Franks, Roberta R. and Hough-Evans, Barbara R. and Britten, Roy J. and Davidson, Eric H. (1988) Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA. Development, 102 (2). pp. 287-299. ISSN 0950-1991. http://resolver.caltech.edu/CaltechAUTHORS:FRAdev88
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A method is described for microinjection of cloned DNA into the zygote nucleus of Lytechinus variegatus. Eggs of this species are unusually transparent, facilitating visual monitoring of the injection process. The initial fate of injected DNA fragments appears similar to that observed earlier for exogenous DNA injected into unfertilized egg cytoplasm. Thus after end-to-end ligation, it is replicated after a lag of several hours to an extent indicating that it probably participates in most of the later rounds of DNA synthesis undergone by the host cell genomes during cleavage. The different consequences of nuclear versus cytoplasmic injection are evident at advanced larval stages. Larvae descendant from eggs in which exogenous DNA was injected into the nuclei are four times more likely (32% versus 8%) to retain this DNA in cell lineages that replicate very extensively during larval growth, i.e. the lineages contributing to the imaginal rudiment, and thus to display greatly enhanced contents of the exogenous DNA. Similarly, 36% of postmetamorphic juveniles from a nuclear injection sample retained the exogenous DNA sequences, compared to 12% of juveniles from a cytoplasmic injection sample. However, the number of copies of the exogenous DNA sequences retained per average genome in postmetamorphic juveniles was usually less than 0.1 (range 0.05-50), and genome blot hybridizations indicate that these sequences are organized as integrated, randomly oriented, end-to-end molecular concatenates. It follows that only a small fraction of the cells of the average juvenile usually retains the exogenous sequences. Thus, even when introduced by nuclear microinjection, the stable incorporation of exogenous DNA in the embryo occurs in a mosaic fashion, although in many recipients the DNA enters a wider range of cell lineages than is typical after cytoplasmic injection. Nuclear injection would probably be the route of choice for studies of exogenous DNA function in the postembryonic larval rudiment.
|Additional Information:||Copyright © 1988 by Company of Biologists. (Accepted 4 November 1987) It is our pleasure to acknowledge Dr R. Andrew Cameron for cell number determinations in squashed preparations of early embryos, Mr Patrick Leahy for culturing sea urchin embryos, larvae and juveniles and Mr Jeffrey Tekanic for his help with genome blot assays. This research was supported by NIH grant HD-05753. R.R.F. was supported by an NIH Postdoctoral Training Grant HD-07257.|
|Subject Keywords:||microinjection, zygote nucleus, mosaic incorporation, sea urchin, DNA, Lytechinus variegatus, Cyllla, cytoskeletal actin|
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|Deposited On:||22 Sep 2008 00:07|
|Last Modified:||26 Dec 2012 10:18|
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