Genetic reshuffling reconstitutes functional expression cassettes in retroviral vectors
Background A major prerequisite for the design of retroviral vectors encoding cell toxic or harmful genes is the possibility to tightly control gene expression, thus limiting activity to the relevant target cells and protecting the packaging cell used for production of recombinant viral particles. M...
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Veröffentlicht in: | The journal of gene medicine 2001-09, Vol.3 (5), p.418-426 |
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creator | Tabotta, Walter Klein, Dieter Hohenadl, Christine Salmons, Brian Günzburg, Walter H. |
description | Background
A major prerequisite for the design of retroviral vectors encoding cell toxic or harmful genes is the possibility to tightly control gene expression, thus limiting activity to the relevant target cells and protecting the packaging cell used for production of recombinant viral particles.
Methods
In the present study a system was developed in which genetic reshuffling during the retroviral life cycle is exploited, allowing reconstitution of functional expression cassettes from separate elements exclusively in transduced target cells. For construction of these murine leukaemia virus (MLV)‐based reconstituting viral vectors (ReCon), a promoterless inverted enhanced green fluorescent protein (EGFP) reporter gene cassette was inserted in place of the U3 region of the 3′ LTR. Subsequently, the human ubiquitin promoter was inserted in the inverse orientation into the R/U5 border of the 5′ LTR of the vector.
Results
PA317 packaging cells stably transfected with ReCon vectors were established and EGFP expression was analysed by fluorescence‐activated cell sorting (FACS). After detection of low‐level background expression, an additional polyadenylation signal was introduced in antisense orientation into the 3′ LTR at the R/U5 border to prevent accidental read‐through transcription from neighbouring cellular promoters. Virus‐containing cell culture supernatants were then used to infect NIH3T3 target cells. EGFP expression, recloning and sequencing of integrated proviruses demonstrated the correct reassembly of the transduced ubiquitin/EGFP transcription unit in these infected cells.
Conclusions
This facile and convenient system should allow production of retroviral vectors encoding potentially toxic proteins, cell cycle inhibitors or inducers of apoptosis, all of which would interfere with vector production if expressed in the retroviral packaging cell. Copyright © 2001 John Wiley & Sons, Ltd. |
doi_str_mv | 10.1002/jgm.209 |
format | Article |
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A major prerequisite for the design of retroviral vectors encoding cell toxic or harmful genes is the possibility to tightly control gene expression, thus limiting activity to the relevant target cells and protecting the packaging cell used for production of recombinant viral particles.
Methods
In the present study a system was developed in which genetic reshuffling during the retroviral life cycle is exploited, allowing reconstitution of functional expression cassettes from separate elements exclusively in transduced target cells. For construction of these murine leukaemia virus (MLV)‐based reconstituting viral vectors (ReCon), a promoterless inverted enhanced green fluorescent protein (EGFP) reporter gene cassette was inserted in place of the U3 region of the 3′ LTR. Subsequently, the human ubiquitin promoter was inserted in the inverse orientation into the R/U5 border of the 5′ LTR of the vector.
Results
PA317 packaging cells stably transfected with ReCon vectors were established and EGFP expression was analysed by fluorescence‐activated cell sorting (FACS). After detection of low‐level background expression, an additional polyadenylation signal was introduced in antisense orientation into the 3′ LTR at the R/U5 border to prevent accidental read‐through transcription from neighbouring cellular promoters. Virus‐containing cell culture supernatants were then used to infect NIH3T3 target cells. EGFP expression, recloning and sequencing of integrated proviruses demonstrated the correct reassembly of the transduced ubiquitin/EGFP transcription unit in these infected cells.
Conclusions
This facile and convenient system should allow production of retroviral vectors encoding potentially toxic proteins, cell cycle inhibitors or inducers of apoptosis, all of which would interfere with vector production if expressed in the retroviral packaging cell. Copyright © 2001 John Wiley & Sons, Ltd.</description><identifier>ISSN: 1099-498X</identifier><identifier>EISSN: 1521-2254</identifier><identifier>DOI: 10.1002/jgm.209</identifier><identifier>PMID: 11601755</identifier><language>eng</language><publisher>Chichester, UK: John Wiley & Sons, Ltd</publisher><subject>3T3 Cells ; Animals ; DNA Primers - chemistry ; enhanced green fluorescent protein ; expression control ; Flow Cytometry ; Gene Expression Regulation ; Gene therapy ; Genetic Vectors ; Green Fluorescent Proteins ; Luminescent Proteins - genetics ; Mice ; Murine leukemia virus ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; ReCon ; reconstituting transcription cassette ; retroviral vector ; Retroviridae - genetics ; Sequence Analysis, DNA ; Terminal Repeat Sequences - genetics ; Transcription, Genetic - genetics ; Transduction, Genetic ; Transgenes - physiology</subject><ispartof>The journal of gene medicine, 2001-09, Vol.3 (5), p.418-426</ispartof><rights>Copyright © 2001 John Wiley & Sons, Ltd.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4099-474f2eec4a3a8c4248e85d2a1d51534e828484075d8758b2634c7b4d29f082dd3</citedby><cites>FETCH-LOGICAL-c4099-474f2eec4a3a8c4248e85d2a1d51534e828484075d8758b2634c7b4d29f082dd3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fjgm.209$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fjgm.209$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,776,780,1411,27901,27902,45550,45551</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11601755$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Tabotta, Walter</creatorcontrib><creatorcontrib>Klein, Dieter</creatorcontrib><creatorcontrib>Hohenadl, Christine</creatorcontrib><creatorcontrib>Salmons, Brian</creatorcontrib><creatorcontrib>Günzburg, Walter H.</creatorcontrib><title>Genetic reshuffling reconstitutes functional expression cassettes in retroviral vectors</title><title>The journal of gene medicine</title><addtitle>J. Gene Med</addtitle><description>Background
A major prerequisite for the design of retroviral vectors encoding cell toxic or harmful genes is the possibility to tightly control gene expression, thus limiting activity to the relevant target cells and protecting the packaging cell used for production of recombinant viral particles.
