A novel retroviral vector system to analyze expression from mRNA with retained introns using fluorescent proteins and flow cytometry
The ability to overcome cellular restrictions that exist for the export and translation of mRNAs with retained introns is a requirement for the replication of retroviruses and also for the expression of many mRNA isoforms transcribed from cellular genes. In some cases, RNA structures have been ident...
Gespeichert in:
| Veröffentlicht in: | Scientific reports 2019-04, Vol.9 (1), p.6467-6467, Article 6467 |
|---|---|
| Hauptverfasser: | , , , , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | 6467 |
|---|---|
| container_issue | 1 |
| container_start_page | 6467 |
| container_title | Scientific reports |
| container_volume | 9 |
| creator | Jackson, Patrick E. H. Huang, Jing Sharma, Monika Rasmussen, Sara K. Hammarskjold, Marie-Louise Rekosh, David |
| description | The ability to overcome cellular restrictions that exist for the export and translation of mRNAs with retained introns is a requirement for the replication of retroviruses and also for the expression of many mRNA isoforms transcribed from cellular genes. In some cases, RNA structures have been identified in the mRNA that directly interact with cellular factors to promote the export and expression of isoforms with retained introns. In other cases, a viral protein is also required to act as an adapter. In this report we describe a novel vector system that allows measurement of the ability of
cis-
and
trans
-acting factors to promote the export and translation of mRNAs with retained introns. One reporter vector used in this system is derived from an HIV proviral clone engineered to express two different fluorescent proteins from spliced and unspliced transcripts. The ratio of fluorescent signals is a measurement of the efficiency of export and translation. A second vector utilizes a third fluorescent protein to measure the expression of viral export proteins that interact with some of the export elements. Both vectors can be packaged into viral particles and be used to transduce cells, allowing expression at physiological levels from the integrated vector. |
| doi_str_mv | 10.1038/s41598-019-42914-3 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_6478720</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>2393006403</sourcerecordid><originalsourceid>FETCH-LOGICAL-c511t-44129a661429883d0ddaefc844dbbf258a76bcd2a2af29225b7c9bd34cb1aaea3</originalsourceid><addsrcrecordid>eNp9UUtrFTEYDaLYUvsHXEjAjZvRvOaRjXApVQvFQtF1yCTf3KbMJNckc-u49oeb21vb6qLZJHAe33dyEHpNyXtKePchCVrLriJUVoJJKir-DB0yIuqKccaeP3ofoOOUrkk5NZOCypfogFNC61o0h-j3CvuwhRFHyDFsXdQj3oLJIeK0pAwTzgFrr8flF2D4uYmQkgseDzFMeLr8usI3Ll_t1Np5sNj5YuMTnpPzazyMcygKAz7jTQwZXIG0twUIN9gsOUxl7PIKvRj0mOD47j5C3z-dfjv5Up1ffD47WZ1XpqY0V0JQJnXT0BK467gl1moYTCeE7fuB1Z1um95YppkemGSs7lsje8uF6anWoPkR-rj33cz9BHa3VsmrNtFNOi4qaKf-Rby7UuuwVY1ou5aRYvDuziCGHzOkrCZX0o2j9hDmpBijXNKGtqxQ3_5HvQ5zLB9ZWFxyQhpBeGGxPcvEkFKE4X4ZStSuZ7XvWZWe1W3Paid68zjGveRvq4XA94RUIL-G-DD7Cds_X9C3rQ</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>2393006403</pqid></control><display><type>article</type><title>A novel retroviral vector system to analyze expression from mRNA with retained introns using fluorescent proteins and flow cytometry</title><source>Nature Open Access</source><source>MEDLINE</source><source>DOAJ Directory of Open Access Journals</source><source>Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals</source><source>PubMed Central</source><source>Alma/SFX Local Collection</source><source>Free Full-Text Journals in Chemistry</source><source>Springer Nature OA Free Journals</source><creator>Jackson, Patrick E. H. ; Huang, Jing ; Sharma, Monika ; Rasmussen, Sara K. ; Hammarskjold, Marie-Louise ; Rekosh, David</creator><creatorcontrib>Jackson, Patrick E. H. ; Huang, Jing ; Sharma, Monika ; Rasmussen, Sara K. ; Hammarskjold, Marie-Louise ; Rekosh, David</creatorcontrib><description>The ability to overcome cellular restrictions that exist for the export and translation of mRNAs with retained introns is a requirement for the replication of retroviruses and also for the expression of many mRNA isoforms transcribed from cellular genes. In some cases, RNA structures have been identified in the mRNA that directly interact with cellular factors to promote the export and expression of isoforms with retained introns. In other cases, a viral protein is also required to act as an adapter. In this report we describe a novel vector system that allows measurement of the ability of
