XB-IMG-78162
Xenbase Image ID: 78162
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Fig. 7. Xrel3 antisense morpholinos (MOxr3) that target the 5′ untranslated regions (UTR) of Xrel3 are specific for Xrel3. (A) Autoradiograph of in vitro translation products in the presence of MOxr3. One nanogram of Xrel3 or XrelA in vitro transcribed RNA was added to rabbit reticulocyte cell-free translation reaction mix with the indicated amounts of morpholinos. (B) Myc-tagged Xrel3 (mtXrel3) RNA lacking the 5′-UTR is not affected by MOxr3 when injected into embryos. Ten embryos were co-injected at the 2-cell stage with 9.2 ng of control morpholinos or MOxr3 along with increasing amounts of mtXrel3 mRNA and assayed for expression of mtXrel3 protein at stage 9 (late blastula) by western blotting. Tubulin was used as a loading control. (C) Embryos at stage 27 injected 9.2 ng of control morpholino develop normally. (D) Embryos injected with 4.6 ng of MOxr3 develop slight developmental abnormalities, including bent body axes and minor head defects, if any. (E) Embryos injected with 9.2 ng of MOxr3 develop extreme defects in body axis development and lack head structures. (F) Rescue of embryos from defects caused by injection of 9.2 ng of MOxr3 by co-injection with and 0.5 ng of synthetic myc-tagged Xrel3. (G) Decrease in the frequency of developmental defects caused by injection of MOxr3 by co-injection with myc-tagged Xrel3 mRNA. The data are pooled from two independent experiments using embryos from two different females. Open bars indicate normal embryos, gray bars indicate mild defects which included normal head and dorsal development but with kinked axes and black bars represent severe defects, as in panel E. (H) Visualization of Lissamine-conjugated (red fluorescence) MOxr3 in embryos at stage 27. Embryos were injected at the 2-cell stage with 9.2 ng MOxr3. (I) Visualization of Lissamine-conjugated (red fluorescence) MOxr3 in embryos at stage 27. Embryos were injected at the 2-cell stage with 4.6 ng MOxr3. (J) Visualization of FITC-conjugated (green fluorescence) control morpholinos in an embryo at stage 27. Embryo was injected at the 2-cell stage with 9.2 ng control morpholino. (K) Visualization of Lissamine-conjugated (red fluorescence) MOxr3 in an embryo at stage 27. Embryo was injected at the 2-cell stage with 9.2 ng MOxr3 and 0.5 ng of myc-tagged Xrel3 synthetic mRNA. (L) Uninjected embryo at stage 27 visualized under fluorescence optimized for FITC to reveal background autofluorescence. (M) Uninjected embryo at stage 27 visualized under fluorescence optimized for Lissamine to reveal background autofluorescence. Image published in: Kennedy MW et al. (2007) Copyright © 2007. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |