Biology subjects

Claassens, J. W. C.

Publications and source records attributed to Claassens, J. W. C..

3 recordsLinked to original sources

bioRxiv2025

Molecular characterization of Cdh12-SCON conditional knockout mice reveals unexpected splicing changes

Functional validation of candidate genes in congenital anomalies of the kidneys and urinary tract (CAKUT) and other disorders is essential for translating genetic discoveries into clinical applications. Conditional knockout mouse models are indispensable for studying gene function in complex organ systems. The Short Conditional intrON (SCON) system accelerates the generation of such models by inserting the artificial SCON into a coding exon. SCON is designed to splice out after transcription, without affecting gene expression. Upon Cre activity, SCON is converted into the {Delta}SCON allele which cannot be spliced out, introducing premature termination codons (PTCs) to inactivate the gene. Previous validation of the SCON system in mice has focused primarily on phenotypic outcomes. Here, we provide a molecular characterization of the SCON system in Cdh12 - a candidate gene implicated in kidney damage in CAKUT. We found that both Cdh12SCON and Cdh12{Delta}SCON alleles caused unintended skipping of the exon downstream of the insertion site, culminating in a frameshift and PTC. Consequently, the Cdh12SCON allele led to a ~25% reduction in mRNA expression, indicating that it was not transcriptionally inert as designed. Despite unintended exon skipping, the Cdh12{Delta}SCON allele still effectively suppressed mRNA expression. These findings reveal previously unrecognized splicing artifacts of the SCON system and underscore the need for transcript-level characterization before utilizing artificial intron-based conditional alleles for functional studies.

genetics↗

bioRxiv2025

Four new Duchenne muscular dystrophy mouse models with clinically relevant exon deletions in the human DMD gene

Mutation specific therapeutic approaches, like exon skipping or gene-editing, hold promise for the treatment of Duchenne muscular dystrophy (DMD). Translatability of preclinical studies investigating these approaches could greatly be improved through the use of humanized mouse models, as these allow preclinical testing of human specific sequences. We developed four novel humanized DMD mouse models with either a deletion of exon 44, 45, 51 or 53 in the human DMD gene, in a mouse dystrophin negative background (mdx mouse; exon 23 nonsense mutation). Our optimized prescreening pipeline allowed us to do so very efficiently with the CRISPR-Cas9 technology. We confirmed either complete lack of dystrophin, or expression of trace levels, which led to development of muscle pathology consisting of muscle fiber de-, and regeneration, inflammation and fibrosis in young adult mice. Intramuscular treatment with vivo-morpholinos targeting a flanking exon induced exon skipping in the DMD strains, which restored the disrupted open reading frame and subsequently dystrophin expression. This validates these models as valuable tools for preclinical studies investigating human sequence specific therapeutic approaches for DMD. Summary statementHumanized Duchenne muscular dystrophy mouse models were created with deletions of exon 44, 45, 51 or 53 in the human DMD gene. These dystrophic models allow preclinical testing of human-specific dystrophin restoring approaches.

neuroscience↗

bioRxiv2025

Genotype-phenotype correlations in Wilms tumor initiation.

Wilms tumor, the most common pediatric kidney cancer, arises from abnormal embryonic kidney development. Therapy resistance and tumor recurrence remain major challenges, likely driven by the presence of Cancer Stem Cells (CSCs). Here, we elucidate the earliest pathogenic events in genetically engineered mouse models exhibiting loss of Wt1 or LIN28B overexpression, two Wilms tumor driver genes. Loss of Wt1 leads to a disturbance of lineage identity of the mutant cells, whereas LIN28B leads to a disturbed transition between uninduced and induced NPC (nephron progenitor cell) state. In both cases the appearance of cells positive for all four Wilms tumor cancer stem cell markers is the result of the tumor initiating mutation. The existence of genotype-phenotype correlations in primary developmental phenotypes and cancer stem cell expression patterns has important implications for therapeutic opportunities and requirements.

developmental biology↗