Rnase free ultrapure water

Morpholino antisense oligos (MOs; Gene Tools) were prepared at a stock concentration of 1 mM according to the manufacturer's instruction. MOs were diluted to 0.3mM and injected into one-cell stage embryos. MOs for targeting zebrafish maeg and dll4 were the same as previously described [22 (link), 40 (link)]. The sequence of standard control MO and itgb1a (ENSDART00000039700) translation-blocking MO are listed in supplemental data (Table S1). Zebrafish maeg and mCherry coding sequence were cloned into PCS2+ vector. The vector template was linearized with NotI Restriction Enzyme (NEB). Sense-capped mRNAs were synthesized with SP6 mMESSAGE mMACHINE Kit (Ambion), purified with RNeasy Mini Kit (Qiagen), and dissolved in RNase free Ultrapure Water (Life Technologies). 2nl maeg and mCherry mixture (1:1) was injected at 100ng/ μl into cytoplasm of 1/2-cell stage zebrafish embryos.

We designed a pair of TALENs targeting the first exon of Maeg using online tools TALE-NT (https://tale-nt.cac.cornell.edu/) [43 (link)]. Left arm and Right arm of Maeg TALEN use FokI heterodimers. The expression plasmids of the TALENs were constructed and linearized with NotI enzyme. TALEN mRNAs were synthesized in vitro using the linearized constructs as templates with SP6 mMESSAGE mMACHINE Kit (Ambion), purified with RNeasy Mini Kit (Qiagen), and dissolved in RNase free Ultrapure water (Life Technologies). Equal amounts (100 ng/μl) of Left and Right TALEN mRNA were injected together into the cytoplasm of 1-cell stage zebrafish embryos.