Figure 2
From: Moderate Nucleoporin 133 deficiency leads to glomerular damage in zebrafish
Splice Morpholinos (MO) targeting nup133 lead to a partial degradation of nup133 mRNAs. (a) Exon structure of Danio rerio (Dr) nup133 around the binding sites of the E3I3 and I3E4 splice morpholinos. Blue arrowheads above the scheme indicate the position of the RT-PCR primers used in (b) and blue/red/orange arrows below indicate the position of primers and RT-qPCR products used in (c). The size of intron 3 is indicated. (b) RT-PCR from total RNAs of 48 hpf uninjected embryos (control) and of embryos coinjected with the two splicing morpholinos (nup133 MO) reveal an additional band caused by retention of intron 3. (c) nup133 mRNA levels relative to actb2 expression was determined by RT-qPCR on 24 and 48 hpf embryos, uninjected, injected with control MO or nup133 MO, or sequentially injected with 3xHA-mCherry-Dr nup133 mRNA (wt mRNA) and nup133 MO. The E3-E4/5 (red) and E3/4-E4/5′ (orange) primer pairs only amplify nup133 mRNA with properly spliced intron 3 (i.e. wt nup133 mRNA not targeted by the morpholinos) while the E1/2-E3 and E7/8-E9 primer pairs (blue bars) recognize both the spliced and unspliced mRNAs.
