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A Rapid FACS-Based Strategy to Isolate Human Gene Knockin and Knockout Clones
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Orientador(es)
Resumo(s)
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.
Descrição
Palavras-chave
Bacterial Proteins Cell Line DNA Flow Cytometry Fluorescent Dyes Gene Targeting Genetic Techniques Genetic Vectors Humans Luminescent Proteins Microscopy, Fluorescence Models, Genetic Promoter Regions, Genetic Gene Knock-In Techniques
Contexto Educativo
Citação
Mata 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.0032646
Editora
PLOS