in vivo DNA Replication Labeling
DNA replication in higher eukaryotes takes place in a well-defined spatial and temporal manner. Large numbers of replication sites are simultaneously active, creating characteristic replication patterns during progression through S phase (Nakamura et al., 1986; O'Keefe et al., 1992). Each site is typically active for ~45 min, forming a replication focus of some 100 kb up to several Mb in size, consisting of a cluster of 1-10 simultanously firing replicons (Berezney et al., 2000). Because the higher order chromatin structures revealed by replication foci persist through all stages of the cell cycle, they are referred to as ~l-Mb chromatin domains. Importantly, replication timing reflects important functional chromatin features, as the generich, transcriptionally active euchromatin replicates during the first half of S phase, whereas gene-poor, heterochromatic sequences are later replicating (Cremer and Cremer, 2001).
In contrast to DNA labeling with halogenated thymidine analogues, which can be detected immunocytochemically after cell fixation and DNA denaturation, usage of fluorescent precursors allows the investigation of dynamic properties of ~l-Mb chromatin domains in living cells. Depending on the time period in S phase when the labeling is accomplished, early and mid to late replication foci can be specifically marked (Schermelleh et al., 2001). Moreover, further proliferation of directly labeled cells results in the random distribution of sister chromatids with labeled and unlabeled chromatin domains during the second mitosis. Subsequent cell generations result in nuclei with few labeled chromosome territories (within a bulk of unlabeled chromatin). These cells are ideally suited to study chromatin domain/chromosome territory movements in vivo in relation to other nuclear components tagged, e.g., with green fluorescent protein (Walter et al., 2003).
Several fluorochrome-coupled deoxynucleotides (dNTPs) are available commercially. The charged phosphate group and the attached fluorophore prevent the uptake of these nucleotides across the cell membrane. Hence, a procedure is required for dNTPs to pass the cell membrane barrier. A well-established and reliable method is microinjection of a nucleotide solution through a thin glass capillary directly into the cytoplasm or nuclei of adherently growing cells. (Ansorge and Pepperkok, 1988; Pepperkok and Ansorge, 1995; Zink et al., 1998). However, this procedure is tedious, requires costly equipment, and allows only labeling of a rather small number of cells in each experiment.
A transient permeabilization that allows the uptake of macromolecules from the surrounding medium can be achieved by a number of methods (for review, see McNeil, 1989), yet not all of them are useful for in vivo replication labeling. Detergents (e.g., Triton X-100, saponin, or digitonin) permeabilize cells effectively, but have detrimental effects on cell viability, whereas more gentle "noninvasive" methods, such as lipofection and osmotic shift, are not efficient in our experience. More suitable regarding loading efficiency and viability are methods creating slight mechanical damage to the cell membrane in the presence of nucleotides. This can be achieved by shaking a labeling solution with small glass beads over a cell layer or by detaching a cell layer with a cell scraper.
The best results in terms of labeling efficiency, reproducibility, and amount of nucleotides needed were obtained by a modified scratch-loading protocol, termed scratch replication labeling. This protocol can be applied on any adherently growing cell line. It allows the labeling of a high number of cells within one experiment with little effort. Most importantly, it does not impair further growth of a large fraction of affected cells (Schermelleh et al., 2001). This protocol has been applied for live cell studies of chromatin domains and chromosome territories (Walter et al., 2003).
II. MATERIALS AND INSTRUMENTATION
Cy3-dUTP (Amersham Bioscience, Cat. No. PA53022), Cy5-dUTP (Amersham Bioscience, Cat. No PA55022), fluorescein-12-dUTP (Roche Cat. No. 1373242), disposable hypodermic needles (e.g., 0.45mm × 25mm), 15 × 15-mm square coverslips, 60/15-mm tissue culture dishes, phase-contrast microscope, appropriate cell culture medium (with HEPES), paper wipes (e.g., Kimwipes lite precision wipes, Kimberly-Clark), fine forceps, live cell chamber with fitting coverslips (e.g., Bioptechs FCS2).
Appropriate cell culture medium (with 25 mM HEPES and supplemented with 10% fetal calf serum and antibiotics); labeling solution: 20µM (Cy3- dUTP) or 50µM (Cy5-dUTP, fluorescein-12-dUTP) in medium.
The scratch procedure creates damage in the cell membrane that may last for only a few seconds ("transient holes"). This allows the uptake of charged macromolecules (such as fluorochrome-coupled dUTPs) from the surrounding medium, to which cells would normally be impermeable. Accordingly, most S-phase cells damaged alongside the scratch line or lifted off from the surface by the needle will incorporate the modified nucleotides.
The fraction of replication-labeled cells depends on the (1) density of scratches applied, i.e., how many cells are affected, and (2) number of cells that are in S phase. Synchronization of cells at the G1/S transition (e.g., by aphidicolin) is recommended to obtain a high yield of labeled cells.
A pool of fluorescent nucleotides is available for DNA replication over a time scale of roughly l h. The labeling pattern therefore resembles a BrdU-labeling experiment with ≤l-h pulse length.
Cy3-dUTP is significantly brighter and more photostable than Cy5- and fluorescein-dUTP and thus is best suited for in vivo observation. In cases where especially high fluorescence intensities are desirable (e.g., for long-term in vivo observations after segregation), a higher concentration of nucleotides (100 µM) is advantageous. We did not note any improvement of label intensities when adding nonfluorescent dNTPs to the labeling solution or by using different labeling buffers (phosphate-buffered saline or Tris-buffered saline instead of medium).
For studies of nascent RNA formation, the scratch protocol can be applied accordingly using BrUTP (5mM in medium) followed by immunofluorescent detection.
Ansorge, W., and Pepperkok, R. (1988). Performance of an automated system for capillary microinjection into living cells. J. Biochem. Biophys. Methods 16, 283-292.
Berezney, R., Dubey, D. D., and Huberman, J. A. (2000). Heterogeneity of eukaryotic replicons, replicon clusters, and replication foci. Chromosoma 108, 471-484.
Cremer, T., and Cremer, C. (2001). Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nature Rev. Genet. 2, 292-301.
McNeil, P. L. (1989). Incorporation of macromolecules into living cells. Methods Cell Biol. 29, 153-173.
Nakamura, H., Morita, T., and Sato, C. (1986). Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus. Exp. Cell Res. 165, 291-297.
O'Keefe, R. T., Henderson, S. C., and Spector, D. L. (1992). Dynamic organization of DNA replication in mammalian cell nuclei: Spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences. J. Cell Biol. 116, 1095-1110.
Pepperkok, R., and Ansorge, W. (1995). Direct visualization of DNA replication sites in living cells by microinjection of fluoresceinconjugated dUTPs. Methods Mol. Cell Biol. 5, 112-117.
Schermelleh, L., Solovei, I., Zink, D., and Cremer, T. (2001). Twocolor fluorescence labeling of early and mid-to-late replicating chromatin in living cells. Chromosome Res. 9, 77-80.
Walter, J., Schermelleh, L., Cremer, M., Tashiro, S., and Cremer, T. (2003). Chromosome order in HeLa cells changes during mitosis and early G1, but is stably maintained during subsequent interphase stages. J. Cell Biol. 160, 685-697.
Zink, D., Cremer, T., Saffrich, R., Fischer, R., Trendelenburg, M. E, Ansorge, W., and Stelzer, E. H. (1998). Structure and dynamics of human interphase chromosome territories in vivo. Hum. Genet. 102, 241-251.
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