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Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step

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Abstract

We have developed technologies that simplify genomic library construction and screening, substantially reducing both the time and the cost associated with traditional library screening methods and facilitating the generation of gene-targeting constructs. By taking advantage of homologous recombination in Escherichia coli, we were able to use as little as 80 bp of total sequence homology to screen for a specific gene from a genomic library in plasmid or phage form. This method, called recombination cloning (REC), takes only a few days instead of the several weeks required for traditional plaque-lift methods. In addition, because every clone in the mouse genomic library we have constructed has a negative selection marker adjacent to the genomic insert, REC screening can generate gene-targeting vectors in one step, from library screening to finished construct. Conditional targeting constructs can be generated easily with minimal additional manipulation.

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Figure 1: Diagrams of λKO vectors.
Figure 2: Library screening and targeting vector construction by homologous recombination using the REC method.
Figure 3: Library screening through the generation of DNA targeting fragments generated in vivo.
Figure 4: Isolation of genomic clones for Pttg1 and trbl using REC.
Figure 5: Generation of conditional targeting constructs.

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  • 27 March 2018

    This article was initially published with an incorrect DOI that did not match the registered version at Crossref. The DOI has been corrected in the article.

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Acknowledgements

We thank M.J. Lombardo, R. Ponder, S.M. Rosenberg and K. Murphy for bacterial strains. This work was supported by NIH and ATP grants to S.J.E. and by a HHMI grant to P.Z. S.J.E. is an Investigator with the Howard Hughes Medical Institute.

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Correspondence to Stephen J. Elledge.

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Zhang, P., Li, M. & Elledge, S. Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step. Nat Genet 30, 31–39 (2002). https://doi.org/10.1038/ng797

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