Femtojet injection system

Morpholinos were designed to block translation by targeting the AUG initiation codon (Gene Tools, Philomath, U.S.A.). The morpholinos used are listed below:

Endoglin-MOs sequence: 5′-GATGAACTCAACACTCGTGTCTGAT-3′.

5-Mispair control MOs sequence: 5′-AAACAgAcCAcATcCTCTTCATcTC-3′.

Off-target effects and specificity of endoglin-MOs were addressed in a commonly used approach, a rescue experiment. Full-length human endoglin mRNA was co-injected with endoglin-MOs to rescue the zebrafish vascular phenotype.
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.

dsRNA was synthesized as previously described (Rouhana et al., 2013 (link)) and resuspended at concentrations ~ 1.5–2 μg/μL. For control injections, 1.5 kb dsRNA derived from ccdB and camR-containing insert of the pJC53.2 vector was used (Collins et al., 2010 (link)). 6-day-old tapeworms were obtained and microinjected with dsRNA using femtotips II via the Femtojet injection system (Eppendorf) to obtain spreading across the first ~3–4 mm anterior of the tapeworm. The spread of injected fluids could be detected by a temporary increase in opacity. 500 hPa injection pressure for 0.3–1 s was used per injection site. Whole tapeworms were cultured in vitro for 3 days, 2 mm anterior fragments were amputated, worms were re-injected with dsRNA on day 6, and cultured in vitro for an additional 9 days before termination.

Just before xenotransplantation, 48-hpf zebrafish were dechorionized using 2 mg pronase (Roche Diagnostics)/ml as described previously [53] (link), anesthetized, and arrayed on a holding sheet. ALDH+ and ALDH- cells (1×10⁶ cells each) were separately suspended in 50 µl of Hanks' balanced salt solution (Life Technologies). The glass needles used to inject the cells were made from a GD-1 glass capillary (Narishige, Tokyo, Japan) using a PP-830 gravity puller (Narishige) and fine-polished with an EG-44 microforge (Narishige). The number of injected cells was counted microscopically by transferring the same volume of injected cells on glass slides using the same glass capillary tubes and injection pressure (FemtoJet, Eppendorf, Hamburg, Germany) in each experiment, as described in a previous study [54] (link). The avascular region of the yolk sac was then injected with a volume of the above-described suspension containing 100–200 cells using the glass needles and the FemtoJet injection system (Eppendorf, Hamburg, Germany). The xenotransplanted zebrafish were subsequently maintained at 32°C.