title: A rapid FACS-based strategy to isolate human gene knockin and knockout clones.
abstract: Gene 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.
authors.affiliation: Instituto Gulbenkian de Ciência, Oeiras, Portugal.
authors.forename: João F
authors.initials: JF
authors.lastname: Mata
authors.forename: Telma
authors.initials: T
authors.lastname: Lopes
authors.forename: Rui
authors.initials: R
authors.lastname: Gardner
authors.forename: Lars E T
authors.initials: LE
authors.lastname: Jansen
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D055879:Gene Knock-In Techniques; D018390:Gene Targeting / Q000379:methods*; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D008957:Models, Genetic; D011401:Promoter Regions, Genetic
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D004247:DNA