Rescue of prpf31 morphant and AD5 phenotype. (A-G) Rescue of prpf31 morphant phenotype by wild-type prpf31, AD5 and SP117. (A) Non-injected control larvae at 48 hpf. (B) Morphants injected with 10 μg/μl (1.2 mM) prpf31 MO show severe malformations, such as reduced brain and eye size, cardiac edema and curved tails. (C) Full rescue of morphant phenotype after co-injection of 573 ng/μl (0.93 μM) prpf31 mRNA. (D) Embryos co-injected with 437 ng/μl (0.89 μM) AD mRNA show high lethality. (E) Embryos co-injected with 325 ng/μl (0.84 μM) SP117 mRNA showed deformations similar to the situation in morphants. (F) Quantitative analysis of rescue efficiency with different MO/RNA combinations. The mean percentage of embryos in each group was calculated from three independent experiments and standard deviation is shown by error bars. (G) Western blot shows reduced endogenous Prpf31 levels after MO injection. Co-injection of prpf31 mRNA results in elevated Prpf31 levels at 24 hpf. (H-O) Co-injection of AD5 with low doses of prpf31 MO and prpf31 RNA. (H) Control embryos at 48 hpf. (I) Morphants injected with 5 μg/μl prpf31 MO. (J) Embryos injected with 200 ng/μl AD5 mRNA. (K) Embryos co-injected with prpf31 MO and AD5 mRNA. (L) Embryos co-injected with 200 ng/μl AD5 mRNA and 262 ng/μl prpf31 mRNA. (M) Quantitative analysis of embryos injected as in H-K. Co-injection of MO and AD5 mRNA results in a significant increase in lethality. (N) Western blot showing almost undetectable endogenous Prpf31 levels after co-injection of 200 ng/μl AD5 mRNA and 5 μg/μl prpf31 MO, but abundant AD5 protein at 11 hpf. (O) Quantitative analysis of embryos injected with 200 ng/μl AD5 and 262 ng/μl prpf31 mRNA. Co-injection of prpf31 mRNA resulted in a partial rescue of the AD5 induced phenotype.
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