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Fig. 4. Exogenous Argonaute2 (Ago2) exacerbates the effects of shRNA-mediated Rx knockdown. (A) Exogenous Ago2 exacerbates the Rx shRNA knockdown phenotype. Embryos were generated by intra-cytosolic sperm injection (ICSI) with Rx shRNA transgene and injected with RNA encoding X. laevis Ago2 RNA. Embryos were co-injected with dsRed Express RNA as a lineage tracer. Embryos were photographed under white light (i, ii, iii), red fluorescence to visualize dsRed lineage tracer (iâ ², iiâ ², iiiâ ²), or blue fluorescence to visualize Rx shRNA transgene (iâ ³, iiâ ³, iiiâ ³). Embryos receiving only the Rx shRNA (panels iâ iii) or Ago2 RNA (panels iâ ³â iiiâ ³) have apparently normal eyes while embryos transgenic for the Rx shRNA and receiving Ago2 RNA in the developing eye exhibited abnormally developed eyes (panels iâ ²â iiiâ ²). (B) Exogenous Ago2 exacerbates the effects of Rx shRNA on Rx expression. Wholemount in situ hybridization using an Rx antisense riboprobe and embryos receiving either the Rx shRNA transgene (ii), Ago2 RNA (iii), or both (iv). Control embryos (i) received neither. These results are presented in graph form in (C). |
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Fig. 1. Rhodopsin and red cone opsin (RCO) are Rx targets. (A–C) Rx is co-expressed with rhodopsin and RCO in photoreceptors. In situ hybridization on sections from paraffin-embedded st 41 tadpoles using probes for Rx (A), rhodopsin (B) or RCO (C). (D, E) Chromatin immunoprecipitation (ChIP) results indicating that myc-tagged Rx (MT-Rx) can bind to the rhodopsin (D) and RCO (E) promoters in vivo. Results are presented as the CT of each sample normalized to the CT of a “no antibody” control. p < 0.003 (XOP), p < 0.002 (RCO). |
