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

Abstract 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 ΔSCON 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 Cdh12 SCON and Cdh12 ΔSCON alleles caused unintended skipping of the exon downstream of the insertion site, culminating in a frameshift and PTC. Consequently, the Cdh12 SCON allele led to a ~25% reduction in mRNA expression, indicating that it was not transcriptionally inert as designed. Despite unintended exon skipping, the Cdh12 ΔSCON 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.