cis-
and
trans
-acting factors to promote the export and translation of mRNAs with retained introns. One reporter vector used in this system is derived from an HIV proviral clone engineered to express two different fluorescent proteins from spliced and unspliced transcripts. The ratio of fluorescent signals is a measurement of the efficiency of export and translation. A second vector utilizes a third fluorescent protein to measure the expression of viral export proteins that interact with some of the export elements. Both vectors can be packaged into viral particles and be used to transduce cells, allowing expression at physiological levels from the integrated vector.</description><identifier>ISSN: 2045-2322</identifier><identifier>EISSN: 2045-2322</identifier><identifier>DOI: 10.1038/s41598-019-42914-3</identifier><identifier>PMID: 31015546</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>13 ; 13/106 ; 13/31 ; 13/44 ; 38 ; 38/47 ; 42 ; 42/35 ; 631/1647/2017/1958 ; 631/337/1645/2052 ; Cell Line ; Flow Cytometry ; Gene expression ; Gene Expression Regulation ; Genetic Vectors ; Green Fluorescent Proteins - biosynthesis ; Green Fluorescent Proteins - genetics ; HIV ; HIV-1 ; Human immunodeficiency virus ; Humanities and Social Sciences ; Humans ; Introns ; Isoforms ; multidisciplinary ; Proteins ; RNA, Messenger - biosynthesis ; RNA, Messenger - genetics ; Science ; Science (multidisciplinary) ; Transduction, Genetic ; Translation</subject><ispartof>Scientific reports, 2019-04, Vol.9 (1), p.6467-6467, Article 6467</ispartof><rights>The Author(s) 2019</rights><rights>The Author(s) 2019. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c511t-44129a661429883d0ddaefc844dbbf258a76bcd2a2af29225b7c9bd34cb1aaea3</citedby><cites>FETCH-LOGICAL-c511t-44129a661429883d0ddaefc844dbbf258a76bcd2a2af29225b7c9bd34cb1aaea3</cites><orcidid>0000-0003-3662-4176</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478720/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478720/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,27901,27902,41096,42165,51551,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31015546$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Jackson, Patrick E. H.</creatorcontrib><creatorcontrib>Huang, Jing</creatorcontrib><creatorcontrib>Sharma, Monika</creatorcontrib><creatorcontrib>Rasmussen, Sara K.</creatorcontrib><creatorcontrib>Hammarskjold, Marie-Louise</creatorcontrib><creatorcontrib>Rekosh, David</creatorcontrib><title>A novel retroviral vector system to analyze expression from mRNA with retained introns using fluorescent proteins and flow cytometry</title><title>Scientific reports</title><addtitle>Sci Rep</addtitle><addtitle>Sci Rep</addtitle><description>The ability to overcome cellular restrictions that exist for the export and translation of mRNAs with retained introns is a requirement for the replication of retroviruses and also for the expression of many mRNA isoforms transcribed from cellular genes. In some cases, RNA structures have been identified in the mRNA that directly interact with cellular factors to promote the export and expression of isoforms with retained introns. In other cases, a viral protein is also required to act as an adapter. In this report we describe a novel vector system that allows measurement of the ability of
cis-
and
trans
-acting factors to promote the export and translation of mRNAs with retained introns. One reporter vector used in this system is derived from an HIV proviral clone engineered to express two different fluorescent proteins from spliced and unspliced transcripts. The ratio of fluorescent signals is a measurement of the efficiency of export and translation. A second vector utilizes a third fluorescent protein to measure the expression of viral export proteins that interact with some of the export elements. Both vectors can be packaged into viral particles and be used to transduce cells, allowing expression at physiological levels from the integrated vector.