AvH International Course “Nuclear Architecture, Chromosome
Territories, Chromatin Dynamics and Genetic Damage“
April 4-7, 2011
Handout for experimental lab work
Preliminary remarks
Studies addressing nuclear architecture of single cells can be achieved by different
approaches. Fluorescence in situ hybridization on 3D preserved nuclei (3D-FISH) in
combination with 3D-microscopy and image reconstruction is a key method to analyze the
spatial arrangement of targeted DNA sequences in the nucleus. Using confocal microscopy
that allows the distinct visualization of three or more different fluorochromes, differently
labeled nuclear targets can be delineated simultaneously within one experiment by multicolor 3D-FISH experiments their spatial interrelationships on the level of individual nuclei can
be analyzed.
Visualization of incorporated nucleotides during S-phase (together with immunodetection of
cell cycle associated markers) is another method for the analysis of nuclear organization.
The entire technique of fluorescence in situ hybridization comprises generation and labeling
of DNA probes or complex probe sets, setup of hybridization solution and hybridization itself,
finally washing and probe detection. Due to time limitations we will get into our lab work with
labeled probes and start our experiments with the setup of the respective hybridization mix.
Probe generation and different labeling procedures (both methods are relatively time
consuming) will be explained in detail in a specific lecture (compare also sections 3.1.1 –
3.1.8 of provided method chapter).
During the course four different 3D FISH experiments will be performed.
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Experiment 1
Background:
Osborne, C et al. (2007) Myc dynamically and preferentially relocates to a transcription
factory occupied by Igh (2007) PLosBiol 5:e192
From Osborne et al. (2007)
Double-label RNA FISH in stimulated
splenic B cells for Igh (red) and and Myc
(green)
Our Rationale: Delineation of MMU chromosome territory #15 (MMU paint probe #15)
together with gene locus of c-Myc (BAC clone containing c-Myc) and an appr. 5 Mb
stretch on both sides of the c-Myc gene (BAC pool). This is a 3-color 3D-FISH
experiment performed on cultivated MMU fibroblasts.
 Visualization of the variable shape of individual CTs, relative positions of the single BAC
(cMYC) and the BAC contig with regard to the respective CT (clearly inside, peripheral
or looping out from the CT); variability in conformation of the BAC contig (extended,
compacted etc). This analysis can provide information on compaction levels of specific
chromatin regions (3 kb of extended naked DNA correspond to approximately 1 µm)
Experiment 1 is performed on cultivated mouse embryonic fibroblasts (3T3)
Experiment 2
Background:
Mahy et al (2002) J Cell Biol 159:753, Küpper et al. (2007) Chromosoma 116: 285
Left: Schematic draft of HSA #11,
painted metaphase chromosome #11
(green) and BAC contig of 11p15.5.
Right: 3D-reconstructions and
maximum projection (insert) of CT11
(green) and the 11p15.5 contig (red)
in a human fibroblast shows
protruding of this segment from the
core territory. (from Küpper et al.
2007)
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Our Rationale: Delineation of HSA chromosome territory #11 (paint probe HSA #11)
together with a BAC clone (RP11-1391J7) from 11p15.5 containing highly expressed genes
AP2A2, POLR2L and TM4SF7. This is a 2-color 3D-FISH experiment performed on
cultivated human fibroblasts.
 The region 11p15.5 is particularly gene dense and ubiquitously highly transcribed on (36
genes/Mb; RIDGE). Looping out of this region from the core CT has been described.
Analysis of conformation and positioning of the specific locus with regard to the respective
CT.
Experiment 2 is performed on cultivated human fibroblasts MRC-5
Experiment 3
Background:
Lander et al. (2001) Nature 409:860; Bolzer et al. (2005) PLoS Biol 3(5): e188
Top left: Scheme of inter-Alu PCR for the delineation of gene dense Alu-enriched segments.
Left: delineation of a banding pattern similar to R-bands on human metaphase chromosomes.
