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Injection of Xenopus eggs before activation, achieved by control of extracellular factors, improves plasmid DNA
1Department of Biology, Brandeis University, Waltham, MA.
Abstract:
Injection of molecular probes into unfertilized Xenopus eggs requires suppression of activation. But the unfertilized egg is poised for activity, and pricking, like sperm penetration, triggers the start of the first cell cycle. Methods of suppressing activation generally rely on introduction of drugs into the cell, but some of these techniques are irreversible. I report here that injection without activation can also be accomplished by simply limiting extracellular free Ca2+ to 1-2 microM. The site of injection heals, but the cortex does not contract. Gentle modification of the vitelline envelope, which causes it to become tougher, improves the rate of healing to about 100%. Healed eggs are stable for hours and can be activated when needed. Injection of a plasmid derived from type 1 bovine papilloma virus revealed that replication occurs only after activation, but preloading the DNA markedly increased the efficiency of first-round replication. DNA interaction with the unactivated egg cytoplasm may therefore be required for efficient replication of exogenous DNA. The new procedures described here are likely to be of general utility.
Insights
Researchers developed a new method for injecting unfertilized Xenopus eggs without triggering activation by controlling extracellular calcium levels. This technique improves egg healing and DNA replication efficiency, offering a stable platform for experimental manipulation.
Area of Science:
- Developmental Biology
- Cell Biology
- Molecular Biology
Background:
- Unfertilized Xenopus eggs activate upon pricking or sperm penetration, initiating the cell cycle.
- Existing methods to suppress activation during injection often use irreversible drugs.
- Controlling extracellular calcium is crucial for maintaining egg quiescence.
Purpose of the Study:
- To develop a non-destructive method for injecting unfertilized Xenopus eggs.
- To investigate the role of extracellular calcium in preventing egg activation.
- To assess the impact of this method on DNA replication efficiency.
Main Methods:
- Limiting extracellular free Ca2+ to 1-2 microM to suppress activation during injection.
- Modifying the vitelline envelope to enhance injection site healing.
- Injecting a bovine papilloma virus-derived plasmid into eggs to study DNA replication.
Main Results:
- Injection without activation was achieved by controlling extracellular Ca2+.
- Injection site healing improved to approximately 100% with vitelline envelope modification.
- Exogenous DNA replication occurred only after activation, but preloading DNA enhanced efficiency.
Conclusions:
- Limiting extracellular Ca2+ provides a reversible method to suppress Xenopus egg activation during injection.
- Enhanced healing and stability allow for prolonged experimental manipulation.
- This technique facilitates efficient exogenous DNA replication in Xenopus eggs, aiding future research.
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