Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System
Recombinase mediated cassette exchange (RMCE) is a powerful tool for targeted insertion of transgenes. Here we describe non-proprietary 'RMCE-in' cell lines as an alternative to the 'Flp-in' system and cell lines. RMCE-in cell lines offer a number of advantages including increase...
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| Veröffentlicht in: | PloS one 2016-08, Vol.11 (8), p.e0161471-e0161471 |
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| creator | Callesen, Morten M Berthelsen, Martin F Lund, Sira Füchtbauer, Annette C Füchtbauer, Ernst-Martin Jakobsen, Jannik E |
| description | Recombinase mediated cassette exchange (RMCE) is a powerful tool for targeted insertion of transgenes. Here we describe non-proprietary 'RMCE-in' cell lines as an alternative to the 'Flp-in' system and cell lines. RMCE-in cell lines offer a number of advantages including increased efficiency of integration of the genetic element of interest (GEI) at a single docking site, lack of bacterial backbone at the docking site both before and after GEI integration, removal of selection and visual markers initially present at the docking site upon GEI integration and the possibility to validate GEI integration by loss of a red fluorescence reporter. Moreover, the RMCE-in cell lines are compatible with GEI donors used for the Flp-in system. We demonstrate a three-step procedure for generating RMCE-in cell lines, (I) RMCE-in transposon and SB10 transposase transfection, (II) clone isolation, and (III) selecting single integrated clones with highest RFP level, which could in principle be used to turn any cell line into an RMCE-in cell line. The RMCE-in system was used as a proof of concept to produce three new RMCE-in cell lines using HEK293, HeLa, and murine embryonic stem (mES) cells. The established RMCE-in cell lines and vector are freely available from the ATCC cell bank and Addgene respectively. |
| doi_str_mv | 10.1371/journal.pone.0161471 |
| format | Article |
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Here we describe non-proprietary 'RMCE-in' cell lines as an alternative to the 'Flp-in' system and cell lines. RMCE-in cell lines offer a number of advantages including increased efficiency of integration of the genetic element of interest (GEI) at a single docking site, lack of bacterial backbone at the docking site both before and after GEI integration, removal of selection and visual markers initially present at the docking site upon GEI integration and the possibility to validate GEI integration by loss of a red fluorescence reporter. Moreover, the RMCE-in cell lines are compatible with GEI donors used for the Flp-in system. We demonstrate a three-step procedure for generating RMCE-in cell lines, (I) RMCE-in transposon and SB10 transposase transfection, (II) clone isolation, and (III) selecting single integrated clones with highest RFP level, which could in principle be used to turn any cell line into an RMCE-in cell line. The RMCE-in system was used as a proof of concept to produce three new RMCE-in cell lines using HEK293, HeLa, and murine embryonic stem (mES) cells. The established RMCE-in cell lines and vector are freely available from the ATCC cell bank and Addgene respectively.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0161471</identifier><identifier>PMID: 27541869</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Bacteria ; Base Sequence ; Biology and Life Sciences ; Biotechnology ; Cell lines ; Clinical medicine ; Cloning ; Deoxyribonucleic acid ; DNA ; DNA Nucleotidyltransferases - metabolism ; Docking ; Fluorescence ; Gene Targeting ; Genes ; Genes, Reporter ; Genetic Vectors ; Genomics - methods ; HEK293 Cells ; HeLa Cells ; Humans ; Insertion ; Integration ; Molecular biology ; Proteins ; Recombinase ; Recombinases ; Recombinases - metabolism ; Recombination, Genetic ; Research and Analysis Methods ; RNA ; Stem cells ; Transfection ; Transgenes ; Transposase</subject><ispartof>PloS one, 2016-08, Vol.11 (8), p.e0161471-e0161471</ispartof><rights>COPYRIGHT 2016 Public Library of Science</rights><rights>2016 Callesen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2016 Callesen et al 2016 Callesen et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c626t-5f551ff958d6635166155f218600d3bd7805cb11d90924ede00686bdac617a983</citedby><cites>FETCH-LOGICAL-c626t-5f551ff958d6635166155f218600d3bd7805cb11d90924ede00686bdac617a983</cites><orcidid>0000-0001-6258-2171</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/PMC4991790/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991790/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,725,778,782,862,883,2098,2917,23849,27907,27908,53774,53776,79351,79352</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27541869$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Hohenstein, Peter</contributor><creatorcontrib>Callesen, Morten M</creatorcontrib><creatorcontrib>Berthelsen, Martin F</creatorcontrib><creatorcontrib>Lund, Sira</creatorcontrib><creatorcontrib>Füchtbauer, Annette C</creatorcontrib><creatorcontrib>Füchtbauer, Ernst-Martin</creatorcontrib><creatorcontrib>Jakobsen, Jannik E</creatorcontrib><title>Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Recombinase mediated cassette exchange (RMCE) is a powerful tool for targeted insertion of transgenes. Here we describe non-proprietary 'RMCE-in' cell lines as an alternative to the 'Flp-in' system and cell lines. RMCE-in cell lines offer a number of advantages including increased efficiency of integration of the genetic element of interest (GEI) at a single docking site, lack of bacterial backbone at the docking site both before and after GEI integration, removal of selection and visual markers initially present at the docking site upon GEI integration and the possibility to validate GEI integration by loss of a red fluorescence reporter. Moreover, the RMCE-in cell lines are compatible with GEI donors used for the Flp-in system. We demonstrate a three-step procedure for generating RMCE-in cell lines, (I) RMCE-in transposon and SB10 transposase transfection, (II) clone isolation, and (III) selecting single integrated clones with highest RFP level, which could in principle be used to turn any cell line into an RMCE-in cell line. 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Here we describe non-proprietary 'RMCE-in' cell lines as an alternative to the 'Flp-in' system and cell lines. RMCE-in cell lines offer a number of advantages including increased efficiency of integration of the genetic element of interest (GEI) at a single docking site, lack of bacterial backbone at the docking site both before and after GEI integration, removal of selection and visual markers initially present at the docking site upon GEI integration and the possibility to validate GEI integration by loss of a red fluorescence reporter. Moreover, the RMCE-in cell lines are compatible with GEI donors used for the Flp-in system. We demonstrate a three-step procedure for generating RMCE-in cell lines, (I) RMCE-in transposon and SB10 transposase transfection, (II) clone isolation, and (III) selecting single integrated clones with highest RFP level, which could in principle be used to turn any cell line into an RMCE-in cell line. The RMCE-in system was used as a proof of concept to produce three new RMCE-in cell lines using HEK293, HeLa, and murine embryonic stem (mES) cells. The established RMCE-in cell lines and vector are freely available from the ATCC cell bank and Addgene respectively.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>27541869</pmid><doi>10.1371/journal.pone.0161471</doi><orcidid>https://orcid.org/0000-0001-6258-2171</orcidid><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; DOAJ Directory of Open Access Journals; Public Library of Science (PLoS); EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Bacteria Base Sequence Biology and Life Sciences Biotechnology Cell lines Clinical medicine Cloning Deoxyribonucleic acid DNA DNA Nucleotidyltransferases - metabolism Docking Fluorescence Gene Targeting Genes Genes, Reporter Genetic Vectors Genomics - methods HEK293 Cells HeLa Cells Humans Insertion Integration Molecular biology Proteins Recombinase Recombinases Recombinases - metabolism Recombination, Genetic Research and Analysis Methods RNA Stem cells Transfection Transgenes Transposase |
| title | Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System |
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