2009, Mangerich, Aswin, Scherthan, Harry, Diefenbach, Jörg, Kloz, Ulrich, Hoeven, Franciscus van der, Beneke, Sascha, Bürkle, Alexander

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.

2008, Mangerich, Aswin

Poly(ADP-ribose) polymerase-1 (PARP-1) uses NAD+ as a substrate to modify various nuclear proteins with the biopolymer poly(ADP-ribose), thereby regulating a variety of cellular processes such as DNA repair, gene transcription, and cell death. These diverse functions on the cellular level are also reflected in the contribution of PARP-1 to multiple physiological and pathophysiological conditions on an organismal level.
While several groups have established PARP-1-deficient mice, PARP-1-overexpressing or hypermorphic mice have not been described to date. The latter, however, should also represent a relevant biological model, the rationale being provided by the fact that the poly(ADP-ribosyl)ation capacity of purified human PARP-1 (hPARP-1) is significantly higher than that of its rodent (rat) orthologue. In this thesis, a novel mouse model with ectopic expression of hPARP-1 (comprising two independent congenic lines) was generated, using gene targeting in embryonic stem (ES) cells. The targeting vector was designed to allow replacement of the murine Parp-1 (mParp-1) coding sequence (32 kb) with its human orthologous sequence (46 kb). Unexpectedly though, site-specific homologous recombination was mimicked by bidirectional extension of the vector homology arms, followed by adjacent integration of the targeting vector, thus leaving the murine locus functional. Related to this phenomenon is the so-called ectopic gene targeting mediated by synthesis-dependent strand annealing (SDSA), which has so far only been described for 'ends-in integration vectors in non-ES cell gene targeting. Therefore, results of this thesis give new insight into the role of SDSA during gene targeting and are of general importance for the design of gene knock-in approaches in mice.
Mutant hPARP-1 ES cells and mice displayed gene-dose-dependent expression levels of hPARP-1, thereby resulting in a moderate overexpression of total PARP-1, while mPARP-1 expression was downregulated to some extent. Consequently, hPARP-1 ES cells exhibited an altered poly(ADP-ribosyl)ation metabolism, but an intact DNA damage response. Phenotypic analyses revealed impaired survival rates in a gene-dose-dependent manner in both sexes of hPARP-1 mice, with females being more affected. Several pathologies were identified in hPARP-1 mice, such as obesity, glomerulopathy, and splenomegaly, all pointing to the development of chronic diseases. Moreover, hPARP-1 mice showed signs of premature aging, such as sporadic kyphosis accompanied by alterations in bone metabolism and impaired regenerative potential of the hair.
In conclusion, this study characterized the occurrence of ectopic gene targeting in murine ES cells transfected with an ends-out gene replacement vector for the first time. Furthermore, the generated hPARP-1 mice represent a novel model system with unexpected, multifaceted phenotypes, which should be instrumental for the elucidation of the role of PARP-1 in health and disease.