Methods
In the present study a system was developed in which genetic reshuffling during the retroviral life cycle is exploited, allowing reconstitution of functional expression cassettes from separate elements exclusively in transduced target cells. For construction of these murine leukaemia virus (MLV)‐based reconstituting viral vectors (ReCon), a promoterless inverted enhanced green fluorescent protein (EGFP) reporter gene cassette was inserted in place of the U3 region of the 3′ LTR. Subsequently, the human ubiquitin promoter was inserted in the inverse orientation into the R/U5 border of the 5′ LTR of the vector.
Results
PA317 packaging cells stably transfected with ReCon vectors were established and EGFP expression was analysed by fluorescence‐activated cell sorting (FACS). After detection of low‐level background expression, an additional polyadenylation signal was introduced in antisense orientation into the 3′ LTR at the R/U5 border to prevent accidental read‐through transcription from neighbouring cellular promoters. Virus‐containing cell culture supernatants were then used to infect NIH3T3 target cells. EGFP expression, recloning and sequencing of integrated proviruses demonstrated the correct reassembly of the transduced ubiquitin/EGFP transcription unit in these infected cells.
Conclusions
This facile and convenient system should allow production of retroviral vectors encoding potentially toxic proteins, cell cycle inhibitors or inducers of apoptosis, all of which would interfere with vector production if expressed in the retroviral packaging cell. Copyright © 2001 John Wiley & Sons, Ltd.</description><subject>3T3 Cells</subject><subject>Animals</subject><subject>DNA Primers - chemistry</subject><subject>enhanced green fluorescent protein</subject><subject>expression control</subject><subject>Flow Cytometry</subject><subject>Gene Expression Regulation</subject><subject>Gene therapy</subject><subject>Genetic Vectors</subject><subject>Green Fluorescent Proteins</subject><subject>Luminescent Proteins - genetics</subject><subject>Mice</subject><subject>Murine leukemia virus</subject><subject>Polymerase Chain Reaction</subject><subject>Promoter Regions, Genetic</subject><subject>ReCon</subject><subject>reconstituting transcription cassette</subject><subject>retroviral vector</subject><subject>Retroviridae - genetics</subject><subject>Sequence Analysis, DNA</subject><subject>Terminal Repeat Sequences - genetics</subject><subject>Transcription, Genetic - genetics</subject><subject>Transduction, Genetic</subject><subject>Transgenes - physiology</subject><issn>1099-498X</issn><issn>1521-2254</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNp10EtP7CAYBmBiNN5z_sHJ5Cx0YapAoYWlmWi9jZdEozvC0K_KnE47Ah3134t2oomJKz7Cwxt4EfpD8D7BmB5MHqf7FMsltE44JQmlnC3HGUuZMCke1tCG9xOMSS6EXEVrhGRx5nwd3RfQQLBm4MA_dVVV2-YxzqZtfLChC-AHVdeYYNtG1wN4nUXn42ZgtPcQPs5tEy8E186ti2QOJrTOb6GVStcethfrJro7ProdniQXV8Xp8PAiMezzcTmrKIBhOtXCMMoECF5STUpOeMpAUMEEwzkvRc7FmGYpM_mYlVRWWNCyTDfRTp87c-1zBz6oqfUG6lo30HZeEUHSLCMiwn8_4KTtXPxUNDKThHEmI9rtkXGt9w4qNXN2qt2bIlh9FK1i0SoWHeXfRVw3nkL57RbNRrDXgxdbw9tvOeqsGPVxSa-tD_D6pbX7r7I8zbm6vyzU2bA4H42ub5RI3wGlqZZ5</recordid><startdate>200109</startdate><enddate>200109</enddate><creator>Tabotta, Walter</creator><creator>Klein, Dieter</creator><creator>Hohenadl, Christine</creator><creator>Salmons, Brian</creator><creator>Günzburg, Walter H.</creator><general>John Wiley & Sons, Ltd</general><general>Wiley Periodicals Inc</general><scope>BSCLL</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>3V.</scope><scope>7QP</scope><scope>7TK</scope><scope>7TM</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>RC3</scope><scope>7QO</scope></search><sort><creationdate>200109</creationdate><title>Genetic reshuffling reconstitutes functional expression cassettes in retroviral vectors</title><author>Tabotta, Walter ; Klein, Dieter ; Hohenadl, Christine ; Salmons, Brian ; Günzburg, Walter H.