Biology subjects

Mort, R.

Publications and source records attributed to Mort, R..

3 recordsLinked to original sources

bioRxiv2026

Nanopore sequencing identifies a new Tyr::CreERT2 allele circulating in existing mouse stocks

The Tyr::CreERT2 transgenic mouse lines are essential tools for conditional gene manipulation in melanocytes and are widely used in melanoma research. Two independent lines are in common use: the Bosenberg line and the Larue line. Precise knowledge of transgene integration sites is critical for designing complex genetic crosses, yet the integration sites for these lines have only recently been characterised by whole genome sequencing. Here we report that a Tyr::CreERT2 mouse stock routinely used in BrafCA;Ptenflox melanoma models carries a previously undescribed integration on Chromosome 1, distinct from the previously reported Chromosome 2 integration. Using long-read nanopore sequencing, we mapped the integration to an intergenic locus between Alppl2 and Alpi, revealing a 2,430 bp genomic deletion at the insertion site. We developed position-specific junction PCR and qPCR assays to genotype this allele and confirmed that offspring are born at Mendelian ratios. Given that this stock is associated with elevated spontaneous melanoma penetrance, accurate characterisation of this allele has direct implications for the many laboratories employing this widely distributed melanoma model. SIGNIFICANCEThis study identifies a previously undescribed Tyr::CreERT2 transgene integration on Chromosome 1 in JAX strain 013590, a line widely used across the melanoma research community. Accurate characterisation of this allele has direct practical implications for the many laboratories that rely on this model for studies of melanoma initiation and progression.

genetics↗

bioRxiv2024

CIZ1 regulates G1 length and the CDK threshold for initiation of DNA replication to prevent DNA replication stress

Eukaryotic cell division is regulated by oscillating CDK activity, which must reach critical CDK threshold activity levels to progress through cell cycle stages. In low mitogen, low CDK environments cells exit the cell cycle into a non-proliferative quiescent state, G0, that plays essential roles in stem cell maintenance and cellular homeostasis. CIZ1 regulates cell cycle and epigenetic programmes, and CIZ1 ablation enhances genomic instability after release from quiescence. Here, we determined the mechanisms that promote genome instability in CIZ1 ablated cells using a combination of Fucci(CA) live cell imaging, cell-free DNA replication assays and DNA combing. Cell cycle dynamics are unaffected in CIZ1-/- (CIZ1 KO) fibroblasts; however, a specific post-quiescent phenotype is observed resulting in a reduced G1 phase and cell cycle length. The reduction in G1 length in CIZ1 KO cells is associated with increased cyclin E1/E2 and A2 expression, and enhanced phosphorylation of Rb leading to early restriction point bypass. CIZ1-/- cells are deficient in cyclin A chromatin binding and required increased cyclin-CDK activity for the initiation of DNA replication, which is associated with DNA replication stress in vitro and in vivo. Significantly, the CDK threshold for initiation of DNA replication was 2-fold higher in CIZ1 KO nuclei than parental controls. Importantly, addition of recombinant CIZ1 in vitro and in vivo promotes recruitment of cyclin A to chromatin and reinstates the CDK threshold for initiation of DNA replication, reversing DNA replication stress and increasing replication fork rates. Loss of CIZ1 is associated with dysregulated cyclin-CDK signalling, resulting in reduced G1 length, an increased CDK activity threshold required to promote initiation of DNA replication that results in DNA replication stress. These data suggest that CIZ1 facilitates recruitment of cyclin-CDK complexes to chromatin and contributes to the mechanisms that determine the threshold CDK activity required for the G1/S transition in post-quiescent cells. Taken together the data support a role for CIZ1 in the prevention of DNA replication stress and maintenance of genome stability.

cell biology↗

bioRxiv2021

Fingerprinting of skin cells by live cell Raman spectroscopy reveals melanoma cell heterogeneity and cell-type specific responses to UVR

Raman spectroscopy is an emerging dermatological technique with the potential to discriminate biochemically between cell types in a label free and non-invasive manner. Here we use live single cell Raman spectroscopy and principal component analysis (PCA) to fingerprint mouse melanoblasts, melanocytes, keratinocytes and melanoma cells. We show the differences in their spectra are attributable to biomarkers in the melanin biosynthesis pathway and that melanoma cells are a heterogeneous population that sit on a trajectory between undifferentiated melanoblasts and differentiated melanocytes. We demonstrate the utility of Raman spectroscopy as a highly sensitive tool to probe the melanin biosynthesis pathway and its immediate response to UV irradiation revealing previously undescribed opposing responses to UVA and UVB irradiation in melanocytes. Finally, we identify melanocyte specific accumulation of {beta}-carotene correlated with a stabilisation of the UVR response in lipids and proteins consistent with a {beta}-carotene mediated photoprotective mechanism. In summary our data show that Raman spectroscopy can be used to determine the differentiation status of cells of the melanocyte lineage and describe the immediate and temporal biochemical changes associated with UV exposure which differ depending on cell type, differentiation status and competence to synthesise melanin.

cell biology↗