Introduction: The podocin encoding NPHS2 is the most frequently mutated gene in steroid-resistant nephrotic syndrome. We aimed to generate an in vivo model to study the interallelic interactions of podocin. The homologue of NPHS2 in C. elegans is
mec-2 with 17 known splice variants according to the Ensembl database. The encoded MEC-2 is expressed in six neurons and is responsible for gentle-touch mechanosensation. Podocin and MEC-2 are members of the stomatin protein family, both contain a PHB domain, both are integral membrane porteins with intracellular C- and N-termin and form oligomers. The MEC-2a isoform is considered to be the canonical one. This isoform shares 45% identity and 83% similarity over 275aa (72%) of podocin (383aa), with primarily different C- and N- terminal regions. We aimed to identify the biologically relevant MEC-2 isoform, which can rescue the diseased phenotype of a
mec-2 mutant strain. Methods: Vectors encoding MEC-2 isoforms under
mec-2 promoter and a selection marker (cbr-unc) were generated. To avoid quantitative differences due to extrachromosomal expression, we implemented the MosSCI (Mos1-mediated Single Copy Insertion) technique to achieve chromosomal integration. Double (
mec-2 and
unc-119) mutant strains were created by crossing. The
mec-2 mutant (Tu37) strain was kindly provided by the laboratory of Prof. M. Chalfie. We used microparticle bombardment to transform worms. The gentle-touch sensation was examined by cat's whiskers in a blinded experiment. RNA expression levels were examined by qualitative and quantitative PCR after total RNA isolation from worms. Results: Strains were successfully transformed with MEC-2a coding vectors. However, no rescue was achieved in strains expressing the MEC-2a isoform. We excluded the biological role of several isoforms based on their absent expression, as determined in total RNA isolated from wild type worms. A complete rescue was achieved with a 16 kb genomic sequence encoding 12 of the 17 isoforms with intact 3' regions. The rescue effect of several selected isoforms is currently investigated. Conclusion: Mutant strains can be successfully generated by the combination of the MosSCI technique and microparticle bombardment. The canonical MEC-2a isoform is not the biologically most relevant, as it cannot rescue the gentle touch sensation defect of the
mec-2 mutant strain.