Xenopus laevis oocytes are used in electrophysiology to make transient expression of injected mRNA fragments. Through the technique of 2-electrodes voltage-clamp we can record the physiological phenomena that originate from the activity of the generated protein that has been embedded in the oocyte membrane. Depending on its activity we will observe hyperpolarization or repolarization of the membrane, in the presence of different external stimuli (different concentrations of anions, amino acids, organic acids, etc.).
In our case, electrophysiology recordings in Xenopus laevis were assayed. A plant's membrane transporter working in an animal cell membrane.
But, in young and old, well-fed, and apparently healthy frogs, sometimes the experiments could not be as brilliant as on other occasions. The oocytes were of poor quality, without showing that polarized two-color differentiation, and after injecting the RNA they exploded most of them. Surviving oocytes showed strange electrical behaviors, mostly related to passive endogenous chloride input channels and ended up lysing soon.
Have you had any similar experiences? If you have an explanation I would be very grateful. We are trying to "dust off" old jobs that were abandoned in a drawer.
I want to thank Dr. Omar Homero Pantoja from IBT-UNAM (Cuernavaca, Mexico) for teaching me during my stay in his electrophysiology laboratory.
#xenopus #oocytes #2EVC #electrophysiology #recordings
Hi Franco,
it sounds for a osmolarity-problem..may be a faulty water system.
check all solutions for correct osmolarity.
best wishes
Hi Berger,
Thank you very much for your comment. Osmolarity was controlled at every moment with a psychrometer equipment, it was the first factor we checked. This is the protocol: The oocytes were obtained by manual dissection of adult frogs (Xenopus laevis). The oocytes were taken and separated with tweezers from the connective tissue surrounding them, and were introduced during 30 minutes and in gentle agitation in collagenase at 0.5 mg/mL (Sigma, Toluca, Mexico) dissolved in calcium-free ND96 solution (NaCl 82.5 mM, KCl 2.5 mM, MgCl2 1 mM and HEPES 5 mM, 240-260 mOsm kg-1 and pH 7.6)....
hi again,
then I have only two more ideas:
Dissolving DNA/RNA in a buffer, not water.
Using sharp, like a injection needle, glass pipettes for the application - check the tip with a microscope.
It's a long time ago, as I measured oocytes...
Wolf Berger thank you very much again,
We also checked different injected volumes of RNA, and also verified the tip of the glass pipettes under the microscope (glass pipettes was prepared at the moment using glass and hot as you probably do). We didn't change the water for buffer, maybe we should had but we didn't. However, oocytes appeared to be ill before injecting anything.
Great job!! -> Article Positive Modulation of Human Nav Channels by Pyrethroids
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