Turbofect in vivo

Chemical transfection: For lipid-based transfection (lipofection), HeLa cells (2 × 10⁴ cells/well) and MJ90 cells (7.5–9 × 10⁴ cells/well) were seeded on a sterile glass coverslip in a 24-well plate to achieve ~80–90% confluence. After 24 h, cells were transfected with plasmids of interest using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol and incubated at 37 °C for an additional 24 h. Turbofect in vivo (Thermo Fisher Scientific, R0533) was used for sponge cell transfection according to the manufacturer’s instructions for in vivo transfections. Sponge cells were transfected with selected vectors, after which they were incubated for 24 h at 8 °C.
Electroporation: MJ90 cells were passaged 1 day before transfection to achieve 50–70% confluency on the day of the experiment. Before electroporation, cells were washed with a PBS and counted. A total of 1 × 10⁷ cells were resuspended in 0.4 mL Opti-MEM (Invitrogen), and 20 μg of plasmid DNA was added to the cells and gently mixed. The cell/DNA mixture was transferred to a 0.4 mm gap cold electroporation cuvette and electroporated under the following conditions: 220 V, 950 μF, and 30 msec. About 1 × 10⁵ of the electroporated cells were transferred into 24-well tissue culture plates with coverslips and incubated in supplemented growth media for 24 h at 37 °C.

For clonal analysis, embryos of st.15 had their roof plate carefully opened. Transfection mix (10 % glucose in 1XPBS, 0.1 μL Turbofect in vivo (Thermo Scientific, USA) containing 1.5 μg AFP plasmid was directly injected to a few hindbrain boundary cells using a fine-pulled glass capillary. Transfection embryos were then electroporated as described above. Next, embryos were incubated for 10–24 h prior to harvesting. Harvested embryos were fixed and stained with Sox2 and 3A10 antibodies and their hindbrains were flat-mounted as described above. For CM-DiI labeling, CM-DiI (C-7000, Molecular Probes) was dissolved in 100 % ethanol to a concentration of 1 mg/mL, which was then further diluted 1:10 in DMSO to working concentration of 10 μg/mL. Labeling was performed in ovo by directly injecting CM-DiI into the hindbrain of st.15 embryos. Hindbrains were harvested 24 h later, fixed and placed on slides for microscopic observation.