a piggybac-based recessive screening method to identify pluripotency regulatorspiggybac-based隐性的筛选方法来识别多能性监管机构.pdfVIP

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a piggybac-based recessive screening method to identify pluripotency regulatorspiggybac-based隐性的筛选方法来识别多能性监管机构.pdf

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a piggybac-based recessive screening method to identify pluripotency regulatorspiggybac-based隐性的筛选方法来识别多能性监管机构

A PiggyBac-Based Recessive Screening Method to Identify Pluripotency Regulators 1 2¤ 1 3 1 Ge Guo , Yue Huang , Peter Humphreys , Xiaozhong Wang , Austin Smith * 1 Wellcome Trust Centre for Stem Cell Research, Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom, 2 Wellcome Trust Sanger Institute, Hinxton, United Kingdom, 3 Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois, United States of America Abstract Phenotype driven genetic screens allow unbiased exploration of the genome to discover new biological regulators. Bloom syndrome gene (Blm) deficient embryonic stem (ES) cells provide an opportunity for recessive screening due to frequent loss of heterozygosity. We describe a strategy for isolating regulators of mammalian pluripotency based on conversion to homozygosity of PiggyBac gene trap insertions combined with stringent selection for differentiation resistance. From a screen of 2000 mutants we obtained a disruptive integration in the Tcf3 gene. Homozygous Tcf3 mutants showed impaired differentiation and enhanced self-renewal. This phenotype was reverted in a dosage sensitive manner by excision of one or both copies of the gene trap. These results provide new evidence confirming that Tcf3 is a potent negative regulator of pluripotency and validate a forward screening methodology to identify modulators of pluripotent stem cell biology. Citation: Guo G, Huang Y, Humphreys P, Wang X, Smith A (2011) A PiggyBac-Based Recessive Screening Method to Identify Pluripotency Regulators. PLoS ONE 6(4): e18189. doi:10.1371/journal.pone.0018189 Editor: Domingos Henrique, Instituto de Medicina Molecular, Portugal Received August 31, 2010; Accepted Februa