General concerns

Antisense morpholino oligo (ASMO) is a viable approach to knock down gene expression in Helobdella embryo. We found translation-blocking ASMO more likely to be successful in generating significant knockdown phenotypes, though there is a reported case of splicing blocker. Nonetheless, considering the paucity in the means of controlling experiments, it is difficult to distinguish specific and non-specific effects of ASMO in leech embryos. It is recommended that a rescue experiment is included as a control to ensure the specificity of ASMO. For such experiment, the ASMO-resistant construct is built by introducing synonymous mutations to the ASMO binding sites of the target gene in the expression construct, and this construct is them co-injected with ASMO. A successful rescue (or even a gain-of-function phenotype) is expected if the observed morphant phenotype is indeed specifically produced by the knockdown of the target gene product.

Preparation and injection

Custom morpholino oligomer is obtained from GeneTools. It arrives in powder form. It is quite stable at room temperature. Before use, it is dissolved in nuclease-free water. A recommended stock concentration is 2.5 mM for Helobdella. In most case, we found injecting at 0.5-1 mM (in micropipette) is sufficient to produce a reproducible and rescuable phenotype in Helobdella embryo. Similar to DNA and RNA, Fast Green has a negative impact on the effectiveness of ASMO, and thus phenol red solution (Sigma P0290), supplemented with fluorescent dextran if necessary, is used as monitor dye for morpholino injection.

We store ASMO stock in small aliquots at -80˚C. This is mainly for minimizing evaporation effect. GeneTools recommends storing morpholino in an air-tight container (microfuge tube sealed with parafilm, for example) at room temperature. It is reported that morpholino precipitates in frozen stock, but we never encounter such a problem with our small-aliquot stocks.