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.
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.
authors.affiliation: Molecular Toxicology Group, Department of Biology, University of Konstanz, Box X911, 78457, Constance, Germany.
authors.forename: Aswin
authors.initials: A
authors.lastname: Mangerich
authors.forename: Harry
authors.initials: H
authors.lastname: Scherthan
authors.forename: Jörg
authors.initials: J
authors.lastname: Diefenbach
authors.forename: Ulrich
authors.initials: U
authors.lastname: Kloz
authors.forename: Franciscus
authors.initials: F
authors.lastname: van der Hoeven
authors.forename: Sascha
authors.initials: S
authors.lastname: Beneke
authors.forename: Alexander
authors.initials: A
authors.lastname: Bürkle
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D003433:Crosses, Genetic; D053595:Embryonic Stem Cells / Q000378:metabolism*; D055879:Gene Knock-In Techniques; D018390:Gene Targeting; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D006579:Heterozygote; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008957:Models, Genetic; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics*; D011995:Recombination, Genetic
chemical_list: C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases