Analyses of Protein Expression

Protein extracts from wild-type and mutant ES cells and mice were analyzed for PARP-1 expression using three different antibodies: (i) monoclonal antibody CII10 that is general-PARP-1-specific, i.e. detecting both human and murine PARP-1; (ii) monoclonal antibody FI23 that is specific for the hPARP-1; and (iii) the novel polyclonal antibody „4595‟ that is specific for mPARP-1 as described in the following section.

4.2.1.2.1 Generation of a Novel Murine-PARP-1-specific Antibody

To evaluate, if the mPARP-1 mRNA transcripts were processed to the full-length protein, a novel mPARP-1-specific polyclonal antibody was generated. For this purpose, a non-conserved 14-amino-acid peptide derived from position 363 to 376 of the mPARP-1 protein was synthesized and used for immunization of two rabbits (Figure 3.3). Figure 4.12 indicates a strong immunoreactivity of serum from rabbit „4595‟ against a murine protein of about 116 kD, presumably mPARP-1. Serum obtained from rabbit „4596‟ resulted in a weak signal from a protein of the same size. Preimmune serum was collected before the immunization of the animals and served as a negative control showing in both cases weak background signals only.

Importantly, no signals were observed with hPARP-1, indicating the specificity for mPARP-1.

Figure 4.12. Validation of the novel murine-PARP-1-specific polyclonal antibody „4595‟.

Western blot analyses showing validation of mPARP-1-specific polyclonal antibodies „4595‟ and „4596‟.

Monoclonal antibodies FI23 and CII10 are specific for human or general PARP-1, respectively. Serum from the second blood collection (2^nd BC) was prepared after the second boost immunization, serum from the final blood collection (final BC) after the third boost immunization. Preimmune serum served as a negative control. Lanes labeled with „m‟ were loaded with 1 × 10⁶ cell equivalents of wild-type murine ES cells, lanes labeled with „h‟ with 200 ng recombinant hPARP-1 protein. L indicates molecular size ladder.

4.2.1.2.2 Expression Analyses in ES Cells

Prior to hPARP-1 expression studies, wild-type ES cells cultured under different conditions, (i.e. on „feeder‟-cell layers or directly on gelatin-coated cell culture dishes) were examined for their differentiation status and PARP-1 expression. Figure 4.13 A shows that under both culture conditions, ES cell colonies exhibited high alkaline phosphatase activity (red staining), a marker for an undifferentiated status of ES cell cultures (Pease et al. 1990). Moreover, under both conditions considerable amounts of PARP-1 were detectable in Western blot analyses. As depicted in Figure 4.13 B, PARP-1 expression was about ten times higher in undifferentiated ES cells compared with ES-cell-derived differentiated cells. Consequently, ES cells were used as an in vitro model to analyze the effects of the hPARP-1 gene targeting on a cellular level.

Figure 4.13. Evaluation of PARP-1 expression under different ES cell culture conditions.

A. Upper panel illustrates differentiation status of wild-type ES cells grown on „feeder‟-cell-coated (left) or gelatin-coated (right) culture dishes as assessed by alkaline phosphatase activity (red staining). Original magnification ×50. Lower panel shows Western blot analysis of PARP-1 expression in wild-type ES cells grown on „feeder‟-cell-coated (F) or gelatin-coated (G) culture dishes using the antibody CII10. Per lane, 1.5 × 10⁶ cell equivalents were loaded. Actin staining served as a loading control. „+‟ indicates recombinant hPARP-1 protein (200 ng) B. Western blot and corresponding densitometric analysis showing different PARP-1 expression levels in wild-type ES cells (ESC) and ES-cell-derived differentiated cells (DC) using the antibody CII10. 5 × 10⁵ cell equivalents were loaded per lane. Tubulin staining served as a loading control and for standardization. Statistical significance was analyzed by Student‟s t test. Means ± SEM, n=3. L indicates molecular weight ladder.

To analyze PARP-1 expression, in particular potential hPARP-1 expression, in mutant ES cells, immunofluorescence and Western blot analyses were performed.

First, total PARP-1 (human plus murine) expression was evaluated using the general-PARP-1-specific monoclonal antibody CII-10. As shown by Western blot analyses (Figure 4.14 A, left-hand panels), mutant ES cell clones exhibited a moderate overexpression of total PARP-1.

However, these differences are not statistically significant. The same tendency was observed in immunofluorescence analysis of the hPARP-1 clone #113 (Figure 4.15 A). Clone #151 served as a negative control for ES cell culture conditions in the presence of the antibiotic G418, since in this case the integration of the targeting vector was incomplete (Figure 4.2 and data not shown).

Second, Immunofluorescence analysis of hPARP-1 clone #113 with t hPARP-1-specific monoclonal antibody (FI23) demonstrated expression of the hPARP-1 in each cell, thus excluding any contamination of the mutant cell cultures with wild-type ES cells (Figure 4.15 B). Western blot analyses with the same antibody confirmed the immunofluorescence results for clone #113 and additionally revealed hPARP-1 expression in hPARP-1 clones #113NeoR-, #225, and #225NeoR- (Figure 4.14 A, right-hand panels). Human PARP-1 expression levels were at a similar level, when comparing NeoR+ and NeoR- clones. Expression levels of hPARP-1 appeared to be higher in clones #113 and #113NeoR- compared with clones #225 and

#225NeoR-. Clone #267, which was also identified to be positive for site-specific homologous recombination, exhibited hPARP-1 expression, too, although to a lesser extent than in clones

#113 and #225 (data not shown). Clone #151 showed no hPARP-1 expression when examined by immunofluorescence and Western blot analyses and therefore served as a control for G418-supplemented ES cell culture conditions in further experiments (Figure 4.15 B and data not shown).

