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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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.
KeywordsES cells Gene targeting Homologous recombination Knock-in mice PARP-1
Double strand break
Diphtheria toxin A
Embryonic stem cell clone
Fluorescence in situ hybridization
Synthesis-dependent strand annealing
We thank Oliver Popp, Gudrun von Scheven, Daniela Gassen, Heidi Henseleit, and Birgitt Planitz for technical help and support, and Prof. Andrew Smith (Edinburgh, UK) for valuable discussion of the data. AM was supported by the ‘Studienstiftung des Deutschen Volkes’ and the ‘Deutsche Forschungs Gemeinschaft’ (DFG) (International Research Training Research Group 1331).
- Nagy A, Gertsenstein M, Vintersten K, Behringer R (2003) Manipulating the mouse embryo, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Sambrook A (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar