a rapid facs-based strategy to isolate human gene knockin and knockout clones快速facs-based策略分离克隆人类基因敲天国和淘汰赛.pdfVIP

A Rapid FACS-Based Strategy to Isolate Human Gene Knockin and Knockout Clones ˜ Joao F. Mata, Telma Lopes, Rui Gardner, Lars E. T. Jansen* ˆ Instituto Gulbenkian de Ciencia, Oeiras, Portugal 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. Citation: 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 Editor: Beth A. Sullivan, Duke University, United States of America Received November 22, 2011; Accepted January 28, 2012; Published February 29, 2012 Copyright: 2012 Mata et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.