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Fig. 4. Wnt11-R antisense morpholino oligomer inhibits translation from Wnt11-R-GFP fusion transcripts. (A) Diagram of the Wnt11-R/GFP fusion (tester) mRNA, indicating binding locations for antisense MO1 and MO2. The ability of MOs to inhibit translation from mRNAs containing Wnt11-R sequences was assayed by coinjecting 500 pg of synthetic tester mRNA with 15 ng of Wnt11-R MO1 into fertilized eggs. (B–D) At about stage 13, embryos were viewed under UV light or standard illumination. Uninjected embryos (B) serve as negative control and do not glow. Embryos injected with tester mRNA (C) fluoresce, confirming the translation of GFP protein. Embryos coinjected with MO1 plus tester mRNA (D) show strongly reduced fluorescence, indicating effective inhibition of translation. The second Wnt11-R antisense MO, MO2, showed no inhibition of GFP expression using this assay (data not shown). (E) Immunoblot detection of GFP protein in whole embryo extracts from uninjected embryos, tester mRNA-injected embryos, and embryos coinjected with tester mRNA and 15 ng of MO1. GFP protein is detected in the tester sample, but is not detectable when translation is inhibited by MO1. Ponceau S staining indicates equal loading of sample. (F) Embryo injected with 15 ng of control MO (contMO) assayed at st. 28 by in situ hybridization for MHCα, showing normal cardiac differentiation. (G) Embryo injected with 15 ng of MO1 also shows normal appearance of differentiation markers when assayed for MHCα transcripts. |
