Gene transfer into whole-mount retinas by the homemade electroporation device.

<p>A–B. Preparation of platinum (Pt) electrodes. Ai. The arrangement of the six slides for the (+) electrode base. The colors of slides represent the slide arrangement in different layers. The numbers in the circles indicate the corresponding sequence to arrange slides. Aii. The Pt foil (15×15 mm with one extra 5×5 mm-overhanging square) was aligned to one side of the 7^th slide with the overhanging square left out. Epoxy glue was applied to attach the Pt foil onto the slide and connect all the slides together. Aiii. The wire was soldered to the edge of the overhanging Pt square. Aiv. The last (8^th) slide was glued onto the (+) electrode base. Bi–ii. The arrangement of the (−) electrode. A pen tube (green) with a diameter of 10 mm was used to attach the same-sized Pt foil by epoxy glue. The electric wire was inserted through the pen tube and soldered onto the Pt foil. C. The setup for electroporation in a horizontal configuration. The retinal explant was placed in a well [with dimensions 12 (length) ×12 (width) ×3 (height) mm] on the (+) electrode. The (−) electrode made contact with solution above the well and covered the retinal explant. The distance between the (+) and (−) electrodes was adjusted by a micromanipulator that held the (−) electrode. D. The P1 rat retinas were transfected with pCMV-HA (vector) or pCMV-HA-Syt I (HA-Syt I) with this electroporation device. The retinal explants were incubated for 72 hr to allow gene expression. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with antibodies indicated on the right (HA or α-tubulin). Only the retinas transfected with HA-Syt I displayed the HA signal with a size of ∼65 kD, which corresponded to the molecular weight of Syt I. Data shown were representative blots from 3 different experiments.