Xenbase Image ID: 84657
Figure S1, related to Figure 1. Design and testing of the Eto2 morpholinos (MOs)
(A) Xenopus laevis has a pseudo-tetraploid genome; genes are therefore present in two �pseudo-allele� forms. Sequence alignment of the beginning of the coding sequence of Eto2 pseudo-alleles A and B and Eto-related genes (top) and of the 5� UTR of Eto2 pseudo-alleles A and B (bottom); the target regions for the �ATG� MO (ETO2-MO) and �5�UTR� MO (ETO2-MO2) are highlighted in yellow and blue respectively. The MOs were designed to have 100% homology to the mRNAs transcribed from both Eto2 pseudo-alleles. The nucleotides in red are conserved between Eto2 and the Eto-related transcripts in the region targeted by Eto2-MO.
The Eto-related transcripts (Eto, Mtgr1 and two transcripts with high similarity to Mtgr1, namely Mtgr1-like1 (MGC68858) and Mtgr1-like2 (IMAGE 5156021), accession numbers are in Supplemental Experimental Procedures, section Probes used for in situ hybridisaiton) are not targeted by the Eto2 MOs. For Mtgr1, Mtgr1-like1 and Mtgr1-like2, the sequences targeted by the MOs are sufficiently divergent to avoid unspecific targeting effects. Of note, 5 mismatches are recommended to avoid unspecific targeting effects (Eisen and Smith 2008). Given that only 3 nucleotides differ between the Eto sequence and the sequence recognised by Eto2-MO, the Eto transcripts could potentially be targeted. However, as detailed in Figure S3, they are not expressed in hematopoietic or somitic tissues and are therefore unlikely to have any function in hematopoiesis. The sequences targeted by Eto2-MO2 are too divergent to be aligned; this MO is therefore very unlikely to interfere with translation of any of the Eto-related mRNAs.
(B) A GFP-reporter mRNA tethered to the 5� region of ETO2 containing the Eto2-MO target sequence (Eto2:GFP) was used to test the efficacy of the MO in vivo. Embryos co-injected with Eto2-MO and Eto2:GFP mRNA had no visible GFP expression (right panel), as opposed to mRNA alone (middle panel). Eto2-MO is therefore able to bind to its intended target sequence in vivo and to block translation.
(C) Expression analysis of the HSC markers Gfi1 and SpiB in Eto2-MO2 injected embryos. Red arrow; DA. Numbers at the bottom of the panels indicate the number of embryos with the given phenotype out of the total number examined. Whole mounts are shown with anterior to the left and dorsal to the top.
Image published in: Leung A et al. (2013)
Image downloaded from an Open Access article in PubMed Central. © 2013 ELL & Excerpta Medica.
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