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

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.

• Publication year

  2012

• Venue

  PLoS ONE

• Publication date

  2012-02-29

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1371/journal.pone.0032646PMID 22393430PMCID 3290580
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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