Third, it is noteworthy that expression levels of mPARP-1, as examined with the mPARP-1-specific antibody „4595‟ (section 4.2.1.2.1), were lower in hPARP-1 ES cells compared to wild-type ES cells (Figure 4.14 B).

Figure 4.14. Western blot analyses of PARP-1 expression in wild-type and hPARP-1 ES cells.

A. Upper panels. Western blot analyses of whole-cell extracts of wild-type (wt) and hPARP-1 ES cell clones #113,

#113NeoR-, #225, and #225NeoR- for general PARP-1 (monoclonal antibody CII10)) and hPARP-1 (monoclonal antibody FI23) expression. A dilution series of 250 ng, 125 ng, 75 ng, 40 ng of recombinant (rec.) hPARP-1 was used as a positive control. Actin served as a loading control and for standardization. A. Lower panels. Signals from lanes loaded with 1 × 10⁶ cell equivalents from three independent blots were analyzed densitometrically. Potential statistical significance was analyzed by Student‟s t test. Means ± SEM, n=3, except for wt, n=6. Data of panel A is adapted from O. Popp, 2007, diploma thesis. B. Detection of mPARP-1 in whole-cell extracts from wild-type and hPARP-1 ES cell clones #225 and #225NeoR- using the polyclonal antibody „4595‟. 1 × 10⁵ cell equivalents (c.e.) were loaded per lane. NeoR- indicates ES cell clones with excised NeoR cassette.

Figure 4.15. Immunofluorescence analyses of PARP-1 expression in wild-type and hPARP-1 ES cells.

Immunofluorescence analyses of wild-type (wt) ES cells and hemizygous hPARP-1 ES cell clone #113. Clone #151 showed incomplete integration of the targeting vector (Figure 4.2) and served as a control for G418-supplemented ES cell culture conditions. COPF5 cells served as an hPARP-1-expressing positive control. A. Total PARP-1 expression analyzed with the general-PARP-1-specific primary antibody CII10. B. Human PARP-1 expression analyzed with the hPARP-1-specific primary antibody FI23 (FITC channel). The Fluorophor-labeled antibody AlexaFluor488 was used as secondary antibody. Nuclei were counterstained by Hoechst DNA staining (Hoechst channel). Images were taken using identical settings. Original magnification ×630. Red bars indicate 10 µm.

4.2.1.2.3 PARP-1 Expression Analyses In Vivo

To analyze PARP-1 expression in vivo, several organs were screened for total PARP-1 expression by Western blot analysis of whole-tissue extracts. High PARP-1 expression levels were detected in pancreatic tissue, the lymphoid organs, i.e. thymus and spleen, and the reproductive organs, i.e. testis, uterus, and ovary. In contrast, moderate PARP-1 expression was detected in brain, lung, liver, and the male accessory glands. Low or no PARP-1 expression was visible in heart, kidney, and intestine (Figure 4.16). Because PARP-1 expression was highest in the spleen, this organ was chosen for further biochemical analyses of hPARP-1 mice.

When analyzing spleen whole-tissue lysates of wild-type mice as well as hemizygous (1×hPARP-1) and homozygous (2×hPARP-1) mutant mice, Western blot results demonstrated an hPARP-1-gene-dose-dependent overexpression of total PARP-1 (Figure 4.17 A and Figure 4.18 A). Depending on the individual experiment, the increase ranged from 25% to 100%, when comparing samples of wild-type mice with those of hPARP-1 homozygotes.

Figure 4.16. Screening for PARP-1 expression in various organs from wild-type mice.

Western blot analysis of whole-tissue lysates from different organs of wild-type mice as indicated. Recombinant (rec.) hPARP-1 (200 ng) served as a positive control. Membrane was probed with the general-PARP-1-specific antibody CII10. Ponceau staining of the membrane served as a loading control. Acc. indicates accessory; L, molecular size ladder.

Moreover, hPARP-1 mice displayed expression of full-length hPARP-1 in an hPARP-1-gene-dose-dependent manner (Figure 4.17 B and Figure 4.18 B). Consistent with results from ES cell analyses, no influence of the NeoR cassette on hPARP-1 expression levels were obvious (Figure 4.18 B), but again human protein expression was higher in line #113NeoR- compared to line

#225NeoR- (Figure 4.18 B).

Murine PARP-1 expression is inversely correlated to hPARP-1 expression in line #225 (Figure 4.17 C and Figure 4.18 C) supporting results from ES cells analysis (section 4.2.1.2.2).

However, this effect was not noticed in lines #225NeoR- and 113NeoR-, possibly due to the low replicate number (n=2, Figure 4.18 C). In none of the lines were there any additional signals from potentially truncated murine or human PARP-1 detectable, indicating that both gene loci were transcriptionally functional.