title: A PiggyBac-based recessive screening method to identify pluripotency regulators.
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.
authors.affiliation: Wellcome Trust Centre for Stem Cell Research, Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Ge
authors.initials: G
authors.lastname: Guo
authors.forename: Yue
authors.initials: Y
authors.lastname: Huang
authors.forename: Peter
authors.initials: P
authors.lastname: Humphreys
authors.forename: Xiaozhong
authors.initials: X
authors.lastname: Wang
authors.forename: Austin
authors.initials: A
authors.lastname: Smith
mesh_terms: D001816:Bloom Syndrome / Q000235:genetics / Q000473:pathology; D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D016366:Open Reading Frames; D039904:Pluripotent Stem Cells / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction