Modification of bacterial artificial chromosomes (BACs) and preparation of intact BAC DNA for generation of transgenic mice

Current Protocols in Neuroscience
Shiaoching Gong, X W Yang

Abstract

BAC transgenesis is a powerful tool for the study of gene expression and gene function in the mouse in vivo. In this unit, detailed protocols are provided for modification (i.e., marker gene insertion, deletion, or point mutation) of BACs by homologous recombination in E. coli. This method utilizes a shuttle vector that allows transient expression of the E. coli RecA gene to support homologous recombination in the BAC host bacteria. In addition, two protocols are provided for purification of BAC DNA for microinjection to generate transgenic mice. Since BAC DNA is prone to degradation, which may introduce positional effects in transgenic mice, two methods are given for purification of intact BAC DNA for subsequent microinjection.

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Citations

Oct 5, 2010·Nature Protocols·Shiaoching GongNathaniel Heintz
Jan 28, 2015·Molecular Psychiatry·J A BrownKároly Mirnics
Aug 16, 2016·The Journal of Clinical Investigation·Ian H KratterSteven Finkbeiner
Jan 5, 2011·Journal of Biomedicine & Biotechnology·Kumaran Narayanan, Qingwen Chen
Jan 13, 2010·Current Protocols in Neuroscience·Qing Xu, Stewart A Anderson

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