of Cre for GAd production. In this study, we constructed the HAd containing enhanced green fluorescent protein gene flanked by two parallel loxP sites (HAd/GFP). The use of HAd/GFP with flow cytometry allows one to select 293Cre cells expressing a high level of Cre without using conventional Western blot analysis. Unlike conventional HAd titration methods such as plaque assay and end‐point dilution assay, it also allows one to monitor rapidly the HAd as byproduct in earlier stages of GAd amplification. Taken together, the use of HAd/GFP with flow cytometry facilitates bioprocess development for efficient GAd production. |
| doi_str_mv | 10.1021/bp034248e |
| format | Article |
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The Cre/ loxP system has been widely used for GAd production. To produce GAd with a low amount of helper adenovirus (HAd) as byproduct, it is indispensable to use 293Cre cells expressing a high level of Cre for GAd production. In this study, we constructed the HAd containing enhanced green fluorescent protein gene flanked by two parallel loxP sites (HAd/GFP). The use of HAd/GFP with flow cytometry allows one to select 293Cre cells expressing a high level of Cre without using conventional Western blot analysis. Unlike conventional HAd titration methods such as plaque assay and end‐point dilution assay, it also allows one to monitor rapidly the HAd as byproduct in earlier stages of GAd amplification. Taken together, the use of HAd/GFP with flow cytometry facilitates bioprocess development for efficient GAd production.</description><identifier>ISSN: 8756-7938</identifier><identifier>EISSN: 1520-6033</identifier><identifier>DOI: 10.1021/bp034248e</identifier><identifier>PMID: 15176899</identifier><identifier>CODEN: BIPRET</identifier><language>eng</language><publisher>USA: American Chemical Society</publisher><subject>Adenoviridae - genetics ; Adenoviridae - isolation & purification ; Adenoviridae - physiology ; Adenovirus ; Biological and medical sciences ; Biotechnology ; Cell Line ; Cell Separation - methods ; CRE protein ; Flow cytometry ; Flow Cytometry - methods ; Fundamental and applied biological sciences. Psychology ; Gene therapy ; Genes, Reporter - genetics ; Genetic Engineering - methods ; Green fluorescent protein ; Green Fluorescent Proteins - genetics ; Helper viruses ; Humans ; Integrases - analysis ; Integrases - genetics ; Integrases - metabolism ; Kidney - virology ; LoxP gene ; Q1 ; Q2 ; Viral Proteins - analysis ; Viral Proteins - genetics ; Viral Proteins - metabolism ; Virus Cultivation - methods ; Western blotting</subject><ispartof>Biotechnology progress, 2004-05, Vol.20 (3), p.913-920</ispartof><rights>Copyright © 2004 American Institute of Chemical Engineers (AIChE)</rights><rights>2004 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4598-7630113e7b500c8277da390bcbe99ba7fbade0809f1884f591cd1abfeae65a293</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1021%2Fbp034248e$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1021%2Fbp034248e$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,777,781,1412,27905,27906,45555,45556</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=15841936$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15176899$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Park, Min Tae</creatorcontrib><creatorcontrib>Hwang, Su-Jeong</creatorcontrib><creatorcontrib>Lee, Gyun Min</creatorcontrib><title>Flow Cytometric Application of Helper Adenovirus (HAd) Containing GFP Gene Flanked by Two Parallel loxP Sites to Evaluation of 293 Cre-Complementing Cell Line and Monitoring of HAd in Gutless Ad Production</title><title>Biotechnology progress</title><addtitle>Biotechnol Progress</addtitle><description>Gutless adenoviruses (GAds), namely, all gene‐deleted adenoviruses, were developed to minimize their immune responses and toxic