Morpholinos were designed to block translation by targeting the AUG initiation codon (Gene Tools, Philomath, U.S.A.). The morpholinos used are listed below:
Capped and polyadenylated full-length mRNA was generated according to Timme-Laragy et al. [15 (link)], including construction of pcDNA plasmids containing human endoglin [19 (link)], zebrafish bmper, zebrafish alk1, zebrafish bmp9 and mCherry (control), linearisation of the plasmids using NheI (New England Biolabs, U.S.A.), synthesis of the mRNA by mMESSAGE mMACHINE T7 Transcription Kit (Thermo Fisher, U.S.A.).
The microinjection was carried out according to Satou et al. [20 (link)]. In brief, 1 cell stage embryos were used as they are the optimal embryos for injection of MOs and mRNA using the Femto Jet injection system (Eppendorf) under a controllable nitrogen pressure. All embryos were injected with 2 ng morpholinos and 500 pg mRNA (rescue experiment). Injected embryos were cultured in acidic sea water at 27°C.
Endoglin-MOs sequence: 5′-GATGAACTCAACACTCGTGTCTGAT-3′.
5-Mispair control MOs sequence: 5′-AAACAgAcCAcATcCTCTTCATcTC-3′.
Capped and polyadenylated full-length mRNA was generated according to Timme-Laragy et al. [15 (link)], including construction of pcDNA plasmids containing human endoglin [19 (link)], zebrafish bmper, zebrafish alk1, zebrafish bmp9 and mCherry (control), linearisation of the plasmids using NheI (New England Biolabs, U.S.A.), synthesis of the mRNA by mMESSAGE mMACHINE T7 Transcription Kit (Thermo Fisher, U.S.A.).
The microinjection was carried out according to Satou et al. [20 (link)]. In brief, 1 cell stage embryos were used as they are the optimal embryos for injection of MOs and mRNA using the Femto Jet injection system (Eppendorf) under a controllable nitrogen pressure. All embryos were injected with 2 ng morpholinos and 500 pg mRNA (rescue experiment). Injected embryos were cultured in acidic sea water at 27°C.
Free full text: Click here
