Related Experiment Video
Updated: Dec 17, 2025
Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids
Published on: May 27, 2017
Nuclear microinjection to assess how heterologously expressed proteins impact Ca2+ signals in Xenopus oocytes
Yaping Lin-Moshier1, Jonathan S Marchant
1Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Abstract:
The Xenopus oocyte is frequently used for heterologous expression and for studying the spatiotemporal patterning of Ca(2+) signals. Here, we outline a protocol for nuclear microinjection of the Xenopus oocyte for the purpose of studying how subsequently expressed proteins impact intracellular Ca(2+) signals evoked by inositol trisphosphate (InsP3). Injected oocytes can easily be identified by reporter technologies and the impact of heterologously expressed proteins on the generation and properties of InsP3-evoked Ca(2+) signals can be resolved using caged InsP3 and fluorescent Ca(2+) indicators.
Insights
This study details a Xenopus oocyte nuclear microinjection protocol to investigate how expressed proteins affect calcium (Ca2+) signals. Researchers can now study protein impacts on inositol trisphosphate (InsP3)-evoked Ca2+ signaling dynamics.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Xenopus oocytes are a model system for heterologous protein expression.
- Studying spatiotemporal patterns of intracellular calcium (Ca2+) signals is crucial in cell signaling research.
- Inositol trisphosphate (InsP3) is a key second messenger in calcium signaling pathways.
Purpose of the Study:
- To establish a protocol for nuclear microinjection in Xenopus oocytes.
- To investigate the impact of heterologously expressed proteins on intracellular Ca2+ signals.
- To analyze how these expressed proteins modulate InsP3-evoked Ca2+ signaling.
Main Methods:
- Nuclear microinjection of Xenopus oocytes.
- Utilizing reporter technologies for identifying injected oocytes.
- Employing caged InsP3 and fluorescent Ca2+ indicators to measure Ca2+ signals.
Main Results:
- The protocol allows for efficient introduction of genetic material into Xenopus oocyte nuclei.
- Heterologously expressed proteins can be studied for their effects on Ca2+ signaling.
- The method enables detailed analysis of InsP3-evoked Ca2+ signal generation and properties.
Conclusions:
- Nuclear microinjection provides a robust method for functional studies in Xenopus oocytes.
- This technique facilitates the investigation of protein function in regulating Ca2+ signaling.
- The protocol enhances the understanding of calcium signal spatiotemporal dynamics and modulation by expressed proteins.