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4099-474f2eec4a3a8c4248e85d2a1d51534e828484075d8758b2634c7b4d29f082dd3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>3T3 Cells</topic><topic>Animals</topic><topic>DNA Primers - chemistry</topic><topic>enhanced green fluorescent protein</topic><topic>expression control</topic><topic>Flow Cytometry</topic><topic>Gene Expression Regulation</topic><topic>Gene therapy</topic><topic>Genetic Vectors</topic><topic>Green Fluorescent Proteins</topic><topic>Luminescent Proteins - genetics</topic><topic>Mice</topic><topic>Murine leukemia virus</topic><topic>Polymerase Chain Reaction</topic><topic>Promoter Regions, Genetic</topic><topic>ReCon</topic><topic>reconstituting transcription cassette</topic><topic>retroviral vector</topic><topic>Retroviridae - genetics</topic><topic>Sequence Analysis, DNA</topic><topic>Terminal Repeat Sequences - genetics</topic><topic>Transcription, Genetic - genetics</topic><topic>Transduction, Genetic</topic><topic>Transgenes - physiology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Tabotta, Walter</creatorcontrib><creatorcontrib>Klein, Dieter</creatorcontrib><creatorcontrib>Hohenadl, Christine</creatorcontrib><creatorcontrib>Salmons, Brian</creatorcontrib><creatorcontrib>Günzburg, Walter H.</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection (ProQuest)</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Genetics Abstracts</collection><collection>Biotechnology Research Abstracts</collection><jtitle>The journal of gene medicine</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Tabotta, Walter</au><au>Klein, Dieter</au><au>Hohenadl, Christine</au><au>Salmons, Brian</au><au>Günzburg, Walter H.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Genetic reshuffling reconstitutes functional expression cassettes in retroviral vectors</atitle><jtitle>The journal of gene medicine</jtitle><addtitle>J. Gene Med</addtitle><date>2001-09</date><risdate>2001</risdate><volume>3</volume><issue>5</issue><spage>418</spage><epage>426</epage><pages>418-426</pages><issn>1099-498X</issn><eissn>1521-2254</eissn><abstract>Background
A major prerequisite for the design of retroviral vectors encoding cell toxic or harmful genes is the possibility to tightly control gene expression, thus limiting activity to the relevant target cells and protecting the packaging cell used for production of recombinant viral particles.
Methods
In the present study a system was developed in which genetic reshuffling during the retroviral life cycle is exploited, allowing reconstitution of functional expression cassettes from separate elements exclusively in transduced target cells. For construction of these murine leukaemia virus (MLV)‐based reconstituting viral vectors (ReCon), a promoterless inverted enhanced green fluorescent protein (EGFP) reporter gene cassette was inserted in place of the U3 region of the 3′ LTR. Subsequently, the human ubiquitin promoter was inserted in the inverse orientation into the R/U5 border of the 5′ LTR of the vector.
Results
PA317 packaging cells stably transfected with ReCon vectors were established and EGFP expression was analysed by fluorescence‐activated cell sorting (FACS). After detection of low‐level background expression, an additional polyadenylation signal was introduced in antisense orientation into the 3′ LTR at the R/U5 border to prevent accidental read‐through transcription from neighbouring cellular promoters. Virus‐containing cell culture supernatants were then used to infect NIH3T3 target cells. EGFP expression, recloning and sequencing of integrated proviruses demonstrated the correct reassembly of the transduced ubiquitin/EGFP transcription unit in these infected cells.
Conclusions
This facile and convenient system should allow production of retroviral vectors encoding potentially toxic proteins, cell cycle inhibitors or inducers of apoptosis, all of which would interfere with vector production if expressed in the retroviral packaging cell. Copyright © 2001 John Wiley & Sons, Ltd.</abstract><cop>Chichester, UK</cop><pub>John Wiley & Sons, Ltd</pub><pmid>11601755</pmid><doi>10.1002/jgm.209</doi><tpages>9</tpages></addata></record> |
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subjects | 3T3 Cells Animals DNA Primers - chemistry enhanced green fluorescent protein expression control Flow Cytometry Gene Expression Regulation Gene therapy Genetic Vectors Green Fluorescent Proteins Luminescent Proteins - genetics Mice Murine leukemia virus Polymerase Chain Reaction Promoter Regions, Genetic ReCon reconstituting transcription cassette retroviral vector Retroviridae - genetics Sequence Analysis, DNA Terminal Repeat Sequences - genetics Transcription, Genetic - genetics Transduction, Genetic Transgenes - physiology |
title | Genetic reshuffling reconstitutes functional expression cassettes in retroviral vectors |
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