Fig. 4

nephrin and podocin mRNA splicing defects induced by morpholinos targeting exon donor sites. RT-PCR was performed from total RNA of single embryos at the indicated stage (24, 48, 72, 96 hpf) with flanking exon primers (M, 10 kb plus DNA marker, Invitrogen). (A) The nephrin morpholino targeting the splice donor of the transmembrane domain coding exon caused an in-frame deletion of this exon, visible as a decrease in amplicon size (272 bp versus 389 bp amino acid sequences are aligned in panel C), or a non-splicing of the adjacent intron, visible as an increase in amplicon size (1070 bp) leading to a truncation after a stretch of non-sense amino acids (C). Both forms of nephrin mRNA are predicted to encode a protein lacking the cytosolic C-terminus of the nephrin. The morpholino effects last for at least 72–96 hpf. (B) The podocin morpholino causes an in-frame deletion of part of the first coding exon (amplicon size 316 bp versus 412 bp leading to a shortened N-terminus of the Podocin molecule (D)).