</description><subject>13</subject><subject>13/106</subject><subject>13/31</subject><subject>13/44</subject><subject>38</subject><subject>38/47</subject><subject>42</subject><subject>42/35</subject><subject>631/1647/2017/1958</subject><subject>631/337/1645/2052</subject><subject>Cell Line</subject><subject>Flow Cytometry</subject><subject>Gene expression</subject><subject>Gene Expression Regulation</subject><subject>Genetic Vectors</subject><subject>Green Fluorescent Proteins - biosynthesis</subject><subject>Green Fluorescent Proteins - genetics</subject><subject>HIV</subject><subject>HIV-1</subject><subject>Human immunodeficiency virus</subject><subject>Humanities and Social Sciences</subject><subject>Humans</subject><subject>Introns</subject><subject>Isoforms</subject><subject>multidisciplinary</subject><subject>Proteins</subject><subject>RNA, Messenger - biosynthesis</subject><subject>RNA, Messenger - genetics</subject><subject>Science</subject><subject>Science (multidisciplinary)</subject><subject>Transduction, Genetic</subject><subject>Translation</subject><issn>2045-2322</issn><issn>2045-2322</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>C6C</sourceid><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNp9UUtrFTEYDaLYUvsHXEjAjZvRvOaRjXApVQvFQtF1yCTf3KbMJNckc-u49oeb21vb6qLZJHAe33dyEHpNyXtKePchCVrLriJUVoJJKir-DB0yIuqKccaeP3ofoOOUrkk5NZOCypfogFNC61o0h-j3CvuwhRFHyDFsXdQj3oLJIeK0pAwTzgFrr8flF2D4uYmQkgseDzFMeLr8usI3Ll_t1Np5sNj5YuMTnpPzazyMcygKAz7jTQwZXIG0twUIN9gsOUxl7PIKvRj0mOD47j5C3z-dfjv5Up1ffD47WZ1XpqY0V0JQJnXT0BK467gl1moYTCeE7fuB1Z1um95YppkemGSs7lsje8uF6anWoPkR-rj33cz9BHa3VsmrNtFNOi4qaKf-Rby7UuuwVY1ou5aRYvDuziCGHzOkrCZX0o2j9hDmpBijXNKGtqxQ3_5HvQ5zLB9ZWFxyQhpBeGGxPcvEkFKE4X4ZStSuZ7XvWZWe1W3Paid68zjGveRvq4XA94RUIL-G-DD7Cds_X9C3rQ</recordid><startdate>20190423</startdate><enddate>20190423</enddate><creator>Jackson, Patrick E. H.</creator><creator>Huang, Jing</creator><creator>Sharma, Monika</creator><creator>Rasmussen, Sara K.</creator><creator>Hammarskjold, Marie-Louise</creator><creator>Rekosh, David</creator><general>Nature Publishing Group UK</general><general>Nature Publishing Group</general><scope>C6C</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>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</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>M2P</scope><scope>M7P</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0003-3662-4176</orcidid></search><sort><creationdate>20190423</creationdate><title>A novel retroviral vector system to analyze expression from mRNA with retained introns using fluorescent proteins and flow cytometry</title><author>Jackson, Patrick E. H. ; Huang, Jing ; Sharma, Monika ; Rasmussen, Sara K. ; Hammarskjold, Marie-Louise ; Rekosh, David</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c511t-44129a661429883d0ddaefc844dbbf258a76bcd2a2af29225b7c9bd34cb1aaea3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>13</topic><topic>13/106</topic><topic>13/31</topic><topic>13/44</topic><topic>38</topic><topic>38/47</topic><topic>42</topic><topic>42/35</topic><topic>631/1647/2017/1958</topic><topic>631/337/1645/2052</topic><topic>Cell Line</topic><topic>Flow Cytometry</topic><topic>Gene expression</topic><topic>Gene Expression Regulation</topic><topic>Genetic Vectors</topic><topic>Green Fluorescent Proteins - biosynthesis</topic><topic>Green Fluorescent Proteins - genetics</topic><topic>HIV</topic><topic>HIV-1</topic><topic>Human immunodeficiency virus</topic><topic>Humanities and Social Sciences</topic><topic>Humans</topic><topic>Introns</topic><topic>Isoforms</topic><topic>multidisciplinary</topic><topic>Proteins</topic><topic>RNA, Messenger - biosynthesis</topic><topic>RNA, Messenger - genetics</topic><topic>Science</topic><topic>Science (multidisciplinary)</topic><topic>Transduction, Genetic</topic><topic>Translation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Jackson, Patrick E. H.</creatorcontrib><creatorcontrib>Huang, Jing</creatorcontrib><creatorcontrib>Sharma, Monika</creatorcontrib><creatorcontrib>Rasmussen, Sara K.