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...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:The journal of gene medicine 2001-09, Vol.3 (5), p.418-426
Hauptverfasser: Tabotta, Walter, Klein, Dieter, Hohenadl, Christine, Salmons, Brian, Günzburg, Walter H.
Format: Artikel
Sprache:eng
Schlagworte:
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
container_end_page 426
container_issue 5
container_start_page 418
container_title The journal of gene medicine
container_volume 3
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
fullrecord <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_18136618</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>18136618</sourcerecordid><originalsourceid>FETCH-LOGICAL-c4099-474f2eec4a3a8c4248e85d2a1d51534e828484075d8758b2634c7b4d29f082dd3</originalsourceid><addsrcrecordid>eNp10EtP7CAYBmBiNN5z_sHJ5Cx0YapAoYWlmWi9jZdEozvC0K_KnE47Ah3134t2oomJKz7Cwxt4EfpD8D7BmB5MHqf7FMsltE44JQmlnC3HGUuZMCke1tCG9xOMSS6EXEVrhGRx5nwd3RfQQLBm4MA_dVVV2-YxzqZtfLChC-AHVdeYYNtG1wN4nUXn42ZgtPcQPs5tEy8E186ti2QOJrTOb6GVStcethfrJro7ProdniQXV8Xp8PAiMezzcTmrKIBhOtXCMMoECF5STUpOeMpAUMEEwzkvRc7FmGYpM_mYlVRWWNCyTDfRTp87c-1zBz6oqfUG6lo30HZeEUHSLCMiwn8_4KTtXPxUNDKThHEmI9rtkXGt9w4qNXN2qt2bIlh9FK1i0SoWHeXfRVw3nkL57RbNRrDXgxdbw9tvOeqsGPVxSa-tD_D6pbX7r7I8zbm6vyzU2bA4H42ub5RI3wGlqZZ5</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>196914549</pqid></control><display><type>article</type><title>Genetic reshuffling reconstitutes functional expression cassettes in retroviral vectors</title><source>MEDLINE</source><source>Wiley Online Library Journals Frontfile Complete</source><creator>Tabotta, Walter ; Klein, Dieter ; Hohenadl, Christine ; Salmons, Brian ; Günzburg, Walter H.</creator><creatorcontrib>Tabotta, Walter ; Klein, Dieter ; Hohenadl, Christine ; Salmons, Brian ; Günzburg, Walter H.</creatorcontrib><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 &amp; 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 &amp; 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 &amp; 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 &amp; 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 &amp; 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 &amp; Calcified Tissue Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Health &amp; 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 &amp; Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health &amp; 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 &amp; Sons, Ltd.</abstract><cop>Chichester, UK</cop><pub>John Wiley &amp; Sons, Ltd</pub><pmid>11601755</pmid><doi>10.1002/jgm.209</doi><tpages>9</tpages></addata></record>
fulltext fulltext
identifier ISSN: 1099-498X
ispartof The journal of gene medicine, 2001-09, Vol.3 (5), p.418-426
issn 1099-498X
1521-2254
language eng
recordid cdi_proquest_miscellaneous_18136618
source MEDLINE; Wiley Online Library Journals Frontfile Complete
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
url https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-13T10%3A05%3A58IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Genetic%20reshuffling%20reconstitutes%20functional%20expression%20cassettes%20in%20retroviral%20vectors&rft.jtitle=The%20journal%20of%20gene%20medicine&rft.au=Tabotta,%20Walter&rft.date=2001-09&rft.volume=3&rft.issue=5&rft.spage=418&rft.epage=426&rft.pages=418-426&rft.issn=1099-498X&rft.eissn=1521-2254&rft_id=info:doi/10.1002/jgm.209&rft_dat=%3Cproquest_cross%3E18136618%3C/proquest_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=196914549&rft_id=info:pmid/11601755&rfr_iscdi=true