|
Visualization of gene expression in living cells
Chromatin structure is thought to play a critical role in gene expression. Using the lac operator/repressor system and two color variants of green fluorescent protein, we developed a system to visualize a gene and its protein product directly in living cells allowing us to examine the spatial organization and timing of gene expression in vivo. Dynamic morphological changes in chromatin structure, from a condensed to an open structure, were observed upon gene activation, and targeting of the gene product/cyan fluorescent protein reporter to peroxisomes was directly visualized in living cells. Interestingly, we found that the integrated gene locus was surrounded by a promyelocytic leukemia (PML) nuclear body. The association was transcription-independent but was dependent upon the direct in vivo binding of specific proteins (EYFP/lac repressor, tetracycline receptor/VP16 transactivator) to the locus. The ability to directly visualize gene expression in living cells provides a powerful system with which to study the dynamics of nuclear events such as transcription, RNA processing, and DNA repair.
This movie shows a stably integrated genetic locus (18 Mb) in a BHK cell. The locus is colored in yellow (YFP-lac repressor). Images were taken in living cells every 10 minutes over a 7 hour period. Approximately 2 hours after the turn on of the locus, by the addition of doxycycline, the protein product is detected as a CFP fusion protein that is targeted to peroxisomes.
From: Tsukamoto, T., N. Hashiguchi, S.M. Janicki, T. Tumbar, A.S. Belmont and D.L. Spector. 2000. Visualization of gene activity in living cells. Nature Cell Biology 2, 871-878.
|
