XB-IMG-86829
Xenbase Image ID: 86829
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Figure 2. Characterization of a X. tropicalis homologue of BCOR and inhibition of translation by MOs
A. Temporal expression of xtBcor was examined in a cDNA series derived from pools of developmentally-staged embryos. Initially, xtBcor was shown to be expressed as a maternal mRNA. Zygotic expression was observed from stage 9, and there after throughout embryogenesis to ~stage 30.
B. Spatial expression of xtBcor was analyzed in a cDNA tissue panel derived from adult female X. tropicalis. Expression of xtBcor was detected in all tissues assayed except for gall bladder and skin.
C. Two adjacent, non-overlapping, translation-blocking MOs were designed to target the 5’ UTR of xtBcor mRNA. As human anti-BCOR antibodies do not recognize xtBcor (data not shown), blocking of xtBcor mRNA translation was confirmed using an in vitro transcription/translation assay. A 5’ fragment of xtBcor mRNA was generated from a PCR template, which included a T7
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29 promoter and a 3’ FLAG tag. The sequence of xtBcor mRNA around the start codon and the
position of the MO binding sites is shown.
D. The 5’ xtBcor mRNA fragment was used as a template in an in vitro translation reaction using a rabbit reticulocyte lysate kit. The reaction was supplemented with xtBcor MO1 or MO2 as appropriate, at 1 μg of MO per 1 μg mRNA template. Using an anti-FLAG antibody, a product of 16 kDa was detected in the presence of xtBcor mRNA. The appearance of this product was completely inhibited in the presence of either xtBcor MO1 or MO2. A control MO had no inhibitory effect. All experiments were carried out using each MO and are derived from at least three independent experiments. Image published in: Hilton EN et al. (2007) Copyright © 2007. This image is reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View |