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  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

Mangerich, A., Scherthan, H., Diefenbach, J., Kloz, U., van der Hoeven, F., Beneke, S., et al. (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. Transgenic Research, 18(2), 261-279. doi:10.1007/s11248-008-9228-x.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-7DCA-4 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-7DCB-2
Genre: Journal Article

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 Creators:
Mangerich, Aswin, Author
Scherthan, Harry1, Author              
Diefenbach, Jörg, Author
Kloz, Ulrich, Author
van der Hoeven, Franciscus, Author
Beneke, Sascha, Author
Bürkle, Alexander, Author
Affiliations:
1Dept. of Human Molecular Genetics (Head: Hans-Hilger Ropers), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1433549              

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Free keywords: ES cells; Gene targeting; Homologous recombination; Knock-in mice; PARP-1
 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.

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Language(s): eng - English
 Dates: 2009-04
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Degree: -

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Title: Transgenic Research
Source Genre: Journal
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Affiliations:
Publ. Info: -
Pages: - Volume / Issue: 18 (2) Sequence Number: - Start / End Page: 261 - 279 Identifier: ISSN: 0962-8819