For microinjection, we used injection buffer (24% glycerol, 20 mM HEPES pH 8.0 and 120 mM KCl). The morpholino (Gene Tools, Philomath, OR, USA) sequences are in the reagent table, and the in-needle concentration with injection buffer was 1.0 mM. Two nonoverlapping translation-blocking morpholinos for Opsin2 and ChAT were used to confirm the function specificity (S1 and S2 Figs). For negative control experiments, we injected a random MO or only injection buffer. The concentrations of MOs in the needles were as follows:
Opsin2-MO1 (0.4 mM), 5’-AGTTTGCCATCTTTGTGTTGCTTCG -3’
Opsin2-MO2 (0.4 mM), 5’-CGCCAATAACCACTGATCACAGTCG -3’
ChAT-MO1 (0.2 mM), 5’-ACGATTAGGCATGTGGTTCATGTAT -3’
ChAT-MO2 (1.0 mM), 5’-TGGAACGTCCAATAGTGGTATTGTA -3’
Gcm-MO, 5’-GCTTTGGACTAACCTTTTGCACCAT -3’ [34 (link)], and
Random-MO (1.0 mM).
Microinjections into fertilized eggs were performed as previously described [60 (link), 61 ]. After microinjection, the embryos were washed with FSW three times and stored with 50 μg/ml kanamycin until the desired stages were reached.
Opsin2-MO1 (0.4 mM), 5’-AGTTTGCCATCTTTGTGTTGCTTCG -3’
Opsin2-MO2 (0.4 mM), 5’-CGCCAATAACCACTGATCACAGTCG -3’
ChAT-MO1 (0.2 mM), 5’-ACGATTAGGCATGTGGTTCATGTAT -3’
ChAT-MO2 (1.0 mM), 5’-TGGAACGTCCAATAGTGGTATTGTA -3’
Gcm-MO, 5’-GCTTTGGACTAACCTTTTGCACCAT -3’ [34 (link)], and
Random-MO (1.0 mM).
Microinjections into fertilized eggs were performed as previously described [60 (link), 61 ]. After microinjection, the embryos were washed with FSW three times and stored with 50 μg/ml kanamycin until the desired stages were reached.
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