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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Mangerich_2008_TransgenicRes_epub.pdf
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2009
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Scherthan, Harry
Diefenbach, Jörg
Kloz, Ulrich
Hoeven, Franciscus van der
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Transgenic Research. 2009, 18(2), pp. 261-279. ISSN 0962-8819. eISSN 1573-9368. Available under: doi: 10.1007/s11248-008-9228-x
Zusammenfassung

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.

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570 Biowissenschaften, Biologie
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ES cells, Gene targeting, Homologous recombination, Knock-in mice, PARP-1
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ISO 690MANGERICH, Aswin, Harry SCHERTHAN, Jörg DIEFENBACH, Ulrich KLOZ, Franciscus van der HOEVEN, Sascha BENEKE, Alexander BÜRKLE, 2009. 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. In: Transgenic Research. 2009, 18(2), pp. 261-279. ISSN 0962-8819. eISSN 1573-9368. Available under: doi: 10.1007/s11248-008-9228-x
BibTex
@article{Mangerich2009cavea-8047,
  year={2009},
  doi={10.1007/s11248-008-9228-x},
  title={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},
  number={2},
  volume={18},
  issn={0962-8819},
  journal={Transgenic Research},
  pages={261--279},
  author={Mangerich, Aswin and Scherthan, Harry and Diefenbach, Jörg and Kloz, Ulrich and Hoeven, Franciscus van der and Beneke, Sascha and Bürkle, Alexander}
}
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