</creatorcontrib><creatorcontrib>Hammarskjold, Marie-Louise</creatorcontrib><creatorcontrib>Rekosh, David</creatorcontrib><collection>Springer Nature OA Free Journals</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>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>Science Database (Alumni Edition)</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 One Sustainability</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</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</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>Science Database</collection><collection>Biological Science Database</collection><collection>Publicly Available Content Database</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 Basic</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Scientific reports</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Jackson, Patrick E. H.</au><au>Huang, Jing</au><au>Sharma, Monika</au><au>Rasmussen, Sara K.</au><au>Hammarskjold, Marie-Louise</au><au>Rekosh, David</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A novel retroviral vector system to analyze expression from mRNA with retained introns using fluorescent proteins and flow cytometry</atitle><jtitle>Scientific reports</jtitle><stitle>Sci Rep</stitle><addtitle>Sci Rep</addtitle><date>2019-04-23</date><risdate>2019</risdate><volume>9</volume><issue>1</issue><spage>6467</spage><epage>6467</epage><pages>6467-6467</pages><artnum>6467</artnum><issn>2045-2322</issn><eissn>2045-2322</eissn><abstract>The ability to overcome cellular restrictions that exist for the export and translation of mRNAs with retained introns is a requirement for the replication of retroviruses and also for the expression of many mRNA isoforms transcribed from cellular genes. In some cases, RNA structures have been identified in the mRNA that directly interact with cellular factors to promote the export and expression of isoforms with retained introns. In other cases, a viral protein is also required to act as an adapter. In this report we describe a novel vector system that allows measurement of the ability of
cis-
and
trans
-acting factors to promote the export and translation of mRNAs with retained introns. One reporter vector used in this system is derived from an HIV proviral clone engineered to express two different fluorescent proteins from spliced and unspliced transcripts. The ratio of fluorescent signals is a measurement of the efficiency of export and translation. A second vector utilizes a third fluorescent protein to measure the expression of viral export proteins that interact with some of the export elements. Both vectors can be packaged into viral particles and be used to transduce cells, allowing expression at physiological levels from the integrated vector.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>31015546</pmid><doi>10.1038/s41598-019-42914-3</doi><tpages>1</tpages><orcidid>https://orcid.org/0000-0003-3662-4176</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 2045-2322 |
| ispartof | Scientific reports, 2019-04, Vol.9 (1), p.6467-6467, Article 6467 |
| issn | 2045-2322 2045-2322 |
| language | eng |
| recordid | cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_6478720 |
| source | Nature Open Access; MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry; Springer Nature OA Free Journals |
| subjects | 13 13/106 13/31 13/44 38 38/47 42 42/35 631/1647/2017/1958 631/337/1645/2052 Cell Line Flow Cytometry Gene expression Gene Expression Regulation Genetic Vectors Green Fluorescent Proteins - biosynthesis Green Fluorescent Proteins - genetics HIV HIV-1 Human immunodeficiency virus Humanities and Social Sciences Humans Introns Isoforms multidisciplinary Proteins RNA, Messenger - biosynthesis RNA, Messenger - genetics Science Science (multidisciplinary) Transduction, Genetic Translation |
| title | A novel retroviral vector system to analyze expression from mRNA with retained introns using fluorescent proteins and flow cytometry |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-08T07%3A18%3A29IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_pubme&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=A%20novel%20retroviral%20vector%20system%20to%20analyze%20expression%20from%20mRNA%20with%20retained%20introns%20using%20fluorescent%20proteins%20and%20flow%20cytometry&rft.jtitle=Scientific%20reports&rft.au=Jackson,%20Patrick%20E.%20H.&rft.date=2019-04-23&rft.volume=9&rft.issue=1&rft.spage=6467&rft.epage=6467&rft.pages=6467-6467&rft.artnum=6467&rft.issn=2045-2322&rft.eissn=2045-2322&rft_id=info:doi/10.1038/s41598-019-42914-3&rft_dat=%3Cproquest_pubme%3E2393006403%3C/proquest_pubme%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2393006403&rft_id=info:pmid/31015546&rfr_iscdi=true |