NDLI: Subcloning plus insertion (SPI)--a novel recombineering method for the rapid construction of gene targeting vectors.

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Subcloning plus insertion (SPI)--a novel recombineering method for the rapid construction of gene targeting vectors.

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Subcloning plus insertion (SPI)--a novel recombineering method for the rapid construction of gene targeting vectors.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Reddy, Thimma R. Kelsall, Emma J. Fevat, Léna M. S. Munson, Sarah E. Cowley, Shaun M.
Description	Author Affiliation: Reddy TR ( Department of Biochemistry, University of Leicester.); Kelsall EJ ( Department of Biochemistry, University of Leicester.); Fevat LM ( Center for Fisheries, Environment and Aquaculture Sciences.); Munson SE ( ES Cell Facility, Centre for Core Biotechnology Services, University of Leicester.); Cowley SM ( Department of Biochemistry, University of Leicester)
Abstract	Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a 'targeting' vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4 kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/52155
Journal	Journal of Visualized Experiments
Issue Number	95
Language	English
Publisher	MyJove Corp.
Publisher Date	2015-01-08
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Gene Targeting Genetic Engineering Escherichia Coli Genetics Recombination, Genetic Research Support, Non-u.s. Gov't Video-audio Media
Content Type	Text
Resource Type	Article

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