Electroporation of Embryos with Fluorescent Proteins

A pmCherry vector (Clontech, Mountain View, CA, USA) was transferred into a pCAGGS (N-R) plasmid [25 (link)] to produce protein under the control of a strong ubiquitous promoter based on the β-actin promoter. Fertilized embryos in the anterior nucleus stage were subjected to electroporation in 30 microL of HBS buffer [20 mM HEPES, pH 7.0–7.6 (Sigma-Aldrich, Saint Louis, MO, USA) and 150 mM NaCl] containing 45 microg of mCherry, siUNC5C with mCherry (ratio of 10:1), Rhox5 with mCherry, siG9a with mCherry, or siNetrin-1 with mCherry. siUNC5C and siNetrin-1 were generated and selected as shown in Table H in S1 File. Three sets of four electric pulses (21 V, duration of 1 ms, interval of 99 ms) were delivered using a CUY21SC electroporator (Nepagene, Chiba, Japan) [27 (link)].