Expression Analysis In vivo: Cell Systems

Abstract

Gene expression involves ribonucleic acid (RNA) synthesis and processing, RNA transport and protein synthesis and localization, which are dynamic processes in time and space. With the advent of nucleic‐acid probe and green fluorescent protein technologies, and developments in real‐time microscopy, it is possible now to study these processes at real time in single cells, meaning that information concerning transcription, protein localization and protein–protein interactions can be obtained from genes whose functions might be known or unknown.

Keywords: expression; real‐time microscopy; green fluorescent protein; fluorescence resonance energy transfer; fluorescence lifetime imaging

Figure 1.

Schematic view of functional antibodies. The variable domains (VHH) of the heavy‐chain antibodies are the smallest functional antigen‐binding domains. VHH can be expressed in cells as intracellular antibodies, which are also called intrabodies. When fused to a fluorescent protein (FP), their binding to intracellular targets can be visualized in living cells.

Figure 2.

Fluorescence resonance energy transfer (FRET) microscopy of living U2OS cells expressing Sp100‐cyan fluorescent protein (CFP) and heterochromatin protein‐1β (HP1β) yellow fluorescent protein (YFP). Images were obtained using: (a) a donor (CFP) filter combination, (b) an acceptor (YFP) filter combination and (c) an FRET filter combination; then (d) after processing these images using an FRET imaging programme, a corrected FRET image is obtained. The relative amount of FRET is indicated by a colour scale. Blue shows FRET at a background level, while yellow and red indicate nuclear compartments where Sp100‐CFP and HP1β‐YFP interact.

close

References

Bastiaens PIH and Squire A (1999) Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends in Cell Biology 9: 48–51.

Chudakov DM, Lukyanov S and Lukyanov KA (2005) Fluorescent proteins as a toolkit for in vivo imaging. Trends in Biotechnology 23: 605–613.

Molenaar C, Marras SA, Slats JCM et al. (2001) Linear 2′‐O‐methyl RNA probes for the visualization of RNA in living cells. Nucleic Acids Research 29: 89.

Rizzo MA, Springer G, Segawa K, Zipfel WR and Pistin DW (2006) Optimization of pairings and detection conditions for measurement of FRET between Cyan and Yellow fluorescent proteins. Microscopy Microanalysis 12: 238–254.

Shaner NC, Steinbach PA and Tsien RY (2005) A guide to choosing fluorescent proteins. Nature Methods 2: 905–909.

Sprague BL and McNally JG (2005) FRAP analysis of binding: proper and fitting. Trends in Cell Biology 15: 84–91.

Tramier M, Zahid M, Mevel JC, Masse MJ and Coppey‐Moisan M (2006) Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on FRET determination by fluorescence lifetime imaging microscopy in living cells. Microscopy Research and Technique 69: 933–939.

Vargas DY, Raj A, Marras SAE, Kramer FR and Tyagi S (2005) Mechanism of mRNA transport in the nucleus. Proceedings of the National Academy of Sciences of the USA 102: 17008–17013.

Verheesen P, de Kluijver A, van Koningsbruggen S et al. (2006) Prevention of oculopharyngeal muscular dystrophy‐associated aggregation of nuclear polyA‐binding protein with a single‐domain antibody. Human Molecular Genetics 15: 105–111.

Xia Z and Liu Y (2001) Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophysical Journal 81: 2395–2402.

Further Reading

BD Biosciences Clontech – References. Incorporating the Tet system in transgenic organisms http://www.clontech.com/tet/Refs/TetRefstrans.shtml

Biology of the Mammary Gland. Animal models to study mammary gland development, physiology, and tumorigenesis http://mammary.nih.gov/models/index.html

Chemistry 2002. The Nobel Prize in Chemistry 2002 http://www.nobel.se/chemistry/laureates/2002/index.html

Chen Y, Mills JD and Periasamy A (2003) Protein localization in living cells and tissues using FRET and FLIM. Differentiation 71: 528–541.

CLONTECHniques Archives. Generation of GFP transgenic mice http://www.clontech.com/archive/JAN96UPD/MouseGFParticle.shtml

Darzacq X, Singer RH and Shav‐Tal Y (2005) Dynamics of transcription and mRNA export. Current Opinions in Cell Biology 17: 332–339.

Dirks RW and Tanke HJ (2006) Advances in fluorescent tracking of nucleic acids in living cells. BioTechniques 40: 489–496.

Giepmans BNG, Adams SR, Ellisman MH and Tsien RY (2006) The fluorescent toolbox for assessing protein localization and function. Science 312: 217–224.

Gordon GW, Berry G, Huan LX, Levine B and Herman B (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophysical Journal 74: 2702–2713.

Kerppola TK (2006) Visualization of molecular interactions by fluorescence complementation. Nature Reviews. Molecular Cell Biology 7: 449–456.

Krouwels IK, Wiesmeijer K, Abraham TE et al. (2005) A glue for heterochromatin maintenance: stable SUV39H1 binding to heterochromatin is reinforced by the SET domain. Journal of Cell Biology 170: 537–549.

Life with 6000 genes. This link points to the original paper describing the genome of Saccharomyces cerevisiaehttp://www.sciencemag.org/cgi/content/full/274/5287/546

Molenaar C, Wiesmeijer K, Verwoerd NP et al. (2003) Visualizing telomere dynamics in living mammalian cells using PNA probes. The EMBO Journal 22: 6631–6641.

Nagy Lab. Samuel Lunenfeld Research Institute. This link points to the database of cre‐transgenic mouse lines, maintained by the Nagy Lab http://www.mshri.on.ca/nagy/cre.htm

Phair RD and Misteli T (2001) Kinetic modelling approaches to in vivo imaging. Nature Reviews Molecular Cell Biology 2: 898–907.

Rothbauer U, Zolghadr K, Tillib S et al. (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nature Methods 3: 887–889.

Saccharomyces Genome Database http://genome‐www.stanford.edu/Saccharomyces/Xenogen Technology Publications. Bibliography http://www.xenogen.com/pubs.html

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Dirks, Roeland W, and Tanke, Hans J(Jul 2007) Expression Analysis In vivo: Cell Systems. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0005679.pub2]