|
Fig. 6
Rescue of T-MPO phenotypes by mutated zebrafish CD mRNA.
A) T-MPO target sequence and corresponding mutated sequence of the in vitro synthesized CD mRNA. B) Western blotting of CD in homogenates of 2 dpf embryos and 4 dpf larvae deriving from control injection (CTRL), injection with T-MPO alone (T-MPO) and injection with T-MPO plus 200 pg/egg of mutant CD mRNA (RESCUED). The filter was incubated with polyclonal antibody against rat CD, stripped and re-probed with monoclonal antibody against β-actin. The densitometry ratio of CD vs actin expression is shown. (a.u. = arbitrary units). Data reproduced in two to three independent experiments. The empty arrow points to CD expressed by the exogenous mRNA. C) Representative image of 4 dpf larvae obtained from control injection (CTRL), injection with T-MPO alone or with T-MPO plus 200 pg/egg of mutant zebrafish CD mRNA (RESCUED). Larvae body length (SL) and oro-anal tract length (SVL) are indicated. The experiment shows that synthesis of protein CD driven by exogenous CD mRNA in T-MPO injected zebrafish was sufficient to rescue a normal phenotype. Scale bar in C is 200 μm. Data presented in this figure have been reproduced in three independent experiments.