EM visualization of transcriptionally active genes after injection into Xenopus oocyte nuclei.
This article has described methods in use in our lab for microinjection of genes into Xenopus oocyte nuclei followed by EM visualization of those genes by the Miller chromatin spreading method. We consider our efforts to be still developing, as we attempt to maximize the visualization of specific, active, mappable genes. One of our main goals at this time is to find a DNA sequence element that will ensure efficient Pol II termination so that the common problem of read-through transcription (as seen in Fig. 6) can be overcome. We currently are testing three different elements reported to have roles in transcription termination. The method is evolving as a unique and valuable approach to study gene expression and RNA processing at the level of individual genes and individual transcripts. Given the ability to manipulate both cis- and trans-acting factors prior to EM visualization, its potential is limited only by the somewhat labor-intensive nature of the method.
Osheim
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Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Beyer
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D000818:Animals; D002467:Cell Nucleus / Q000648:ultrastructure; D015870:Gene Expression; D008845:Microinjections; D008854:Microscopy, Electron / Q000379:methods*; D009865:Oocytes / Q000648:ultrastructure*; D014158:Transcription, Genetic; D014982:Xenopus laevis