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A Rapid FACS-Based Strategy to Isolate Human Gene Knockin and Knockout Clones

2012-02-29Journal article Open access
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dc.contributor.authorMata, João F.
dc.contributor.authorLopes, Telma
dc.contributor.authorGardner, Rui
dc.contributor.authorJansen, Lars E. T.
dc.date.accessioned2016-05-18T18:19:15Z
dc.date.available2016-05-18T18:19:15Z
dc.date.issued2012-02-29
dc.description.abstractGene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted clones obviating the need for selection and outgrowth of drug resistant clones. Importantly, the use of a promoter-less, ATG-less construct greatly facilitates the recovery of correctly targeted cells. Using this method we report successful gene targeting in up to 94% of recovered human somatic cell clones. We create functional EYFP-tagged knockin clones in both transformed and non-transformed human somatic cell lines providing a valuable tool for mammalian cell biology. We further demonstrate the use of this technology to create gene knockouts. Using this generally applicable strategy we can recover gene targeted clones within approximately one month from DNA construct delivery to obtaining targeted monoclonal cell lines.pt_PT
dc.description.sponsorshipMarie Curie Reinstallation grant: (PIRG02-GA-2007-224874); FCT grants: (BIA-BCM/100557/2008, BIA-PRO/100537/2008); EMBO installation grant.pt_PT
dc.identifier.citationMata JF, Lopes T, Gardner R, Jansen LET (2012) A Rapid FACS-Based Strategy to Isolate Human Gene Knockin and Knockout Clones. PLoS ONE 7(2): e32646. doi:10.1371/journal.pone.0032646pt_PT
dc.identifier.doi10.1371/journal.pone.0032646pt_PT
dc.identifier.urihttp://hdl.handle.net/10400.7/609
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherPLOSpt_PT
dc.relation.publisherversionhttp://journals.plos.org/plosone/article?id=10.1371/journal.pone.0032646pt_PT
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/pt_PT
dc.subjectBacterial Proteinspt_PT
dc.subjectCell Linept_PT
dc.subjectDNApt_PT
dc.subjectFlow Cytometrypt_PT
dc.subjectFluorescent Dyespt_PT
dc.subjectGene Targetingpt_PT
dc.subjectGenetic Techniquespt_PT
dc.subjectGenetic Vectorspt_PT
dc.subjectHumanspt_PT
dc.subjectLuminescent Proteinspt_PT
dc.subjectMicroscopy, Fluorescencept_PT
dc.subjectModels, Geneticpt_PT
dc.subjectPromoter Regions, Geneticpt_PT
dc.subjectGene Knock-In Techniquespt_PT
dc.titleA Rapid FACS-Based Strategy to Isolate Human Gene Knockin and Knockout Clonespt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.citation.endPage8pt_PT
oaire.citation.issue2pt_PT
oaire.citation.startPage1pt_PT
oaire.citation.titlePLoS ONEpt_PT
oaire.citation.volume7pt_PT
rcaap.rightsopenAccesspt_PT
rcaap.typearticlept_PT

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