Xenbase Image ID: 159727

Figure S5. Validation of Morpholino injections. (A) C9MOatg1 does not target Xenopus laevis casp9 RNA. Western blot analysis of exogenous casp9 levels after depletion with the translation blocking morpholino C9MOatg1. Embryos were injected at 2-cell stage with 10 ng C9MOatg1 and 10 ng CoMO alone or combined with 250 pg RNA coding for Xenopus laevis casp9 (Myc-tagged) or Xenopus tropicalis casp9 (Myc-tagged). The functionality of C9MOatg1 was proven by the clear reduction in Xtcasp9 protein levels upon depletion while the four mismatches in the morpholino target sequence of the Xlcasp9 mRNA seem to be sufficient for blocking the binding with the morpholino and make the Xlcasp9 transcripts suited for rescue experiments. (B) Splice blocking morpholino C9MOE2I2 retained intron I in the X. tropicalis casp9 transcripts. RT-PCR analysis of Xtcasp9 levels after depletion with the splice blocking morpholino (C9MOE2I2). Embryos were injected at 2-cell stage with 20 ng C9MOE2I2 or 20 ng CoMO and lysed at stage 20. Blockage of the exon2 and intron2 boundary by the morpholino resulted in a intron1 retention, which was noticed by the 579 bp fragment. The intron retention causes a shift in the open reading frame of Xtcasp9 and results in an incorrectly translated protein and a premature stop. (NI, non injected) Image published in: Tran HT et al. (2017) Copyright © 2017. Image reproduced with permission of the Publisher, The Company of Biologists Ltd. Larger Image Printer Friendly View

Return to previous page