Figure 5.

Disruption of pkd2 expression with splice donor antisense MO oligonucleotides. (A) Diagram of Polycystin-2 protein in the cell membrane showing membrane topology and C-terminal motifs associated with Polycystin-2 function. (B) Targeting splice donor sites of exons 3, 5, 12, and 13 with antisense MO oligonucleotides resulted in the production of internal in-frame deletions (exons 3, 5, and 13 MO) and a C-terminal truncation (exon 12 MO). Transmembrane domains of Polycystin-2 are depicted in gray. (C) The efficacy of the injected MO was quantified at 24-, 48-, and 72-h intervals by RT-PCR. Polycystin-2 exon 3 donor MO (MOex3) caused a 39–amino acid in-frame deletion of part of the first transmembrane domain as detected by the presence of a smaller, internally deleted RT-PCR product. Some recovery of normal Polycystin-2 mRNA was observed by 72 hpf. Similar results were observed for all splice donor targeted MO. (D) Control embryos at 72 hpf. (E) Histologic section of normal pronephros at 72 hpf. (F) MOex3-injected embryo showing axis curvature and hydrocephalus (arrow; 97%, 941 of 967 embryos). (G) Histologic section showing cystic pronephric tubules (*) in MOex3-injected embryo. (H) MOex12-injected embryos and section (I) of the cystic pronephros. (J) MOex13-injected embryos showing severe axis curvature and kidney cysts (arrow). (K) Histologic section of embryo in J showing kidney cyst (*). (L) Antisense MO deletions in the Polycystin-2 protein sequence predicted on the basis of nucleotide sequence of RT-PCR products amplified from MO-injected embryos. In-frame deletions are shown in gray for MOex3, MOex5, and MOex13. MOex12 induced a nonsplicing event that resulted in a stop codon immediately after exon 12 in the cDNA (shown in red *). Transmembrane domains (tm) and the PKD1 binding homology domain are highlighted in brown. Membrane targeting motifs in the cytoplasmic C-terminus including the phosphofurin acidic cluster sorting protein–1 (PACS-1)-binding acidic cluster are underlined.