3.1. The generation of a tamoxifen-inducible ARTD1 deficient mouse
The options for studying ARTD1 gene function in vivo are currently limited when using the classical Artd1-/- mouse generated by Zhao-Qi Wang221. Although ARTD1 plays important functions during DNA repair it is not essential for murine development222. Interestingly, the
Artd1/2 double knock-out is embryonically lethal13, suggesting a compensatory mechanism(s)
and partially overlapping function of ARTD1 and ARTD2. To further investigate this hypothesis and specifically to investigate whether double deficiency of Artd1/2 is only important during development or also during adulthood we generated first an tamoxifen- inducible Artd1-/- strain with the plan to cross this strain later into the Artd2-/- strain, allowing us to delete ARTD1 in these mice (but also in isolated cells) at any desired time-point.
To generate the tamoxifen-inducible Artd1-/- strain we crossed Artd1flox/flox mice with B6.129-Gt(ROSA)26Sortm1(cre/ERT2)Tyj from the Jackson Laboratory (Strain ID #008463). Having generated the strain (genotyping results not shown) we next assessed the recombination efficiency in vivo. Three mice received 1 mg tamoxifen by i.p. injection for five consecutive days. Similarly, two control animals were vehicle-treated (PBS). Ten days after the last tamoxifen administration, the mice were euthanized and lung, liver, spleen and testis were isolated. Whole cell protein extracts were generated and 150 µg of protein loaded on a SDS-PAGE gel. ARTD1 levels were quantified by Western Blot using tubulin as a loading control (Figure 16).
Figure 16. Tamoxifen administration to Rosa26-CreERT2+ Artd1flox/flox mice efficiently deletes
Artd1. One milligram Tamoxifen or PBS (vehicle) was administrated i.p. into Rosa26-CreERT2+ Artd1flox/flox mice for five consecutive days. Whole cell lysates (150 µg protein) of the indicated organs were subjected to Western Blot analysis and stained with antibodies against ARTD1 or tubulin.
In all analyzed tissues, ARTD1 levels markedly decreased upon tamoxifen administration, although tissue specific differences in ARTD1 reduction were observed. The strongest tamoxifen-induced ARTD1 reduction was observed in testis, moderately in lung, liver and spleen. Notably, the absolute expression of ARTD1 was the highest in liver, moderately in testis and lung and the lowest in the spleen. Together these data indicate that a tamoxifen- inducible Artd1-/- mouse strain was successfully generated. Different layers of complexity can account for the observed tissue-specific differences. First, the injected tamoxifen might not reach any given target cell in the mouse. Second, translocation of the ERT2 protein into the nucleus of the target cell affects the expression of the Cre-recombinase. Lastly, the expressed Cre has to assess and recombine the floxed locus on the DNA. Depending on the tissue, the chromatin environment of specific loci might vary and influencing Cre recombination efficiency. Whether the observed reduction in the individual organs is sufficient to investigate phenotypical changes in vivo remains to be investigated. For example, ischemia-reperfusion injuries may be a first disease model to determine the functional impact on ARTD1 reduction. Next, we assessed Cre-mediated ARTD1 recombination efficiency not only in vivo, but also
in vitro. To do so, BMDM of either Artd1flox/flox or Rosa26-CreERT2+ Artd1flox/flox mice were
differentiated for 10 days. On day 5, the culture medium was supplemented with either 20 µM 4-Hydroxytamoxifen (4-OHT), or vehicle (PBS). Whole cell extracts were generated and ARTD1 levels quantified by Western Blot (Figure 17). The quantification of the ARTD1 levels revealed a successful reduction of ARTD1 in 4-OHT treated Rosa26-CreERT2+
Artd1flox/flox BMDM. The ARTD1 levels were below the detection limit, suggesting that the
recombination of the ARTD1 locus is very efficient if tamoxifen is readily available as is the case in vitro. * - Lung Liver - + + + * Tamoxifen Spleen Testis - - + + + - - + + + - - + + + Tamoxifen ARTD1 Tubulin ARTD1 Tubulin
Figure 17. Tamoxifen efficiently deletes Artd1 in Rosa26-CreERT2+ Artd1flox/flox BMDM. BMDM were differentiated for 10 days. On day 5, the medium was supplemented with 20 µM Hydroxytamoxifen (4-OHT) or PBS. Whole cell lysates (150 µg protein) were subjected to Western Blot analysis and stained with antibodies against ARTD1 or tubulin.
3.2. Towards the generation of an ARTD1/2 double knock-out mouse
As already indicated above Artd1−/−/Artd2−/− double mutant mice are not viable and die at the onset of gastrulation, thus demonstrating the crucial role of PARylation during embryonic development222. To study the involvement of PARylation during development and adulthood, we generated a tamoxifen-inducible Artd1−/− mouse in an Artd2−/− background. Hence, tamoxifen administration would induce a partial (see 3.1 and Figure 16) double knock-out in
vivo. After having successfully combined all three alleles (Rosa26-CreERT2, Artd1flox/flox and
Artd2−/−) in mice, the animals were administrated 1 mg tamoxifen or vehicle (PBS) for 5
consecutive days. After another 10 days to allow for recombination and residual ARTD1 to be degraded, the mice were viable and did not show any phenotypic abnormalities. Thus, new breeding pairs of tamoxifen-treated and vehicle-treated mice were generated. A significantly lower reproduction, if any at all, was observed over at least 3 generations in tamoxifen-treated mice, compared to normal litter sizes of vehicle treated animals. Additionally, we noticed no macroscopic differences of tamoxifen- and vehicle treated animals during aging up to 8 months.
The data suggests, that indeed ARTD1 and ARTD2 are only necessary during embryonic development, but dispensable during adulthood under basal conditions. It remains very interesting to learn how these animals would behave during different disease models or under (genotoxic) stress conditions.
4-OHT - + - +
Artd1flox/flox
Rosa26-CreERT2+ Artd1flox/flox
+ + + + - - - - ARTD1 Tubulin