SummaryPICH is a DNA translocase necessary for the resolution of ultrafine anaphase DNA bridges and to ensure the fidelity of chromosomal segregation. Here, we report the generation of an animal model deficient for PICH that allowed us to investigate its physiological relevance. Pich KO mice lose viability during embryonic development due to a global accumulation of DNA damage. However, despite the presence of chromosomal instability, extensive p53 activation, and increased apoptosis throughout the embryo, Pich KO embryos survive until day 12.5 of embryonic development. The absence of p53 failed to improve the viability of the Pich KO embryos, suggesting that the observed developmental defects are not solely due to p53-induced apoptosis. Moreover, Pich-deficient mouse embryonic fibroblasts exhibit chromosomal instability and are resistant to RASV12/E1A-induced transformation. Overall, our data indicate that PICH is essential to preserve chromosomal integrity in rapidly proliferating cells and is therefore critical during embryonic development and tumorigenesis.

The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific genomic locus, but limits the detection of its correct genomic integration by standard PCR methods. Genomic Long Range PCR (LR-PCR), using primers adjacent to the homology arms, has been used as an alternative to radioactive-based Southern blot screenings. However, LR-PCRs are often difficult and render many false positive and false negative results. Here, we propose an alternative screening method based on the detection of a genetic modification at the mRNA level, which we successfully optimized in two mouse models. This screening method consists of a reverse-transcription PCR (RT-PCR) using primers that match exons flanking the targeting construct. The detection of the expected modification in this PCR product confirms the integration at the correct genomic location and shows that the mutant mRNA is expressed. This is a simple and sensitive strategy to screen locus-specific recombination of targeting constructs which can also be useful to screen KO and KI mutant mice or cell lines including those generated by CRISPR/Cas9.

Replication Stress (RS) is a type of DNA damage generated at the replication fork, characterized by single-stranded DNA (ssDNA) accumulation, and which can be caused by a variety of factors. Previous studies have reported elevated RS levels in aged cells. In addition, mouse models with a deficient RS response show accelerated aging. However, the relevance of endogenous or physiological RS, compared to other sources of genomic instability, for the normal onset of aging is unknown. We have performed long term survival studies of transgenic mice with extra copies of the Chk1 and/or Rrm2 genes, which we previously showed extend the lifespan of a progeroid ATRhypomorphic model suffering from high levels of RS. In contrast to their effect in the context of progeria, the lifespan of Chk1, Rrm2 and Chk1/Rrm2 transgenic mice was similar to WT littermates in physiological settings. Most mice studied died due to tumors-mainly lymphomas-irrespective of their genetic background. Interestingly, a higher but not statistically significant percentage of transgenic mice developed tumors compared to WT mice. Our results indicate that supraphysiological protection from RS does not extend lifespan, indicating that RS may not be a relevant source of genomic instability on the onset of normal aging.

AbstractReplication Stress (RS) is a type of DNA damage generated at the replication fork, characterized by single-stranded DNA (ssDNA) accumulation, and which can be caused by a variety of factors. Previous studies have reported elevated RS levels in aged cells. In addition, mouse models with a deficient RS response show accelerated aging. However, the relevance of endogenous or physiological RS, compared to other sources of genomic instability, for the normal onset of aging is unknown. We have performed long term survival studies of transgenic mice with extra copies of the Chk1 and/or Rrm2 genes, which we previously showed extend the lifespan of a progeroid ATR-hypomorphic model suffering from high levels of RS. In contrast to their effect in the context of progeria, the lifespan of Chk1, Rrm2 and Chk1/Rrm2 transgenic mice was similar to WT littermates in physiological settings. Most mice studied died due to tumors -mainly lymphomas-irrespective of their genetic background. Interestingly, a slightly higher percentage of transgenic mice developed tumors compared to WT mice. Our results indicate that supraphysiological protection from RS does not extend lifespan, indicating that RS may not be a relevant source of genomic instability on the onset of “normal” aging.