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Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons
Published on: December 10, 2009
An automated system for intracellular and intranuclear injection
Ron C Hogg1, Florence Bandelier, Audrey Benoit
1Department of Neurosciences Fondamentales, Medical Faculty, University of Geneva, Switzerland. Ronald.Hogg@medecine.unige.ch
Journal of Neuroscience Methods
|February 5, 2008
Summary
An automated system efficiently injects Xenopus oocytes with genetic material, advancing membrane protein studies. This high-throughput method significantly boosts the expression of ion channels and transporters for research.
Area of Science:
- Biochemistry
- Molecular Biology
- Membrane Physiology
Background:
- The Xenopus oocyte system is vital for studying cellular proteins, especially membrane proteins like ion channels and transporters.
- Manual injection limits throughput for large-scale studies and mutant analysis.
- Automated technologies are emerging, increasing demand for high-volume oocyte manipulation.
Purpose of the Study:
- To develop and validate an automated system for high-throughput oocyte injection.
- To improve efficiency and consistency in expressing membrane proteins in Xenopus oocytes.
- To assess the expression rates of ion channels and transporters following automated injection.
Main Methods:
- Development of an automated injection system for Xenopus oocytes.
- Oocytes housed in microplates with conical wells for automated handling.
- Intracellular injection of mRNA and intranuclear injection of cDNA coding for ion channels.
- Application of the system to zebrafish embryos.
Main Results:
- The automated system achieves injection rates of up to 600 oocytes per hour.
- Intracellular mRNA injection resulted in nearly 100% positive expression of ligand-gated ion channels.
- Intranuclear cDNA injection yielded a >50% expression rate.
- Successful application to zebrafish embryo injection.
Conclusions:
- The automated system significantly enhances the efficiency of Xenopus oocyte expression for membrane protein research.
- This technology supports large-scale functional screening and structure-function studies of ion channels and transporters.
- The method shows potential for broader applications in other cell types and model organisms.