effects for a successful gene delivery tool in gene therapy. The Cre/ loxP system has been widely used for GAd production. To produce GAd with a low amount of helper adenovirus (HAd) as byproduct, it is indispensable to use 293Cre cells expressing a high level of Cre for GAd production. In this study, we constructed the HAd containing enhanced green fluorescent protein gene flanked by two parallel loxP sites (HAd/GFP). The use of HAd/GFP with flow cytometry allows one to select 293Cre cells expressing a high level of Cre without using conventional Western blot analysis. Unlike conventional HAd titration methods such as plaque assay and end‐point dilution assay, it also allows one to monitor rapidly the HAd as byproduct in earlier stages of GAd amplification. Taken together, the use of HAd/GFP with flow cytometry facilitates bioprocess development for efficient GAd production.</description><subject>Adenoviridae - genetics</subject><subject>Adenoviridae - isolation & purification</subject><subject>Adenoviridae - physiology</subject><subject>Adenovirus</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Cell Line</subject><subject>Cell Separation - methods</subject><subject>CRE protein</subject><subject>Flow cytometry</subject><subject>Flow Cytometry - methods</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene therapy</subject><subject>Genes, Reporter - genetics</subject><subject>Genetic Engineering - methods</subject><subject>Green fluorescent protein</subject><subject>Green Fluorescent Proteins - genetics</subject><subject>Helper viruses</subject><subject>Humans</subject><subject>Integrases - analysis</subject><subject>Integrases - genetics</subject><subject>Integrases - metabolism</subject><subject>Kidney - virology</subject><subject>LoxP gene</subject><subject>Q1</subject><subject>Q2</subject><subject>Viral Proteins - analysis</subject><subject>Viral Proteins - genetics</subject><subject>Viral Proteins - metabolism</subject><subject>Virus Cultivation - methods</subject><subject>Western blotting</subject><issn>8756-7938</issn><issn>1520-6033</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kc2O0zAUhSMEYsrAghdAd4NgFgE7aeJ4GaJpilSgGopYWk5yg8w4dsZOptOH5J1IaFXYsLIlf-dHPkHwkpJ3lET0fdWTeBktM3wULGgSkTAlcfw4WGQsSUPG4-wieOb9T0JIRtLoaXBBE8rSjPNF8Gul7R6Kw2A7HJyqIe97rWo5KGvAtrBG3aODvEFj75UbPbxd580VFNYMUhllfkC52kKJBmGlpbnFBqoD7PYWttJJrVGDtg9b-KoG9DBYuL6Xejz7RzyGwmFY2K7X2KEZZssCtYaNmjylaeCTNWqwbn6YG-UNKAPlOGj0fmoGW2ebsZ4dnwdPWqk9vjidl8G31fWuWIebL-XHIt-E9TLhWcjSmFAaI6sSQuosYqyRMSdVXSHnlWRtJRuc_oq3NMuWbcJp3VBZtSgxTeRU-TJ4c_Ttnb0b0Q-iU76eSkuDdvSCTTEkStN0Iq-OZO2s9w5b0TvVSXcQlIh5PHEeb2JfnVzHqsPmL3laawJenwDpa6lbJ02t_D9ctqQ8nkPJkdsrjYf_J4oPu-3Nn-skCY8S5Qd8OEukuxUpi1kivn8uBeE3abFipUji31VfwKA</recordid><startdate>20040501</startdate><enddate>20040501</enddate><creator>Park, Min Tae</creator><creator>Hwang, Su-Jeong</creator><creator>Lee, Gyun Min</creator><general>American Chemical Society</general><general>American Institute of Chemical Engineers</general><scope>BSCLL</scope><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></search><sort><creationdate>20040501</creationdate><title>Flow Cytometric Application of Helper Adenovirus (HAd) Containing GFP Gene Flanked by Two Parallel loxP Sites to Evaluation of 293 Cre-Complementing Cell Line and Monitoring of HAd in Gutless Ad Production</title><author>Park, Min Tae ; Hwang, Su-Jeong ; Lee, Gyun Min</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4598-7630113e7b500c8277da390bcbe99ba7fbade0809f1884f591cd1abfeae65a293</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Adenoviridae - genetics</topic><topic>Adenoviridae - isolation & purification</topic><topic>Adenoviridae - physiology</topic><topic>Adenovirus</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Cell Line</topic><topic>Cell Separation - methods</topic><topic>CRE protein</topic><topic>Flow