Cuy701p2

The ratiometric pH sensor pHluorin (9 (link)) was used to generate the expression vector pCAGG-pHluorin-IRES2-Td-Tomato. Full length pHluorin sequence was sub-cloned in pENTR-1A vector (Invitrogen), and inserted into a pCAGGS-IRES2-tdt-RFA destination vector using the Gateway system (Invitrogen). Chicken embryos ranging from stage 6HH to stage 7HH were prepared for EC culture. A DNA solution (1.0-5.0 μg/μl) was microinjected in the space between the vitelline membrane and the epiblast at the anterior primitive streak level which contains the precursors of the paraxial mesoderm. In vitro electroporations were carried out with five successive square pulses of 8V for 50ms, keeping 4mm distance between anode and cathode using Petri dish type electrodes (CUY701P2, Nepa Gene, Japan) and a CUY21 electroporator (Nepa Gene, Japan).

pCAGG-H2B-Venus and pCAGG-H2B-RFP have been described (Denans et al., 2015 ). Chicken embryos ranging from stage 6HH to stage 7HH were prepared for EC culture. A DNA solution (1.0–5.0 μg/μl) was microinjected in the space between the vitelline membrane and the epiblast surrounding the anterior primitive streak level, which contains the precursors of the paraxial mesoderm. In vitro electroporations were carried out with five successive square pulses of 8V for 50ms, keeping 4mm distance between anode and cathode using Petri dish type electrodes (CUY701P2, Nepa Gene, Japan) and a CUY21 electroporator (Nepa Gene, Japan). This procedure only labels the superficial epiblast layer. For time-lapse analysis, after electroporation, embryos were re-incubated in a humidified incubator until they reached stage 9HH at 38 °C, then embryos were transferred to the microscope stage for time-lapse imaging.