Middle and Right: enrichment of Alu enriched regions in the nuclear interior both in flat HFb and
spherical lymphocytes
Our Rationale: Delineation of Alu enriched sequences using an “inter Alu” amplified
probe. This 3D FISH experiment on human fibroblasts should be run simultaneously on
metaphase preparations. (Slide with methanol/Acetic acid fixed metaphase spreads will be
provided).
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 Alu sequences belong to the family of “Short interspersed nucleotide elements” (SINEs) in
the human genome and represent approximately 10% of the human genome. They are
enriched in gene-dense DNA segments (R-bands) and thus indicate gene dense regions
of the human genome. The probe used here was amplified by primers designed to amplify
DNA located between two oppositely oriented Alu sequences.
primer CL-1: 5´ TCC CAA AGT GCT GGG ATT ACA G 3´
primer CL-2: 5´CTG CAC TCC AGC CTG GG 3`
(for amplification conditions see appendix)
With this experiment we will exemplify the ”transformation” of a segmental arrangement
along metaphase chromosomes into a polarized arrangement in 3D preserved interphase
nuclei.
Experiment 3 is performed on cultivated human fibroblasts MRC-5 and on a human
metaphase slide
Experiment 4
Background: Solovei et al. (2009) Cell
Left: schematic draft of a mouse
chromosome
with
typical
acrocentric conformation.
Right: Rod cell nucleus of mouse
with
one
typically
central
chromocenter. Bottom: bipolar cell
nucleus
with
several
chromo-
centers (red = MMS; green is
telomere.
Our Rationale: Delineation of all pericentromeric regions of the mouse by using a mouse
major satellite probe (MMS) and of mouse telomeres.
MSM primer: 5.’-GCGAGAAAACTGAAAATCAC and 5.’-TCAAGTCGTCAAGTGGATG
see section 3.1.8 of chapter. For the specific amplification of telomeres see appendix.
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 In mouse all chromosomes are centromeric, resulting in a close proximity of one telomere
and the centromere. In mouse cell nuclei (peri)centromeric regions tend to form clusters
( chromocenters, most pronounced in e.g. differentiated rod nuclei.
Experiment 4 is performed on cultivated MEF (3T3) and optionally on metaphase
preparations. For the delineation of the different telomere size between mouse and
human a human metaphase slide can be used in addition.
Experiment 5
(Scratch Replication Labeling)
Background: Schermelleh et al (2001) Chromos. Res. 9: 77-80; O´Keefe et al (1992)
JCB 116: 1095 - 1110
Depending on the moment of replication during S-phase, chromatin can mainly be
subclassified into early, mid and late replicated chromatin. Each stage offers a typical pattern
if DNA during ongoing synthesis is labeled (see figure below).
Exemplification of early, mid and late replication patterns in Hela cells (green – DNA
counterstain; red – replicating DNA labeled with Cy3-dUTPs during S-phase).
 During early S-phase replicating DNA is found throughout the nucleoplasm, excluding the
nucleoli. Early replicated chromatin is consistent with the localization of euchromatin. Mid
replicated chromatin predominantly occupies the periphery of the nucleus and of the
nucleoli, which both represent heterochromatin. During late S-phase replication occurs
primarily at a few large regions.
Our rationale: Cells growing on coverslips will be covered with cell culture medium, which
contains direct labeled fluorochromes (in our case Cy3-dUTPs). To guarantee access of
Cy3-dUTPs to the cells, the plasma membrane will be injured by scratching with a hollow
needle. Sufficient Cy3-dUTPs can now diffuse through the created holes. Concerned cells in
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S-phase will incorporate the labeled nucleotides instead of dTTPs resulting in a microscopic
detectable replication pattern.
Experiment 5 is performed on cultivated Hela cells growing on coverslips.
Monday, April 4, 14:00 - 17:30
Experiment 1 – 4:
Precipitation of DNA probes and Setup of hybridization solution
A hybridization area covered by 12 x 12 mm coverslip requires 3 µl of hybridization mixture.
We prepare a final volume of 12 µl hybridization solution, sufficient for 4 hybridizations (or 3
hybridizations on 15 x 15 mm cover slips respectively).
Approximately 40 – 80 ng DNA/µl hybridization solution is used for non-repetitive (single
copy) probes. Since exact measurement of DNA probe concentration may be somewhat
tedious, as a rule of thumb we recommend to use 2 – 3 µl of (DOP) labeled PCR-product or
of a nick translated probe per 1µl hybridization solution in case of chromosome painting
probes or locus-specific probes. For repetitive probes this amount can be reduced to about
10-20 ng/µl hybridization solution. The concentration of unlabeled competitor DNA (e.g. Cot1DNA) added to the probe for suppression of nonspecific hybridization depends on the
frequency of repetitive sequences in the probe and should be around 10 up to 50-fold of the
concentration of the probe DNA. However, in case of complex probe mixtures it is assumed
that probes suppress each other and the amount of Cot-1 DNA can be reduced to around 5
fold. In case of smaller or larger hybridization areas the required amount should be adjusted
accordingly.
1. Mix in 1,5ml tube all labeled DNA-probes that will be hybridized together and in case of
experiment #1 and #2 COT-1 DNA.
Experiment #1
Probe (conc)
Labeled paint probe MMU #15 –DNP (25 ng/µl)
cMYC–NT – Bio (generated from MDA) (40 ng /µl)
Contig around cMYC – NT –DIG (generated from
pre-pool after MDA) (30 ng / µl)
Mouse COT-1 DNA (500 ng / µl)
amount/12 µl
hybmix
30 µl
25 µl
30 µl
100 µl
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Experiment #2
Probe (conc)
Paint HSA #11 – DNP (20 ng/µl)
amount/12 µl
hybmix
40 µl
BAC RP11-1391J7 NT – Bio (30 ng / µl)
30 µl
Human COT-1 DNA (1 µg / µl)
60 µl
Experiment #3
Probe (conc)
Alu probe – Bio (after label PCR) (20 ng/µl)
Salmon sperm DNA (1 mg/ µl)
This DNA is used for better precipitation in case of
small amounts of DNA
amount/12 µl
hybmix
50 µl
2 µl
Experiment #4
Probe (conc)
Telomere specific probe –Bio (20 ng /µl)
amount/12 µl
hybmix
30 µl
major mouse sat (Pancentromer probe) (20 ng / µl) 20 µl
DNP
Unlabeled Genomic BAC DNA unspecified
40µl
This DNA is used for better precipitation in case of
small amounts of DNA
2. Mix probe DNA with ice-cold 100%EtOH ( 2,5 x volume = 500 µl)
 at least 1h, at -20°C or at -80°C.
 (During this precipitation step we start with fixation and pretreatment of cells (see below)
3. Spin down at 13.000rpm  20min.
4. Discard supernatant and dry the pellet (using vacuum centrifuge if available).
5. Resuspend the pellet in 50% formamide/2xSSC/10% dextran sulfate as follows: resolve
the pellet in the appropriate amount of 100% formamide (shake at 37°C) leave O/N on
shaker
 step 6 is performed on Tuesday as the first act
6. add equal volume of 4xSSC/20% dextran sulfate. Briefly mix and incubate at 37oC  10
min. Probes are now ready for hybridization.
(Compare also section 3.1. 9 of provided method chapter)
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Fixation and pre-treatment of cultivated mouse cells (MEF 3T3)
(Compare also section 3.2 – 3.2. 1 of provided method chapter)
1. Rinse cover slip with grown cells grown briefly in 2-3 changes of 1xPBS (formula as
used for cell culture) at 37C.
2. Fix in 4% paraformaldehyde in 1xPBS (freshly made, pH 7.0 or defroze an aliquot),
RT  10min.
3. Wash in 1xPBS with 0.01% TritonX-100, RT  3 x 3min.
4. Incubate in 0.5% TritonX-100/1xPBS, RT  20 min.
5. Incubate in 20% glycerol in 1xPBS, RT  minimum 60min
 During this incubation step go back to step 3 for probe precipitation
6. Freeze by dipping the slide into liquid nitrogen (~30sec) and thaw on a piece of paper
towel. As soon as the frozen layer disappears, put the cover slip back to 20%
Glycerol/PBS  repeat 4x.
7. Wash in 1xPBS  3 x 10min.
8. Incubate in 0.1M HCl  5min, RT3.
9. Incubate in 2xSSC  2 x 3min.
10. Incubate in 50% formamide (pH = 7.0)/2xSSC O/N before proceeding with
hybridization and keep at 4C (please wear gloves when working with FA and use a
hood!).
If we decide to perform a pepsin digestion we continue with the following steps:
11. Equilibrate slides (kept in 50% formamide /2xSSC) in 2xSSC, RT  2min.
12. Equilibrate slides in 1xPBS, RT  3min.
13. Incubate in pepsin (0.005% in 0.01N HCl). Warm up 0.01N HCl to 37°C in a bottle
and add pepsin (30 - 50 µl of 10% stock solution) just before use, shake well and
pour pepsin solution in a coplin jar  incubation in pepsin 3 to 5min
14. Incubate in 1xPBS/50mM MgCl2 to inactivate pepsin, RT  2x5min.
15. Post-fix in 1% parformaldehyde/1xPBS, RT  10 min.
16. Wash in 1x PBS, RT  5 min.
17. Wash in 2xSSC  2x5min, then return slides to 50% formamide/2xSSC
Tuesday, April 5: 14:00 - 17:30
Experiment 5: Scratch replication labeling
 Scratching procedure
1. Already prepared: Hela cells growing on 18x18 cover slips. Cells should be in a well
growing state at approximately 80% confluence.
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2. Take cover slip with forceps, carefully wipe the rim and the bottom side at a Kleenex
and place the cover slip in a new Petri dish with the cell side up (cells should not dry
out, continue work immediately).
3. Pipette 7 µl of the Cy3-dUTP solution (1mM stock solution diluted 1:10 in appropriate
cell culture medium) in the center of the cover slip and distribute the solution by
moving the Petri dish.
4. Scratch the cells with a hollow needle (0.45 x 25 mm).
Work speedy but properly since Cy3-dUTPs are light sensitive and cells do not replicate
well when they cool down or the pH shifts.
5. Carefully and slowly (cells are partially detached by scratching but normally will seed
down again) add 2 ml of pre-warmed culture medium to the Petri dish and place it for
1 h in the incubator (37°C, 5% CO2).
During incubation:
Experiment 1 – 4:
Denaturation of probes and cells and hybridization
(compare also section 3.2.4.)
1. Place 3 µl of hybridization solution on a conventional microscopic slide
2. Take a cover slip with cells out of the 50% formamide/2xSSC, quickly drain the
excess of fluid off the slide and put the cover slip (cells down!) on the hybridization
field
3. Wipe off the excess fluid around the cover slip and seal with rubber cement; let the
rubber cement dry completely.
4. Place slides on a hot block at 76°C to denature cellular and probe DNA  2 min.
Keep this time and temperature strictly.
5. Perform hybridization in a metal box floating in a 37oC water bath overnight.
Continue Experiment 5:
 Cell fixation with formaldehyde (for following steps protect specimen from direct light)
6. Discard medium, fill up Petri dish with 4% formaldehyde/1x PBS and incubate for 10
min. at room temperature.
7. Discard formaldehyde and fill up Petri dish with 1x PBS for washing (2x 3min.).
In principle, cells now could be stored in 1x PBS at 4°C over night without lost of quality.
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 Counterstain with TOPRO-3
8. Incubate cells 5 min. in 0.5%Triton/1x PBS in the Petri dish.
9. Prepare 50 µl of TOPRO-3 solution (1 µM/1x PBS) on a microscopic slide, carefully
take the cover slip with forceps out of the Petri dish, wipe the rim at a Kleenex and
place the cover slip with the cell side on the TOPRO-3 drop and incubate 5 - 10 min.
(light proof; longer incubation may lead to strong overstaining).
