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.
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.
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.
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
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
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.
II. MATERIALS AND
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
dUTP) or 50µM
(Cy5-dUTP, fluorescein-12-dUTP) in
- Seed cells on small coverslips (15 × 15 mm) and
grow them until they reach near confluence.
- Pick up the coverslip with fine forceps, drain
excess medium, dry the bottom side of the coverslip
briefly with a wipe, and place it into an empty tissue
culture dish. This will prevent the coverslip from
sliding during the subsequent scratching procedure.
- Add 8-10 µl of the labeling solution onto the coverslip
and distribute it evenly over the cells by gently
tilting the dish. Surface tension will prevent the solution
from running off the coverslip.
- With the tip of a hypodermic needle, apply parallel
scratches into the cell layer. For a high fraction of
labeled cells, scratches should be performed a few cell
diameters apart from each other and cover the complete
coverslip (Fig. 1). For optimal coverage, the procedure
can be performed under a low magnification
phase-contrast microscope (e.g., 5× objective lens). The
procedure should not take longer than a few minutes
to avoid drying of the cells.
- Add 5ml prewarmed medium and incubate
further. Exchange medium after 30-60min to remove
- To obtain nuclei with segregated labeled and
unlabeled chromosome territories, the cells are harvested
by trypsinization some hours later and cultivated
further for two or more cell cycles prior to live
cell observation. The day before live cell observations
are carried out, seed cells on coverslips fitting the live
cell chamber (Fig. 2).
|FIGURE 1 (A) Scratch replication labeling of human neuroblastoma cells. Arrowheads indicate scratches
in the monolayer applied with the tip of a hypodermic needle (top left) in the presence of Cy3-dUTP. (B)
Cells were fixed with 4% formaldehyde two hours after the scratching procedure and counterstained with
4', 6-diamidino-2-phenylindole (DAPI, blue). Numerous cells display Cy3-dUTP labeled nuclei (red) along
the scratch lines. Bar: 100µm.
|FIGURE 2 (A) Living HeLa cells with green fluorescent protein (GFP)-tagged histone H2B reveal green
fluorescent nuclear chromatin. The cell culture was scratch labeled with Cy3-dUTP 5 days prior to observation
in the live cell chamber. Numerous cells display nuclei with a few Cy3-1abeled chromosome territories/
~1-Mb chromatin domains. The framed cells are shown at higher resolution in the inset. Bars: 10µm.
(B) Confocal midsection of a HeLa cell expressing histone H2B-GFP fixed 3 days after labeling with Cy3-
dUTP shows segregated chromosome territories/~1-Mb chromatin domains at high resolution. (Inset)
GFP-labeled chromatin of the same nucleus.
Bar: 5 µm.
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
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
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
in medium) followed by immunofluorescent
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