Improved preparation of Xenopus oocytes for patch-clamp recording

We report a new technique for improving the efficiency of single-channel recording in the Xenopus oocyte expression system. Conventional methods of oocyte preparation (adequate for two-electrode voltage-clamp) often leave residual adhesions between the vitelline and plasma membranes. As a result, removal of the vitelline membrane for patch-cramp recording produces microscopic damage to the oocyte plasma membrane that interferes with the formation of high-resistance electrical seals. This problem has been alleviated by a three-phase method of oocyte preparation: (1) a strong initial enzymatic digestion using both collagenase and hyaluronidase, immediately followed by (2) exposure to hypertonic media and mechanical defolliculation, (3) pre-shrinkage and selection of only those oocytes displaying clear separation between vitelline and plasma membranes. Following this selection process, oocytes were returned to solutions of normal osmolarity, incubated overnight at 19 degrees C, and then injected with RNA for expression studies. Comparison of oocytes prepared by different methods indicated that use of these three steps significantly improved the success of high-resistance seal formation, without compromising oocyte survivability or the efficiency of channel expression.