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In JoVE (1)
Other Publications (3)
Articles by Alison Claybon in JoVE
JoVE Neuroscience
Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium
A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.
Other articles by Alison Claybon on PubMed
PARP1 Suppresses Homologous Recombination Events in Mice in Vivo
Recent studies suggest that PARP1 inhibitors, several of which are currently in clinical trial, may selectively kill BRCA1/2 mutant cancers cells. It is thought that the success of this therapy is based on immitigable lethal DNA damage in the cancer cells resultant from the concurrent loss or inhibition of two DNA damage repair pathways: single-strand break (SSB) repair and homologous recombination repair (HRR). Presumably, inhibition of PARP1 activity obstructs the repair of SSBs and during DNA replication, these lesions cause replication fork collapse and are transformed into substrates for HRR. In fact, several previous studies have indicated a hyper-recombinogenic phenotype in the absence of active PARP1 in vitro or in response to DNA damaging agents. In this study, we demonstrate an increased frequency of spontaneous HRR in vivo in the absence of PARP1 using the p(un) assay. Furthermore, we found that the HRR events that occur in Parp1 nullizygous mice are associated with a significant increase in large, clonal events, as opposed to the usually more frequent single cell events, suggesting an effect in replicating cells. In conclusion, our data demonstrates that PARP1 inhibits spontaneous HRR events, and supports the model of DNA replication transformation of SSBs into HRR substrates.
Mouse WRN Helicase Domain Is Not Required for Spontaneous Homologous Recombination-Mediated DNA Deletion
Werner syndrome is a rare disorder that manifests as premature aging and age-related diseases. WRN is the gene mutated in WS, and is one of five human RecQ helicase family members. WS cells exhibit genomic instability and altered proliferation, and in vitro studies suggest that WRN has a role in suppressing homologous recombination. However, more recent studies propose that other RecQ helicases (including WRN) promote early events of homologous recombination. To study the role of WRN helicase on spontaneous homologous recombination, we obtained a mouse with a deleted WRN helicase domain and combined it with the in vivo pink-eyed unstable homologous recombination system. In this paper, we demonstrate that WRN helicase is not necessary for suppressing homologous recombination in vivo contrary to previous reports using a similar mouse model.
A Conditional Mouse Model for Measuring the Frequency of Homologous Recombination Events in Vivo in the Absence of Essential Genes
The ability to detect and repair DNA damage is crucial to the prevention of various diseases. Loss of function of genes involved in these processes is known to result in significant developmental defects and/or predisposition to cancer. One such DNA repair mechanism, homologous recombination, has the capacity to repair a wide variety of lesions. Knockout mouse models of genes thought to be involved in DNA repair processes are frequently lethal, making in vivo studies very difficult, if not impossible. Therefore, we set out to develop an in vivo conditional mouse model system to facilitate investigations into the involvement of essential genes in homologous recombination. To test our model, we measured the frequency of spontaneous homologous recombination using the pink-eyed unstable mouse model, in which we conditionally excised either Blm or full-length Brca1 (breast cancer 1, early onset). These two genes are hypothesized to have opposing roles in homologous recombination. In summary, our in vivo data supports in vitro studies suggesting that BLM suppresses homologous recombination, while full-length BRCA1 promotes this process.
