Biology subjects

Linssen, M. M.

Publications and source records attributed to Linssen, M. M..

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

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↗

bioRxiv2021

OX40 agonism enhances efficacy of PD-L1 checkpoint blockade by shifting the cytotoxic T cell differentiation spectrum

Immune checkpoint therapy (ICT) has the potency to eradicate cancer but the mechanisms that determine effective versus non-effective therapy-induced immune responses are not fully understood. Here, using high-dimensional single-cell profiling we examined whether T cell states in the blood circulation could predict responsiveness to a combined ICT, sequentially targeting OX40 costimulatory and PD-1 inhibitory pathways, which effectively eradicated syngeneic mouse tumors. Unbiased assessment of transcriptomic alterations by single-cell RNA sequencing and profiling of cell-surface protein expression by mass cytometry revealed unique activation states for therapy-responsive CD4+ and CD8+ T cells. Effective ICT elicited T cells with dynamic expression of distinct NK cell and chemokine receptors, and these cells were systemically present in lymphoid tissues and in the tumor. Moreover, NK cell receptor-expressing CD8+ T cells were also present in the peripheral blood of immunotherapy-responsive cancer patients. Targeting of the NK cell and chemokine receptors in tumor-bearing mice showed their functional importance for therapy-induced anti-tumor immunity. These findings provide a better understanding of ICT and highlight the use of dynamic biomarkers on effector CD4+ and CD8+ T cells to improve cancer immunotherapy.

immunology↗