Episomal replication of cloned DNA injected into the fertilised ovum of the hen, Gallus domesticus.
We describe preliminary experiments to analyse the fate of cloned DNA microinjected into the cytoplasm of the chick fertilised ovum. The reporter gene construct pRSVcat was injected into the germinal disc before the first cleavage division, and the chick embryos were cultured for up to 7 days using the method of Perry (Nature 331:70-72, 1988). Linear plasmid molecules ligated rapidly after injection to form high-molecular-weight DNA molecules consisting mainly of random concatemers of the injected plasmid. Recombination involving circular molecules resulted in head-to-tail multimers of the plasmid. Some of the DNA was lost after injection, but the remainder was replicated approximately 20-fold during the first 24 h of development. Between days 1 and 7 in culture, the DNA was gradually lost and diluted out as the embryos developed. By day 7 in culture plasmid DNA was detectable in only 30% of the cultures analysed. No evidence for chromosomal integration of the exogenous DNA was obtained, suggesting that the plasmid DNA persisted episomally. Expression of the reporter gene construct pRSVcat was detected in day 2 and day 7 embryos.
Sang
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AFRC Institute of Animal Physiology and Genetics Research, Roslin, Midlothian, United Kingdom.
Perry
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MM
D000818:Animals; D030801:Animals, Genetically Modified; D002645:Chickens / Q000235:genetics*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000008:administration & dosage / Q000378:metabolism*; D004261:DNA Replication; D005260:Female; D008845:Microinjections; D010957:Plasmids; D015183:Restriction Mapping; D015053:Zygote / Q000378:metabolism*
D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase