Department Developmental Biology
Principal investigator Cristina Gontan
E-mail address m.gontanpardo@erasmusmc.nl
Establish a human XCI system and identify skewing due to X-linked mutations
Asymptomatic female carriers of mutations in genes like RNF12 linked to Neurodevelopmental Disorders (NDD), exhibit significant skewing in X Chromosome Inactivation (XCI). This skewing results in the inactivation of the X-chromosome carrying the mutated allele. We propose that this phenomenon is driven by the action of the ubiquitin ligase RNF12 during human XCI, rather than being a result of negative secondary selection during neurogenesis.
In this study, We aim to validate this hypothesis by generating a dual fluorescent reporter system to monitor XCI in female induced pluripotent stem cells. Confirming this hypothesis would significantly enhance our understanding of this phenomenon related to NDD mutations and shed light on XCI-skewing mechanisms in general.
Techniques
- Cell culture of hiPSCs
- Generation of transgenic lines using CRISPR-Cas9
- Molecular cloning
- DNA and RNA isolation
- Fluorescent in situ hybridization (FISH)
- Immunofluorescence
- Western blotting
- Microscopy
- Neuronal differentiation
Further reading
Frints, S.G.M., et al. (2019). Molecular Psychiatry, 24, 1748–1768. DOI: 10.1038/s41380-018-0065-x.
Identifying RNF12 substrates and mutations mediating NDD in RNF12 male patients
Supervisor: Kyra Swildens, k.swildens@erasmusmc.nl
RNF12 mutations cause NDD in male patients, probably because RNF12 target-proteins are not/less degraded and affect brain development. Here I aim to identify RNF12 target-proteins during neurogenesis. To explore future therapeutic avenues, I will test whether antisense RNA-mediated knock-down, with antisense oligonucleotides (ASOs), of RNF12 targets complements (potential) defects observed during neurogenesis of RNF12 mutant PSCs. For diagnostic and scientific purposes, I will test new RNF12 patient mutations for their effect on RNF12 activity.
Techniques
- Cell culture of hiPSCs and neuronal differentiation
- Generation of transgenic lines using CRISPR-Cas9
- Molecular cloning
- DNA and RNA isolation
- Fluorescent in situ hybridization (FISH)
- Immunofluorescence
- Microscopy
Further reading
Frints, S.G.M., et al. (2019). Molecular Psychiatry, 24, 1748–1768. DOI: 10.1038/s41380-018-0065-x.