Supplementary Materials and Methods
Immunofluorescence, RNA FISH and DNA FISH on mouse embryos and testis cells
The preparation of pre-implantation embryos, immunofluorescence and RNA FISH were carried
out as described previously6. Probes used for RNA FISH were overlapping lambda clones for
Chic121; a genomic BAC probe (RP24-148H21) for Smcx; and a cDNA probe for Ube1x (a kind
gift from M. Sugimoto and N. Takagi). To identify the transgenic locus or a chromosome, DNA
FISH was performed following immunofluorescence and/or RNA FISH. Images of embryos were
first acquired, immediately following IF / RNA FISH. The coverslips carrying the embryos were
then recovered in dH2O and incubated in Rnase A (100 ug/ml) in 2x SSC at 37 °C for 1h,
before a brief rinse in 2x SSC, dehydration through an ethanol series (70%, 90%, 100%) and air
drying. The DNA was then denatured in 70% formamide, 2x SSC for 10 min at 80 °C and
dehydrated again through an ice-cold ethanol series. Hybridization with an Xic probe (YAC PA2) or a transgene specific YAC vector probe (pYAC) has been described previously18.
Hybridization and revelation of the chromosome paint probes were performed according to
manufacturer’s instructions (Cambio). Cover slips were counterstained with DAPI (1ug/ml),
mounted and viewed under the fluorescence microscope.
Mouse testes were isolated from 6-7-week –old male transgenic mice. Seminiferous
tubules were minced into small fragments with small scissors in PBS to release spermatogenic
cells, which were then transferred onto a glass coverslip in a drop of PBS and allowed to adhere
by aspirating the excess medium prior to fixation. Specimen preparation for immunofluorescence
or RNA FISH was carried out as described above. Primary antibody incubation was overnight at
4 °C, followed by a secondary antibody incubation of 30 min at RT. After washing in PBS,
preparations were post-fixed in 4% paraformaldehyde for 10 min at RT and rinsed in PBS.
Preparations were incubated in 0.7% Triton X-100, 0.1M HCl for 10 min on ice. They were then
washed twice in 2x SSC for 10 min at RT. DNA FISH with paint probes for chromosomes 13 and
X was carried out as described above, expect that the denaturation step was for 30 min at 80 °C;
for pYAC4 and YAC PA-2 probes denaturation was for 5 min at 75°C.
Preparation of metaphase spreads from ear fibroblasts and DNA FISH
Ear fibroblasts were collected from ear tip tissue derived from male mice that were produced by
setting up Tg 53 hemizygous intercrosses. Pieces of ear tip tissue were attached to the bottom of
plastic tissue culture dishes and left to dry at room temperature. About 30 min later, DMEM with
10 % FBS was added. The ear tip tissues was cultured in a humidified atomosphere of 5%
CO2 /95% air at 37 °C for 7-10 days. After 2 to 3 passages, metaphase chromosome preparation
and DNA FISH were performed as described previously (S1, 18), with a denaturation step of 5
min at 75 °C
3D Microscopy
Confocal image series were recorded on a LEICA TCS SP2 laser scanning microscope using the
wavelengths 364 nm (Ar), 488 nm (Ar) and 543 nm (HeNe). Widefield 3D stacks were
performed on a Delta Vision (Applied Precision Inc.) and deconvolved using Delta Vision
sofware (Ratio method, 7 iterations). All optical sections were acquired by using a 100x objective
and separate by 0.2 µm.
Replication Timing analysis
For late replication timing assays, embryos (late blastocysts) were placed in Eagle’s minimum
essential medium (MEM) with 10% fetal bovine serum(FBS) containing 5-bromo-2’-deoxyuridine (BrdU, 7.5 ug/ml) and incubated for 6 h. For early replication timing assays S2, the late
blastocysts were incubated in the presence of BrdU (7.5 ug/ml) for 1h and then transfered to
MEM + 10% FBS with no BrdU, and incubated for another 9 hr. Incubation times were
established in preliminary experiments, and were carried out based on previously published
blastocyst cell cycle timesS3. Colcemid (0.03 ug/ml) was added to the medium for the last 1.5-2.0
h of incubation in all cases. Chromosome slides were prepared as described previouslyS3.
Chromosome preparations were treated with 0.01% pepsin in 0.01N HCl at 37 °C for 7.5 min
and then dehydrated in an ethanol series (70, 90 and 100%). Denaturation was then performed in
70% formamide/2xSSC for 2 min at 70°C followed by dehydration in an ice-cold ethanol series.
After air-drying for 1h, the slides were incubated with blocking solution ( 1% BSA, 0.1% Tween
20 /PBS) for 10 min and then with mouse monoclonal anti-BrdU antibody (Becton Dickinson),
diluted 1:200 in blocking solution, at 37°C for 30 min. The slides were then washed in PBST
(0.1% Tween 20, PBS) three times for 5 min each and incubated for 30 min with Alexa Fluor 488
goat anti mouse IgG (Molecular Probes) appropriately diluted with blocking solution. After three
further washes with PBST, the slides were mounted and the coverslips temporarily sealed. Slides
were examined under the fluorescence microscope and selected images were captured. Coverslips
were then removed in dH2O and the slides were washed in PBST, dehydrated in ethanol series,
dried, denatured in 70% formamide/2xSSC for 2 min at 80°C and dehydrated in an ice-cold
ethanol series, and subjected to chromosome painting with paint probes for Ch 13 or X (Cambio).
Slides were stained with DAPI, mounted and viewed under the fluorescence microscope.
Supplementary References
S1. Popescu, P., Hayes, H. & Dutrillaux, B (Eds.). Techniques in animal cytogenetics. SpringerVerg. (1998).
S2. Sugawara O., Takagi N. & Sasaki M. Allocyclic early replicating X chromosome in mice:
genetic inactivity and shift into a late replicator in early embrogenesis. Chromosoma. 88,133138 (1983).
S3. Dyban, A.P. An improved method for chromosome preparations from preimplantation
mammalian embryos, oocytes or isolated blastocysts. Stain Technol. 58, 69-728 (1983).
S4. Mukherjee, Anil.B. Cell cycle analysis and X-chromosome inactivation in the developing
mouse. Proc Natl Acad Sci USA. 73, 1608-1611 (1976).