Targeted Knockdown of Islet2a in Zebrafish

Antisense MO oligonucleotides (Gene Tools, Philomath, OR) were injected into one-cell stage zebrafish embryos. We used a previously published translation blocking antisense, T-MO [15 (link)], directed to the start codon of the islet2a mRNA sequence (5’- GGATGCGGTAGAATATCCACCATAC-3’) at a concentration of 5 mg/ml. This T-MO differs from but partially overlaps with another T-MO previously used to perturb Islet2a function [17 (link)]. We used a control MO (Ctl MO) with 5-base pair mismatches compared to the T-MO (5’-GaATGCGcTAcAATATCCAgCAaAC-3’) at a concentration of 5 mg/ml. In addition, a splice blocking MO, Sp-MO (10 mg/ml) was designed to a sequence overlapping the splice junction between intron 1–2 and exon 2 (5’-CAGACTTCTCTGGATATGGAAAGCA-3’; S1 Fig). Comparisons of the MO and islet gene sequences supports specificity of the MOs targeting islet2a (Table 1 and S1 Table) [28 (link), 29 (link)]. The T-MO and Sp-MO produced similar motor neuron morphological results and we report results obtained with T-MO.