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Fig. 3. Generation of Rx knockdown embryos. (A) A portion of X. laevis Rx1A was selected as a target for development of an shRNA. Alignment of the shRNA target sequence with corresponding regions of X. laevis Rx2A and mouse Rx. (B) Tadpoles transgenic for the Rx shRNA plasmid appeared normal. Bright light or UV-light views of transgenic tadpoles at st 41. (C) Retinas from Rx shRNA transgenic tadpoles appeared histologically normal. Hematoxylin and eosin stained sections of paraffin-embedded wild type (left panel) or Rx shRNA transgenic tadpole. (D) Rx shRNA transgenic tadpoles have reduced levels of both Rx1A and Rx2A as determined by quantitative RT-PCR (qRT-PCR) performed using total RNA purified from isolated tadpole heads (st 41). (E) Rx1A is expression is reduced by in situ hybridization performed using 8 μM sections of paraffin-embedded tadpoles at st 38, 41, and 45 (from left to right). The reduction in Rx expression appears to increase as development progresses. |
