3 Materials and Methods
5.2 Establishment of Models to Study the Effects of hPARP-1 Gene Targeting
5.2.1 ES Cells as an In Vitro Model to Study PARP-1 in General
Mutant hPARP-1 ES cell clones, #113, #225, and #267 were obtained as „intermediates‟ during the generation of hPARP-1 mice. To evaluate the suitability of ES cells for the in vitro study of the effects of hPARP-1 gene targeting, ES cells were characterized with respect to PARP-1 expression and poly(ADP-ribosyl)ation capacity.
PARP-1 expression in ES cells was about ten times higher than in ES-cell-derived differentiated cells (Figure 4.13 B). High expression levels of genome maintenance proteins like PARP-1 in ES cells are plausible: As mentioned above, DNA damage accumulates in the
genomes of somatic cells, due to imperfect DNA repair, possibly leading to cancer and aging.
However, imperfect DNA repair is intolerable in cells of the germ line and ES cells, because these cells give rise to a whole organism and are crucial for the persistence of a species. In accordance with this, a substantial body of evidence shows that ES cells possess stress defense and DNA repair mechanisms that are superior to those of somatic cells, ensuring the integrity of their genomes throughout successive generations (Cervantes et al. 2002; Saretzki et al. 2004).
High PARP-1 expression levels in ES cells as identified in this thesis are in line with this notion.
High PARP-1 expression levels indicated that mutant ES cells could represent a suitable in vitro model to study the potential ectopic hPARP-1 expression and its effects on poly(ADP-ribosyl)ation metabolism. To validate this, optimal culture conditions were assessed by culturing wild-type ES cells either on „feeder‟ cell layers or directly on gelatin-coated culture plates.
Under both conditions ES cells preserved their undifferentiated state and expressed PARP-1 at similar levels (Figure 4.13 A). To avoid any dilution effects caused by co-culture with „feeder‟
cells, ES cells used for in vitro experiments were cultured directly on gelatin-coated culture plates. Under those conditions, ES cells produced poly(ADP-ribose) in a dose-dependent manner and the intracellular NAD+ content decreased by 20% upon X-irradiation of up to 25 Gy, indicating an efficacious PARP activation potential (Figure 4.20 & Figure 4.22 A).
Since ES cells displayed high PARP-1 expression levels and dose-dependent poly(ADP-ribose) formation upon genotoxic stimuli, they indeed represent a suitable model to study PARP-1 and poly(ADP-ribosyl)ation in vitro. Moreover, ES cells are preferable to cancer cell lines, as the latter have acquired large numbers of mutations across the whole genome.
However, when comparing different ES cell sub-lines, these have to be of similar, preferably low passage number and great care must be taken to ensure equal cell densities, because ES cell cultures are sensitive and particularly prone to respond with differentiation. In the present work, all of those factors have been taken into account.
5.2.2 Establishment of hPARP-1 ES Cell Clones
During screening of G418-resistant ES cell clones for site-specific homologous recombination of the targeting vector, one clone (#151) was identified that showed only partial integration of the targeting vector and no hPARP-1 expression (Figure 4.2 and data not shown). This clone was used as an additional negative control for G418-supplemented ES cell culture conditions during in vitro experiments.
As it was reported that transcription of the NeoR cassette can interfere with the transcription of flanking genes (Nagy 2000; Nagy 2003), the NeoR cassette was excised in vitro in ES cell clones #113 and #225, thus giving rise to ES cell lines #113NeoR- and #225NeoR- (Figure 4.10). These lines were cultured without G418 and were included in in vitro experiments.
An attempt to generate homozygous hPARP-1 ES cell clones by increasing the selection pressure for the NeoR cassette (Lefebvre et al. 2001; Nagy 2003) resulted in the isolation of clone #225C1, which was identified by qPCR to carry two gene copies of hPARP-1 (Figure 4.8;
O. Popp, 2007, diploma thesis). Fluorescence in situ hybridization confirmed this result, but moreover revealed that the two gene copies were caused by duplication of chromosome 1 resulting in an isochromosome 1 (Figure 4.6). Therefore, clone #225C1 was not suitable for further experiments. However, this outcome again validated the reliability of the hPARP-1 qPCR in addition to test breeding of hPARP-1 homozygotes with wild-type mice (Figure 4.7).
5.2.3 Establishment of hPARP-1 Mouse Lines
As mentioned above, mutant ES cell clones #113 and #225 gave rise to two independent mouse lines. Hemizygous hPARP-1 mice of line #225 of the first filial generation (F1) were intercrossed in order to establish a cohort (F2) consisting of wild-type mice as well as hPARP-1 hemizygotes and homozygotes in the expected Mendelian ratio (Table 4.3). This cohort had a B6;129P2 mixed genetic background and was used for detailed biochemical and phenotypic characterization (Figure 3.7). In line #113, intercrossing of hPARP-1 hemizygotes of F1 was not possible, as only three hemizygous hPARP-1 females were obtained.
To excise the NeoR cassette in vivo, mice of lines #113 and #225 (F1) were crossed with EIIa-Cre transgenic mice, which ubiquitously express Cre recombinase. The NeoR cassette was successfully excised in both lines (Figure 4.10). Because Cre recombinase can cause genomic instability triggered by cryptic loxP sites in the mouse genome (Thyagarajan et al. 2000;
Loonstra et al. 2001; Semprini et al. 2007), the Cre cassette was outcrossed by mating hPARP-1 hemizygotes (F2) with wild-type C57BL/6 mice. The descending lines were designated
#113NeoR- and #225NeoR- (Figure 3.7).
On the one hand, hPARP-1 hemizygotes of lines #113NeoR- and #225NeoR- (F3) were intercrossed and gave rise to two cohorts, #113NeoR- and #225NeoR- (F4), consisting of wild-type mice as well as hPARP-1 hemizygotes and homozygotes in the expected Mendelian ratio (Table 4.4). These mice were in an 87.5% C57BL/6 genetic inbred background and were used for additional biochemical and phenotypic characterization (Figure 3.7).
On the other hand, hPARP-1 hemizygotes of lines #113NeoR- and #225NeoR- (F3) were successively crossed with wild-type C57BL/6 mice in order to establish coisogenic lines (F10, 99.9% C57BL/6). The C57BL/6 inbred strain was chosen for backcrossing to transfer the mutant genotype on a well-defined background to distinguish effects of the targeted mutation from the natural strain phenotype. The C57BL/6 strain is currently the best studied inbred strain with respect to genetics and phenotypic analyses (Ward 2000). Furthermore, use of C57BL/6 mice as breeding partners of chimeras allowed to isolate 129P2/OlaHsd ES cell contribution in
chimeric offspring via determination of animal coat color. At the time of this writing, two independently obtained congenic hPARP-1 lines (F5, 96.9% C57BL/6) have been generated (Figure 3.7).