Femtotip 2

We used commercial Cas9 mRNA (Thermo, USA) and custom synthesized gRNAs. The microinjection procedure was conducted with a microscope (Eclipse TE2000; Nikon, Tokyo, Japan) and micromanipulator (Narishige, Tokyo, Japan) with holding and injection pipettes. We used Femtotip II (Eppendorf, Hamburg, Germany) as an injection pipette. The concentrations of Cas9 RNA and each gRNA were 20 ng/μL and 10 ng/μL, respectively, following a previous report [23 (link)]. RNA was injected into fertilized eggs at 2 hours after IVF.

Experiments were performed using the Casper zebrafish stain (ZIRC, Oregon, USA). Adult fish aged 6 months to 2 years were crossed to produce embryos. Embryos were cultured in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄) at 28 °C. Dechorionated 2-day post fertilization (dpf) zebrafish embryos were anaesthetized with 0.003% tricaine (Sigma-Aldrich) and positioned in 3% methylcellulose on a dish coated with 1% agarose. Cancer cells expressing the tdTomato protein, either wild type or ANXA2 deficient, were treated with versene solution and resuspended in PBS containing 1% phenol red at the density of 4 × 10⁷ cells per mL. The cell suspension was loaded into borosilicate glass capillary needles (Femtotip II, Eppendorf, Hamburg, Germany) and injections were performed using a pump (Femtojet 4i; Eppendorf) and micromanipulator (Phymep, Paris, France). Around 500 cells/embryos were injected above the duct of cuvier in perivitelline space of the embryo. After checking the implantation with mammalian cells, zebrafish embryos were maintained at 35.5 °C. Tumor imaging is done at 3-, 28- and 52-h post injection (hpi).

As previously reported (Sepulveda-Rincon et al. 2016 (link)), the lipophilic tracer CM-DiI (Chloromethyl DiI, Molecular probes) was dissolved in olive oil at a final concentration of 2 mg/mL. Prior to labelling, the FemtoTip II (Eppendorf, Hamburg, Germany) was backfilled with the dye. Mouse embryos at 2-cell stage were placed in HEPES buffered medium at 37°C during the micromanipulation. The injections were performed on an inverted microscope (Leica DMI3000 B) using Eppendorf TransferMan NK 2 micromanipulators with a coupled Eppendorf FemtoJet microinjector. The micropipette was pushed through the zona pellucida and pressed against one of the blastomere membranes, and then a microdrop (≈5 pL) was deposited.

All small interfering RNAs (siRNAs) were purchased from GenePharma. Ago2-siRNAs was designed to specifically target Ago2 and on the basis of 30%-52% GC content and avoiding of internal repeats (5′–3′). Ago2-siRNA1: GCAAAGAUCGCAUCUUUAATT; Ago2-siRNA2: GCCAGUGAUCGAGUUUGUUTT; Ago2-siRNA3: GCAGAAACACACCUACCUUTT; the scrambled siRNA used as negative control. All siRNAs were modified with 2′Fluoro rU/C to increase their annealing temperature. The prediction of off-target effects was described in result. To perform microinjection, zygote-stage embryos were placed in 150 µg/mL hyaluronidase (Sigma) to digest the outer granule cells. SiRNAs was centrifuged at 12,000 rpm for 10 minutes at 4 °C and placed at 4 °C for use. Then, siRNA microinjection was carried out with an Eppendorf FemtoJet microinjector and Narishige NT-88NE micromanipulators. For injection, a glass capillary Femtotip II (Eppendorf) was loaded with 2 μL of 10 µM siRNA by a microloader (Eppendorf), and the solution was injected into the cytoplasm in a 100 μL drop of M2 medium (Sigma) plus 5 μg/mL cytochalasin B (Sigma). The injection volume was approximately 2–5 pL. The injection conditions consisted of 250 hPa injection pressure, 60 hPa compensation pressure and 0.7 s injection time. Immediately after microinjection, embryos were cultured in KSOM medium at 37 °C in 5% CO₂.

Sperm RNA from 4 HC and 4 EE animals was isolated as described above. RNAs were pooled and set at 0.5 ng/μL in 250 μL 0.1 mM EDTA, 5 mM (pH 7.4) based on a previous publication reporting significant effects at this concentration (Gapp et al., 2014 (link)). 3- to 4-week-old C57BL/6JRj females were super-ovulated with pregnant male serum gonadotropin (7.5 U, Intervet) and human chorionic gonadotropin (7.5 U, Intervet) and mated with C57BL/6JRj males. Donor females were sacrificed by cervical dislocation on the day of plug, and fertilized eggs were collected. RNA was injected into the cytoplasm of zygotes at the pronuclear stage using an Eppendorf Femtojet and Femtotip II capillaries with constant flow under visual control on an inverted microscope using a 40× air objective and differential interference contrast (DIC) optics. Injected zygotes were transferred into the oviduct of pseudo-pregnant NMRI fosters bilaterally. The offspring generated from these injections was allowed to grow in a standard cage until 3 month of age before behavioral testing.