cytometry</topic><topic>Flow Cytometry - methods</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene therapy</topic><topic>Genes, Reporter - genetics</topic><topic>Genetic Engineering - methods</topic><topic>Green fluorescent protein</topic><topic>Green Fluorescent Proteins - genetics</topic><topic>Helper viruses</topic><topic>Humans</topic><topic>Integrases - analysis</topic><topic>Integrases - genetics</topic><topic>Integrases - metabolism</topic><topic>Kidney - virology</topic><topic>LoxP gene</topic><topic>Q1</topic><topic>Q2</topic><topic>Viral Proteins - analysis</topic><topic>Viral Proteins - genetics</topic><topic>Viral Proteins - metabolism</topic><topic>Virus Cultivation - methods</topic><topic>Western blotting</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Park, Min Tae</creatorcontrib><creatorcontrib>Hwang, Su-Jeong</creatorcontrib><creatorcontrib>Lee, Gyun Min</creatorcontrib><collection>Istex</collection><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><jtitle>Biotechnology progress</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Park, Min Tae</au><au>Hwang, Su-Jeong</au><au>Lee, Gyun Min</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Flow Cytometric Application of Helper Adenovirus (HAd) Containing GFP Gene Flanked by Two Parallel loxP Sites to Evaluation of 293 Cre-Complementing Cell Line and Monitoring of HAd in Gutless Ad Production</atitle><jtitle>Biotechnology progress</jtitle><addtitle>Biotechnol Progress</addtitle><date>2004-05-01</date><risdate>2004</risdate><volume>20</volume><issue>3</issue><spage>913</spage><epage>920</epage><pages>913-920</pages><issn>8756-7938</issn><eissn>1520-6033</eissn><coden>BIPRET</coden><abstract>Gutless adenoviruses (GAds), namely, all gene‐deleted adenoviruses, were developed to minimize their immune responses and toxic effects for a successful gene delivery tool in gene therapy. The Cre/ loxP system has been widely used for GAd production. To produce GAd with a low amount of helper adenovirus (HAd) as byproduct, it is indispensable to use 293Cre cells expressing a high level of Cre for GAd production. In this study, we constructed the HAd containing enhanced green fluorescent protein gene flanked by two parallel loxP sites (HAd/GFP). The use of HAd/GFP with flow cytometry allows one to select 293Cre cells expressing a high level of Cre without using conventional Western blot analysis. Unlike conventional HAd titration methods such as plaque assay and end‐point dilution assay, it also allows one to monitor rapidly the HAd as byproduct in earlier stages of GAd amplification. Taken together, the use of HAd/GFP with flow cytometry facilitates bioprocess development for efficient GAd production.</abstract><cop>USA</cop><pub>American Chemical Society</pub><pmid>15176899</pmid><doi>10.1021/bp034248e</doi><tpages>8</tpages></addata></record> |
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| subjects | Adenoviridae - genetics Adenoviridae - isolation & purification Adenoviridae - physiology Adenovirus Biological and medical sciences Biotechnology Cell Line Cell Separation - methods CRE protein Flow cytometry Flow Cytometry - methods Fundamental and applied biological sciences. Psychology Gene therapy Genes, Reporter - genetics Genetic Engineering - methods Green fluorescent protein Green Fluorescent Proteins - genetics Helper viruses Humans Integrases - analysis Integrases - genetics Integrases - metabolism Kidney - virology LoxP gene Q1 Q2 Viral Proteins - analysis Viral Proteins - genetics Viral Proteins - metabolism Virus Cultivation - methods Western blotting |
| title | Flow Cytometric Application of Helper Adenovirus (HAd) Containing GFP Gene Flanked by Two Parallel loxP Sites to Evaluation of 293 Cre-Complementing Cell Line and Monitoring of HAd in Gutless Ad Production |
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