A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1 |
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Authors: | Aswin Mangerich Harry Scherthan Jörg Diefenbach Ulrich Kloz Franciscus van der Hoeven Sascha Beneke Alexander Bürkle |
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Institution: | (1) Molecular Toxicology Group, Department of Biology, University of Konstanz, Box X911, 78457 Constance, Germany;(2) Bundeswehr Institute of Radiobiology, Munich, Germany;(3) German Cancer Research Center, Transgenic Core Facility, Heidelberg, Germany |
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Abstract: | Here we report an approach to generate a knock-in mouse model using an ‘ends-out’ gene replacement vector to substitute the
murine Parp-1 (mParp-1) coding sequence (32 kb) with its human orthologous sequence (46 kb). Unexpectedly, examination of mutant ES cell clones
and mice revealed that site-specific homologous recombination was mimicked in three independently generated ES cell clones
by bidirectional extension of the vector homology arms using the endogenous mParp-1-flanking sequences as templates. This was followed by adjacent integration of the targeting vector, thus leaving the endogenous
mParp-1 locus functional. A related phenomenon termed ‘ectopic gene targeting’ has so far only been described for ‘ends-in’ integration-type
vectors in non-ES cell gene targeting. We provide reliable techniques to detect such ectopic gene targeting which represents
an unexpected caveat in mouse genetic engineering that should be considered in the design and validation strategy of future
gene knock-in approaches.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. |
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Keywords: | ES cells Gene targeting Homologous recombination Knock-in mice PARP-1 |
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