Tweezertrode

After anesthesia, the mouse was placed in the supine position, and the tongue was gently externalized by using the rubber-tipped forceps. A piece of paper tissue (~4 by 4 mm²) soaked with calcium green dextran (5% w/v) was placed on the anterior dorsal surface of the tongue and then electric pulses (5 Vpp, 2 Hz, pulse width: 100 ms, duration: 10 s) from a function generator were applied through a tweezer-type electrode (Tweezertrode, Harvard Apparatus) for electrophoresis (Supplementary Fig. S5). The stained tongue was rinsed with artificial saliva several times to remove nonspecifically bound dyes in the epithelium. Imaging experiments were conducted 1 to 2 days after the calcium loading procedure.

IUE was performed as described previously (14 (link)) on E11.5, E13.5, and E14.5 timed pregnancies. Briefly, dams were anesthetized with ketamine/xylazine cocktail, and the uterine horns were exposed by a midline laparotomy. One to two microliters of plasmid, or plasmid combination, mixed with 0.1% fast green dye (Sigma-Aldrich) in phosphate buffer was injected into the lateral ventricles using a pulled glass micropipette and a picoinjector (PLI-100, Harvard Apparatus). Final plasmid or plasmid mixture concentration was between 3 and 6 μg/μl. The anode of a tweezertrode (1-mm diameter for E11.5, 3-mm diameter for E13.5, and 5 mm diameter for E14.5, Harvard Apparatus) was placed over the dorsal telencephalon above the uterine muscle, and four pulses (50 V for E11.5, 35 V for E13.5, and E14.5, 50-ms duration separated by 950-ms intervals) were applied with a BTX ECM830 square pulse generator (Harvard Apparatus). Following electroporation, the uterine horns were replaced into the abdomen, and the cavity was filled with warm 0.9% saline before suturing the abdominal muscle and skin separately. Dams were then placed into a clean cage for recovery and monitoring. These procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Boston University School of Medicine.

In utero electroporation (IUE) was performed on timed pregnant CD-1 dams at embryonic day 14.5 (Gilbert and Man, 2016 (link)). Dams were anesthetized via i.p. injection of a ketamine/xylazine mixture. The fur over the abdomen was shaved and cleaned with 70% ethanol. An incision (2.5 cm) in the abdominal cavity was made to expose the uterine horns. 1–2 μL of plasmid DNA mixed with 0.1% fast green dye (Sigma-Aldrich; cat. #F7258) was injected into the ventricle through the uterine wall and amniotic sac using a pulled-glass micropipette. The plasmid vectors were used at a final concentration of 2–3 μg/μL. The anode of a Tweezertrode (Harvard Apparatus, Holliston, MA) was placed over the dorsal telencephalon above the uterine muscle, and four 35 V pulses (50 ms duration separated by a 950 ms interval) were delivered from a BTX ECM830 pulse generator (Harvard Apparatus). The gap between the electrodes was filled with warm saline before electric pulses. After electroporation, the uterine horns were returned to the abdomen, and the cavity was filled with a warm saline solution. The surgical incisions were closed with sutures. The dams were then placed in a clean cage and monitored closely during recovery (about 2 hours). The pups were allowed to mature with the mother until the time as indicated.