Journal ArticleDOI
An improved recombineering approach by adding RecA to lambda Red recombination.
Junping Wang,Mihail Sarov,Jeanette M.J. Rientjes,Jun Fu,Heike Hollak,Harald Kranz,Wei Xie,A. Francis Stewart,Youming Zhang +8 more
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TLDR
It is reported that transient RecA co-expression enhances the total numer of successful recombinations in bacterial artificial chromosomes (BACs), mostly because the E. coli host is more able to survive the stresses of DNA transformation procedures.Abstract:
Recombineering is the use of homologous recombination in Escherichia coli for DNA engineering. Of several approaches, use of the lambda phage Red operon is emerging as the most reliable and flexible. The Red operon includes three components: Redalpha, a 5' to 3' exonuclease, Redbeta, an annealing protein, and Redgamma, an inhibitor of the major E. coli exonuclease and recombination complex, RecBCD. Most E. coli cloning hosts are recA deficient to eliminate recombination and therefore enhance the stability of cloned DNAs. However, loss of RecA also impairs general cellular integrity. Here we report that transient RecA co-expression enhances the total number of successful recombinations in bacterial artificial chromosomes (BACs), mostly because the E. coli host is more able to survive the stresses of DNA transformation procedures. We combined this practical improvement with the advantages of a temperature-sensitive version of the low copy pSC101 plasmid to develop a protocol that is convenient and more efficient than any recombineering procedure, for use of either double- or single-stranded DNA, published to date.read more
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Journal ArticleDOI
A conditional knockout resource for the genome-wide study of mouse gene function.
William C. Skarnes,Barry Rosen,Anthony P. West,Manousos Koutsourakis,Wendy Bushell,Vivek Iyer,Alejandro O. Mujica,Alejandro O. Mujica,Mark G. Thomas,Jennifer Harrow,Tony Cox,David A. Jackson,Jessica Severin,Jessica Severin,Patrick J. Biggs,Patrick J. Biggs,Jun Fu,Michael Nefedov,Pieter J. de Jong,A. Francis Stewart,Allan Bradley +20 more
TL;DR: High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.
Journal ArticleDOI
Recombineering: a homologous recombination-based method of genetic engineering
TL;DR: Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in Escherichia coli that circumvents the need for most standard in vitro cloning techniques.
Journal ArticleDOI
Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting
Jun Fu,Xiaoying Bian,Shengbaio Hu,Shengbaio Hu,Hailong Wang,Hailong Wang,Fan Huang,Fan Huang,Philipp Martin Seibert,Alberto Plaza,Liqiu Xia,Rolf Müller,A. Francis Stewart,Youming Zhang +13 more
TL;DR: Direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination mechanistically distinct from conventional recombineering mediated by Redαβ from lambda phage or truncated versions of RecET is described.
Journal ArticleDOI
A Genome-Scale Resource for In Vivo Tag-Based Protein Function Exploration in C. elegans
Mihail Sarov,John I. Murray,Kristin Schanze,Andrei Pozniakovski,Wei Niu,Karolin Angermann,Susanne Hasse,Michaela Rupprecht,Elisabeth Vinis,Matthew Tinney,Elicia Preston,Andrea Zinke,Susanne Enst,Tina Teichgraber,J. Janette,Kadri Reis,Stephan Janosch,Siegfried Schloissnig,Radoslaw K. Ejsmont,C. Slightam,Xiao Xu,Stuart K. Kim,Valerie Reinke,A. Francis Stewart,Michael Snyder,Robert H. Waterston,Anthony A. Hyman +26 more
TL;DR: A genome-scale transgenic platform for in vivo expression of fluorescent- and affinity-tagged proteins in Caenorhabditis elegans under endogenous cis regulatory control is built, generating a resource of 14,637 genomic DNA transgenes, which covers 73% of the proteome.
Journal ArticleDOI
Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A
TL;DR: Examination of microtubule nucleation during spindle assembly shows that microtubules fail to nucleate from chromosomes, and chromosome nucleation is not essential for bipolarity during human cell mitosis when centrosomes are present.
References
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Journal ArticleDOI
One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products
TL;DR: A simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s), which should be widely useful, especially in genome analysis of E. coli and other bacteria.
Journal ArticleDOI
Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.
TL;DR: The tight regulation of the PBAD promoter is exploited to study the phenotypes of null mutations of essential genes and the use of pBAD vectors as an expression system is explored.
Journal ArticleDOI
An efficient recombination system for chromosome engineering in Escherichia coli
TL;DR: A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA using a defective lambda prophage, which will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.
Journal ArticleDOI
Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector.
Hiroaki Shizuya,Bruce Birren,Ung-Jin Kim,Valeria Mancino,Tatiana Slepak,Yoshiaki Tachiiri,Melvin I. Simon +6 more
TL;DR: Because of high cloning efficiency, easy manipulation of the cloned DNA, and stable maintenance of inserted DNA, the BAC system may facilitate construction of DNA libraries of complex genomes with fuller representation and subsequent rapid analysis of complex genomic structure.
Journal ArticleDOI
A new logic for DNA engineering using recombination in Escherichia coli
TL;DR: A straightforward way to engineer DNA in E. coli using homologous recombination is described in this article, which uses RecE and RecT and is transferable between different E coli strains.