Single Cell Manipulations
In order to visualize chromatin in living cells, several groups employ the lac operator (lacO)/lac repressor (LacI) system. Naturally, the lacO sequence is part of the E. coli lac operon regulating the bacterial lactose transport and metabolism. However, it can be amplified and stably integrated in multiple copies into eukaryotic genomes resulting in a chromosomal binding site array for LacI. Upon transfection of LacI fused to a fluorescence protein like the green fluorescence protein (GFP), the integration can be detected in a conventional light microscope (Figure 1). Using this technique a specific genomic locus can be observed in fixed as well as in living cells, thus allowing to address the dynamics of chromatin. In our lab, several clones with stable lacO integrations derived from the osteosarcoma cell line U2OS have been established. Most of the lacO arrays are integrated at telomeric regions, which enabled us to use these cells for studies on the dynamics of telomeres .
Figure 1: Schematic representation of the lacO/LacI system for labeling chromatin in living cells. Arrays of the lacO repeats are integrated into the genome of the cell. The lac repressor LacI binds with high affinity to these arrays. Thus, transfection of LacI fused to a fluorescence protein like GFP visualizes these genomic loci.
Together with Heinrich Leonhardt (LMU Munich) the system was further developed in order to recruit proteins of interest to the lacO labeled chromatin and investigate the effect of the recruitment. Here, a lac repressor LacI fused to a GFP-binding protein (GBP) and red fluorescent protein (RFP) is transfected into cells containing lacO integrations. The LacI component mediates the binding to the lacO labeled chromatin region. A co-transfected GFP-protein of interest is then tethered via the GBP-GFP interaction. If this triggers the subsequent accumulation of another protein, this can be detected by immunofluorescence staining against that latter factor resulting in a co-localization signal. Accordingly, this approach was termed fluorescence three-hybrid assay (Figure 2).
Figure 2: Schematic representation of the fluorescence three-hybrid system. The GFP-binding protein (GBP) is bound to the chromosomal lacO array by its fusion to the LacI repressor. This construct can additionally be tagged with an RFP-domain. A co-transfected GFP fusion protein is recruited via the GBP-GFP interaction. Thereby induced accumulations of endogenous proteins can subsequently be detected by immunofluorescence. Modified from .
Microinjection is a very useful technology, used for the first time in 1979. It allows injecting small volumes of various molecules directly into specific parts of living cells via a glass capillary. We are using a computer assisted microinjection system as depicted bellow (Figure 3). It is easy to handle, highly accurate and fast.
Figure 3: The system consists of (i) an inverted microscope equipped with (ii) a micromanipulator, its controller, (iii) the injection pressure system, which controls the selected compensation pressure, injection pressure and injection time, (iv) a computer with a user friendly program that allows a complete on screen control of the whole system. Conveniently, the position to be microinjected is indicated per mouse click and the program does the rest. Any needed adjustment can be done via the program. Picture and further information can be found on the website www.AIS2.com.
Using this system, we microinjected various enzymes directly into cell nuclei of mammalian cells and observed their respective effects on genome organization, as depicted in the following picture (Figure 4).
Figure 4: Confocal optical sections of NIH 3T3 mouse fibroblasts microinjected with a mix containing enzymes as indicated or the corresponding storage buffer (control). The DNA staining clearly reveals the specific chromatin re-arrangement taking place after DNase I, Proteinase K or RNase A as compare to the control sample. RNase H did not seem to have any effect. Scale bars, 10 Ám.
Jegou T, Chung I, Heuvelmann G, Wachsmuth M, Görisch SM, Greulich-Bode K, Boukamp P, Lichter P and Rippe K. (2009).
Dynamics of telomeres and PML nuclear bodies in a telomerase negative human cell line.
Molecular Biology of the Cell 20, 2070-2082.
Chung I, Leonhardt H and Rippe K. (2011).
De novo assembly of a PML nuclear subcompartment occurs via multiple pathways and induces telomere elongation.
Journal of Cell Science 124, 3603-3618.
Caudron-Herger M, Muller-Ott K, Mallm J-P, Marth C, Schmidt U, Fejes-Toth K and Rippe K. (2011).
Coding RNAs with a non-coding function: maintenance of an open chromatin structure.
Nucleus 2, 410-42411.
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