Site-specific cassette exchange and germline transmission with mouse ES cells expressing φC31 integrase

Since the publication of the first edition of Gene Targeting: A Practical Approach in 1993 there have been many advances in gene targeting and this new edition has been thoroughly updated and rewritten to include all the major new techniques. It provides not only tried-and-tested practical protocols but detailed guidance on their use and applications. As with the previous edition Gene Targeting: A Practical Approach 2e concentrates on gene targeting in mouse ES cells, but the techniques described can be easily adapted to applications in tissue culture including those for human cells. The first chapter covers the design of gene targeting vectors for mammalian cells and describes how to distinguish random integrations from homologous recombination. It is followed by a chapter on extending conventional gene targeting manipulations by using site-specific recombination using the Cre-loxP and Flp-FRT systems to produce 'clean' germline mutations and conditionally (in)activating genes. Chapter 3 describes methods for introducing DNA into ES cells for homologous recombination, selection and screening procedures for identifying and recovering targeted cell clones, and a simple method for establishing new ES cell lines. Chapter 4 discusses the pros and cons or aggregation versus blastocyst injection to create chimeras, focusing on the technical aspects of generating aggregation chimeras and then describes some of the uses of chimeras. The next topic covered is gene trap strategies; the structure, components, design, and modification of GT vectors, the various types of GT screens, and the molecular analysis of GT integrations. The final chapter explains the use of classical genetics in gene targeting and phenotype interpretation to create mutations and elucidate gene functions. Gene Targeting: A Practical Approach 2e will therefore be of great value to all researchers studying gene function.

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Analysis of GATA and its related factors using in vitro differentiation of mouse ES cells

Blood Cells Molecules and Diseases ◽

10.1016/j.bcmd.2006.10.097 ◽

2007 ◽

Vol 38 (2) ◽

pp. 161

Author(s):

Toru Nakano ◽

Jie Zheng ◽

Daijiro Sugiyama ◽

Hilo Yen ◽

Kenji Kitajima

Keyword(s):

Es Cells ◽

In Vitro Differentiation ◽

Related Factors ◽

Mouse Es Cells

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Hoxa1 and TALE proteins display cross-regulatory interactions and form a combinatorial binding code on Hoxa1 targets

10.1101/092296 ◽

2016 ◽

Author(s):

Bony De Kumar ◽

Hugo J. Parker ◽

Ariel Paulson ◽

Mark E. Parrish ◽

Irina Pushel ◽

...

Keyword(s):

Es Cells ◽

Hox Proteins ◽

Binding Motifs ◽

Functional Roles ◽

Regulatory Interactions ◽

Mouse Es Cells ◽

Genome Wide ◽

A Genome ◽

Combinatorial Interactions ◽

Insight Into

AbstractHoxa1 has diverse functional roles in differentiation and development. We have identified and characterized properties of regions bound by Hoxa1 on a genome-wide basis in differentiating mouse ES cells. Hoxa1 bound regions are enriched for clusters of consensus binding motifs for Hox, Pbx and Meis and many display co-occupancy of Pbx and Meis. Pbx and Meis are members of the TALE family and genome-wide analysis of multiple TALE members (Pbx, Meis, TGIF, Prep1 and Prep2) show that nearly all Hoxa1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins defines distinct classes of Hoxa1 targets and indicates a role as cofactors in modulating the specificity of Hox proteins. We also discovered extensive auto- and cross-regulatory interactions among the Hoxa1 and TALE genes. This study provides new insight into a regulatory network involving combinatorial interactions between Hoxa1 and TALE proteins.

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Physiological low oxygen during development induces neural differentiation from mouse ES cells and mouse iPS cells

Neuroscience Research ◽

10.1016/j.neures.2010.07.1082 ◽

2010 ◽

Vol 68 ◽

pp. e244

Author(s):

Masataka Fujita ◽

Tea-Sun Kim ◽

Sachiyo Misumi ◽

Yoshitomo Ueda ◽

Hitoo Nishino ◽

...

Keyword(s):

Neural Differentiation ◽

Es Cells ◽

Ips Cells ◽

Low Oxygen ◽

Mouse Es Cells

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Baf53a is involved in survival of mouse ES cells, which can be compensated by Baf53b

Scientific Reports ◽

10.1038/s41598-017-14362-4 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 7

Author(s):

Bo Zhu ◽

Atsushi Ueda ◽

Xiaohong Song ◽

Shin-ichi Horike ◽

Takashi Yokota ◽

...

Keyword(s):

Es Cells ◽

Mouse Es Cells

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Cbx2 stably associates with mitotic chromosomes via a PRC2- or PRC1-independent mechanism and is needed for recruiting PRC1 complex to mitotic chromosomes

Molecular Biology of the Cell ◽

10.1091/mbc.e14-06-1109 ◽

2014 ◽

Vol 25 (23) ◽

pp. 3726-3739 ◽

Cited By ~ 23

Author(s):

Chao Yu Zhen ◽

Huy Nguyen Duc ◽

Marko Kokotovic ◽

Christopher J. Phiel ◽

Xiaojun Ren

Keyword(s):

Molecular Mechanisms ◽

Es Cells ◽

Epigenetic Inheritance ◽

Polycomb Group ◽

Mitotic Chromosomes ◽

Mouse Es Cells ◽

C Terminus ◽

Independent Mechanism ◽

Cellular Identity ◽

Quantitative Fluorescence

Polycomb group (PcG) proteins are epigenetic transcriptional factors that repress key developmental regulators and maintain cellular identity through mitosis via a poorly understood mechanism. Using quantitative live-cell imaging in mouse ES cells and tumor cells, we demonstrate that, although Polycomb repressive complex (PRC) 1 proteins (Cbx-family proteins, Ring1b, Mel18, and Phc1) exhibit variable capacities of association with mitotic chromosomes, Cbx2 overwhelmingly binds to mitotic chromosomes. The recruitment of Cbx2 to mitotic chromosomes is independent of PRC1 or PRC2, and Cbx2 is needed to recruit PRC1 complex to mitotic chromosomes. Quantitative fluorescence recovery after photobleaching analysis indicates that PRC1 proteins rapidly exchange at interphasic chromatin. On entry into mitosis, Cbx2, Ring1b, Mel18, and Phc1 proteins become immobilized at mitotic chromosomes, whereas other Cbx-family proteins dynamically bind to mitotic chromosomes. Depletion of PRC1 or PRC2 protein has no effect on the immobilization of Cbx2 on mitotic chromosomes. We find that the N-terminus of Cbx2 is needed for its recruitment to mitotic chromosomes, whereas the C-terminus is required for its immobilization. Thus these results provide fundamental insights into the molecular mechanisms of epigenetic inheritance.

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