A PiggyBac-Based Recessive Screening Method to Identify Pluripotency Regulators

被引：48

作者：

Guo, Ge ^{[1
]}

Huang, Yue ^{[2
]}

Humphreys, Peter ^{[1
]}

Wang, Xiaozhong ^{[3
]}

Smith, Austin ^{[1
]}

机构：

[1] Univ Cambridge, Dept Biochem, Wellcome Trust Ctr Stem Cell Res, Cambridge CB2 1QW, England

[2] Wellcome Trust Sanger Inst, Hinxton, S Cambs, England

[3] Northwestern Univ, Dept Biochem Mol Biol & Cell Biol, Evanston, IL USA

来源：

PLOS ONE | 2011年 / 6卷 / 04期

基金：

英国医学研究理事会; 英国生物技术与生命科学研究理事会; 英国惠康基金;

关键词：

STEM-CELL DIFFERENTIATION; SIGNALING PATHWAYS; ES CELLS; NANOG; TRANSCRIPTION; MAINTAINS; ENTRY; TCF3;

D O I：

10.1371/journal.pone.0018189

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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.

引用

页数：11

共 34 条

[1] A new retroviral vector, CA1, to identify and select for cells expressing an inserted gene in vitro and in vivo [J].

Abram, CL ;

Page, MJ ;

Edwards, PAW .

GENE, 1997, 196 (1-2) :187-189

[2] CellProfiler: image analysis software for identifying and quantifying cell phenotypes [J].

Carpenter, Anne E. ;

Jones, Thouis Ray ;

Lamprecht, Michael R. ;

Clarke, Colin ;

Kang, In Han ;

Friman, Ola ;

Guertin, David A. ;

Chang, Joo Han ;

Lindquist, Robert A. ;

Moffat, Jason ;

Golland, Polina ;

Sabatini, David M. .

GENOME BIOLOGY, 2006, 7 (10)

[3] TRANSPOSON MUTAGENESIS OF BACULOVIRUSES - ANALYSIS OF TRICHOPLUSIA-NI TRANSPOSON IFP2 INSERTIONS WITHIN THE FP-LOCUS OF NUCLEAR POLYHEDROSIS VIRUSES [J].

CARY, LC ;

GOEBEL, M ;

CORSARO, BG ;

WANG, HG ;

ROSEN, E ;

FRASER, MJ .

VIROLOGY, 1989, 172 (01) :156-169

[4] Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells [J].

Chambers, I ;

Colby, D ;

Robertson, M ;

Nichols, J ;

Lee, S ;

Tweedie, S ;

Smith, A .

CELL, 2003, 113 (05) :643-655

[5] Nanog safeguards pluripotency and mediates germline development [J].

Chambers, Ian ;

Silva, Jose ;

Colby, Douglas ;

Nichols, Jennifer ;

Nijmeijer, Bianca ;

Robertson, Morag ;

Vrana, Jan ;

Jones, Ken ;

Grotewold, Lars ;

Smith, Austin .

NATURE, 2007, 450 (7173) :1230-U8

[6] Tcf3 is an integral component of the core regulatory circuitry of embryonic stem cells [J].

Cole, Megan F. ;

Johnstone, Sarah E. ;

Newman, Jamie J. ;

Kagey, Michael H. ;

Young, Richard A. .

GENES & DEVELOPMENT, 2008, 22 (06) :746-755

[7] SPLINKERETTES IMPROVED VECTORETTES FOR GREATER EFFICIENCY IN PCR WALKING [J].

DEVON, RS ;

PORTEOUS, DJ ;

BROOKES, AJ .

NUCLEIC ACIDS RESEARCH, 1995, 23 (09) :1644-1645

[8] Efficient transposition of the piggyBac resource (PB) transposon in mammalian cells and mice [J].

Ding, S ;

Wu, XH ;

Li, G ;

Han, M ;

Zhuang, Y ;

Xu, T .

CELL, 2005, 122 (03) :473-483

[9] Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells [J].

Guo, G ;

Wang, W ;

Bradley, A .

NATURE, 2004, 429 (6994) :891-895

[10] Oct4 and LIF/Stat3 Additively Induce Kruppel Factors to Sustain Embryonic Stem Cell Self-Renewal [J].

Hall, John ;

Guo, Ge ;

Wray, Jason ;

Eyres, Isobel ;

Nichols, Jennifer ;

Grotewold, Lars ;

Morfopoulou, Sofia ;

Humphreys, Peter ;

Mansfield, William ;

Walker, Rachael ;

Tomlinson, Simon ;

Smith, Austin .

CELL STEM CELL, 2009, 5 (06) :597-609

← 1 2 3 4 →