10. Rinse only briefly (not more than a few seconds) in 4xSSC/0.2%Tween (otherwise
TOPRO-3 will be washed out).
11. Mount cover slip in antifade (Vectashield) by putting a tiny drop of Vectashield on a
new microscopic slide, remove excessive antifade with Kleenex and seal the rims
with nail polish. Place the cover slip right in the center of the slide.
12. Slides are now ready for microscopic inspection.
Wednesday, April 6: 12:00 – 13:00 and 14:00 - 17:30
Washing steps and detection of hybridized slides
(compare also section 3.2.5.)
1. After hybridization, peal off rubber cement, gently remove the cover slip and transfer
cells to 2xSSC. In case that the cover slip cannot be striped off easily, incubate briefly
in 2xSSC and try again. All following washing steps should be performed e.g. in 6-or
12 well plates with marked slots. In order to avoid drying up of cells, cover slips
should quickly transferred from one well into the next.
2. Wash in 2xSSC at 37°C, shaking  3x3min.
3. Wash in 0.1xSSC at 60°C (stringent washes), shaking  3x5min.
4. Rinse briefly in 4xSSC/0.2%Tween.
5. Block in 4xSSC/0.2%Tween + 4% BSA (bovine serum albumin) at 37°C  10min15min.
6. Dilute the required antibodies or avidin-conjugates to the appropriate working
concentration in 4xSSC/0.2%Tween + 1% BSA in a dark 1.5 ml tube. Provide 50 µl of
antibody solution per slide. In case of 4 slides an amount of 200 -250 µl I reasonable.
Pipet all antibodies for 1. layer together and all antibodies for 2nd layer together. Keep
antibody dilutions in dark and on ice.
Experiment #1
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DNA-probe
label
1. (ab) layer
2nd (ab) layer
MMU CT #15
DNP
Rab-anti-DNP (1:200)
G-anti
–rabbit-TR
(1:75)
cMYC
Bio
Avidin Alexa 488 (1:200)
----
Contig around cMYC
Dig
Mouse-anti-Dig (1 :500)
G-anti-mouse-Cy3
(1:200)
Experiment #2
DNA-probe
label
1. (ab) layer
2nd (ab) layer
HSA CT #11
DNP
Rab-anti-DNP (1:200)
G-anti
–rabbit-Cy3
(1:200)
BAC RP11-1391J7
Bio
Avidin Alexa 488 (1:200)
----
Experiment #3
DNA-probe
label
1. (ab) layer
2nd (ab) layer
Alu probe – Bio
Bio
Avidin Alexa 488 (1:200)
----
Experiment #4
DNA-probe
label
1. (ab) layer
2nd (ab) layer
Telomere probe
Bio
Avidin Alexa 488 (1:200)
----
Pancentromere
DNP
Mouse-anti-Dig (1 :500)
G-anti
probe
mouse
Cy3
(1:200)
7. Prepare a dark moist chamber for antibody incubation. Put 50 µl primary antibody
dilution on a microscopic slide and gently cover with the cover slip with cells (observe
the correct side!). Incubate at RT or 37°C  45 min. Take care that all subsequent
steps are performed under light protection
 Lunch break?
8. Wash cover slips in 4xSSC/0.2%Tween, shaking  3x3min.
9. Incubate with the appropriate concentration of secondary antibody (second layer) in a
dark moist chamber at 37°C  30 - 45 min.
10. Wash in 4xSSC/0.2%Tween, shaking  3x3min.
11. For DNA counterstaining use T0PR0-3 (1 µM) at RT  5 -10 min (light proof; longer
incubation may lead to a strong overstaining).
12. Rinse only briefly (not more than a few seconds) in 4xSSC/0.2%Tween (otherwise
TOPRO-3 will be washed out).
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13. Mount cover slip in antifade (Vectashield) by putting a tiny drop of Vectashield on a
microscopic slide, remove excessive antifade and seal with nail polish. Place the
cover slip right in the center of the slide.
14. Slides are now ready for microscopic inspection.
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