Abstract
How transcription of individual genes is regulated in a single, intact, three-dimensionally organized cell nucleus remains mysterious. Recently, live cell imaging has become an essential tool to dissect the in vivo mechanisms of gene transcription. It not only examines functions of transcription factors at their gene targets within the chromatin context, but it also provides a non-disruptive approach for observing the dynamics of a transcription cycle in real time. However, the identification of any endogenous gene loci and their associated transcription factors remains technically difficult. Here, we describe the method of imaging the transcriptional dynamics of heat shock genes in Drosophila polytene chromosomes in living salivary gland tissues by multiphoton microscopy (MPM). This method has provided the experimental capability to visualize the assembly and dynamics of individual transcription factors and regulators and to dissect their functions at their endogenous gene targets in living cells.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Animals
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Chromatin / metabolism
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Cross-Linking Reagents
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Drosophila melanogaster / genetics*
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Drosophila melanogaster / metabolism
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Fluorescence Recovery After Photobleaching / methods
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Gene Dosage
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Genes, Insect
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Genetic Vectors
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Green Fluorescent Proteins / analysis
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Heat-Shock Proteins / analysis
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Heat-Shock Proteins / genetics*
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Heat-Shock Proteins / metabolism
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Kinetics
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Microscopy, Fluorescence, Multiphoton / methods
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Organisms, Genetically Modified
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Recombinant Fusion Proteins / analysis*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Research Design
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Salivary Glands / metabolism
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Transcription Factors / analysis
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Transcription Factors / genetics
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Transcription Factors / metabolism
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Transcription, Genetic* / physiology
Substances
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Chromatin
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Cross-Linking Reagents
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Heat-Shock Proteins
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Recombinant Fusion Proteins
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Transcription Factors
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Green Fluorescent Proteins