Injection of Xenopus eggs before activation, achieved by control of extracellular factors, improves plasmid DNA replication after activation.
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.
Wangh
L J
LJ
Department of Biology, Brandeis University, Waltham, MA.
D000818:Animals; D015139:Blotting, Southern; D052688:Bovine papillomavirus 1 / Q000235:genetics*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002471:Cell Transformation, Neoplastic; D004261:DNA Replication; D005260:Female; D007700:Kinetics; D008845:Microinjections; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D027383:Papillomaviridae / Q000235:genetics*; D010957:Plasmids; D013489:Suppression, Genetic; D014817:Vitelline Membrane / Q000187:drug effects / Q000502:physiology
D000001:Calcimycin; D002118:Calcium