The Roboocyte: Automated cDNA/mRNA Injection and Subsequent TEVC Recording on Xenopus Oocytes in 96-Well Microtiter Plates

Abstract

Membrane-bound neurotransmitter receptors and ion channels are among the most numerous and important drug targets, and electrophysiological methods are the gold standard for the study of their functional properties and their response to drugs. However, electrophysiological measurements are usually performed one at a time by highly skilled individuals, and secondary functional screening is often hampered by this lack of throughput. Accordingly, the use of automated procedures to increase the efficiency of electrophysiological techniques is of great interest. Among the many different electrophysiological techniques that have been described, two electrode voltage clamp recording (TEVC) from Xenopus oocytes seems particularly suitable for the implementation of automated measurement systems. Here, we describe a workstation that was expressly developed for this purpose. The Roboocyte is the first (and the only currently available) instrument that automatically performs both cDNA (or mRNA) injection and subsequent TEVC recording on Xenopus oocytes plated in a standard 96-well microtiter plate. This paper describes the scientific background of the oocyte expression system for drug screening and the development of the Roboocyte. Then, some technical details of the Roboocyte system are presented and, finally, results obtained with the Roboocyte are discussed with regard to increased throughput compared with manually performed experiments. Further information can be obtained at www.roboocyte.com.

An Integrated Field-Effect Microdevice for Monitoring Membrane Transport in Xenopus laevis Oocytes via Lateral Proton Diffusion

“…This procedure requires delicate glass microelectrodes and a high degree of micromanipulation, either by a human operator or precision robotics. [2], [3]. In the whole cell patch clamp, electrical access to the cytosol is gained by sealing the tip of a glass microelectrode to the membrane and applying suction to eventually rupture the cell membrane within the electrode.…”

The Patch Clamp Technique in Ion Channel Research

“…Second, in order to be able to control the cell's membrane potential, a low-resistance electrical pathway must form through the cell wall that covers the pore. Basically, three strategies fulfilling these criteria are being developed for automation of the technique: one is to simulate human behavior in manipulating the glass pipettes into contact with the cell to form the seal in the twoelectrode voltage clamp configuration for oocytes [23,24], a second is a patch clamp method with arrays of pipettes into which the cells are placed and by suction the seal is produced [25], and the last is based on replacement of the pipette altogether by a planar glass or silicon chip with micro apertures to form the seal [26,27]. Fig.…”

Heterologous Protein Expression in the Xenopus Oocyte

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Depending on the equipment used, this step commonly involves back-filling the needle with mineral oil, breaking the tip, and then front-filling with injection solution. Different microinjectors are available, ranging from handheld injectors (for mRNA injection), to widely used and recommended plunger-based systems (e.g., Drummond Nanoject II and III), and even automated injectors (Schnizler et al 2003; Papke and Stokes 2010). Any stably mounted microinjector that reproducibly dispenses nanoliter volumes is suitable.

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