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

Area of Science:

• Genetics and Genomics
• Developmental Biology
• Molecular Biology

Background:

• Functional validation of candidate genes is vital for understanding congenital anomalies of the kidneys and urinary tract (CAKUT) and other genetic disorders.
• Conditional knockout mouse models are essential tools for studying gene function in complex biological systems.
• The Short Conditional intrON (SCON) system is designed to facilitate the rapid generation of conditional knockout mouse models by inserting an artificial intron into coding exons.

Purpose of the Study:

• To provide a molecular characterization of the SCON system's performance in the context of Cdh12, a candidate gene implicated in CAKUT.
• To assess the unintended molecular consequences of SCON system insertion on gene expression at the mRNA level.

Main Methods:

• Generation of Cdh12 conditional alleles using the SCON system in mice.
• Molecular characterization focusing on mRNA expression analysis of the engineered alleles.
• Assessment of splicing patterns and identification of premature termination codons (PTCs).

Main Results:

• Both Cdh12^SCON and Cdh12^ΔSCON alleles resulted in unintended skipping of the exon downstream of the SCON insertion site, leading to frameshift and PTC.
• The Cdh12^SCON allele caused an approximate 25% reduction in mRNA expression, indicating it was not transcriptionally inert as designed.
• Despite unintended exon skipping, the Cdh12^ΔSCON allele effectively suppressed Cdh12 mRNA expression.

Conclusions:

• The SCON system, while intended to be transcriptionally inert before Cre activation, can lead to artefactual splicing events, including unintended exon skipping.
• Transcript-level characterization is essential to validate engineered alleles and ensure accurate interpretation of functional studies.
• Findings underscore the need for rigorous molecular validation across various gene-targeting strategies, including artificial intron-based systems.