Dr Michael Fejtl reports on the automation of cDNA/mRNA injection and subsequent TEVC recording in Xenopus oocytes plated in 96-well micro-titer plates.
Oocytes of the claw frog Xenopus laevis have been widely used as an expression system to study the functional and pharmacological properties of ion channels/receptors using the two-electrode-voltage-clamp technique (TEVC).
Conventional electrophysiological techniques allow the recording of only several cells per day. The Roboocyte enables reseachers to inject cDNA or mRNA and record ion currents in oocytes in a
96-well dish automatically. Thus several hundred data points can be gathered per day which increases the speed and throughput tremendeously.
Four processes are accomplished automatically with the Roboocyte: u cDNA/mRNA injection. u TEVC recording. u Wash cycles. u Automated perfusion paradigms.
The system is cost-effective and runs 24 hours a day. A prototype has been developed at the BAYER AG in Germany and is running in their laboratories for some years.
Several applications can be conducted with the Roboocyte, and it is routinely used for expression screening, drug screening of agonists, antagonists, co-agonists, expression cloning, screening of mutated or chimeric receptors or ion-channels, and generation of dose-response curves.
The Roboocyte consits of a magnetic platform, ca. 40 x 40 cm, a mounting support for 96-well plates, two independent z-axes for injection, and the TEVC recording and in-between wash, respectively. Each plate will be indentified by a bar-code. Thus a mismatch between plates is virtually impossible. A standard 8- or 16-valve system and a multiple perfusion port system, (i.e. Gilson) can be attached to accommodate every users need.
A proprietary newly developed digital TEVC- amplifier is used to measure currents up to a range of 32 µA and is housed in the Roboocyte. The fully computer-controlled amplifier does not need adjustment knobs or switches.
The z-axis for TEVC holds a plug-and play measuring head (TEVC probe), comprised of 2 pre-pulled glass electrodes (ca. 700 kOhm resistance),
2 Ag/AgCl reference electrodes, and two perfusion ports for solution in- and outlet.
The TEVC probe lasts several days and can be easily replaced in case of broken or clogged electrodes.
The windows-based software handles the injection of the oocytes, the TEVC measurement, the wash cycle of the cells, and serves as the interface for other important features: u Determination of the reference point of a 96 well plate. u Chart and zoom function of the current traces. u A scripting tool to define user-specific recording sessions (the scripts are saved and can be recalled for future recordings). u A history mode to recall a previously recorded plate to visualise all the successful recordings and define a region for min/max measurements of the currents.
Data management is assured so that each well has its specific finger-print. All the data gathered from a well is linked to the wells ID.
Thus no mismatch between wells is possible. ASCII export and a link to popular data bases will be provided as well. u
ENQUIRY No 104
Dr Michael Fejtl is CSO with Multi Channel Systems MCS GmbH, Reutlingen, Germany. www.multichannelsystems.com or www.roboocyte.com