Supplementary Methods
Construction of pCX-cre plasmid. The coding sequence of Cre (XhoI-MluI blunted fragment
from pBS185, Invitrogen) was subcloned in the EcoRI site (blunted) of pCX-EYFP1 (a gift from A.
Nagy, Samuel Lunenfeld Research Institute, Toronto, CA), replacing EYFP.
Cell culture. Male R1 ES cells2 (provided by A. Nagy, Samuel Lunenfeld Research Institute,
Toronto, CA) were grown on irradiated DR-4 mouse embryonic fibroblats (MEFs made from DR-4
mouse strain3) or on gelatin coated dishes, in a media (DMEM high glucose with L-glutamine and
pyruvate (Invitrogen), 15% fetal calf serum (Invitrogen),1.5 x 10-4 M -monothioglycerol (Sigma)
and 1x10-4 M non-essential amino acids (Invitrogen)) supplemented with 1000 U ml-1 of ESGRO
(Chemicon) or leukemia inhibitory factor (LIF, conditioned media from transfected COS cells).
Serum replacement (Invitrogen) was used for extended period of growth on gelatin. The number
of replating ES cells was estimated by counting the colonies included in 0.09 cm2 areas (n=3
minimum). ES cells were differentiated into embryonic bodies as described 4, after one passage
or more on gelatinized dishes. Briefly, single cells suspensions (between 5x10 3 - 5x105 cells)
were plated in bacterial-grade 35 mm dishes without LIF (IMDM (Sigma), 15% fetal calf serum
(HyClone), 5% serum-free and protein-free media for hybridoma culture (Invitrogen), 2 x 10-3 M Lglutamine (Invitrogen), 50 ug/ml ascorbic acid (Sigma) and 3x10-4 M -monothioglycerol).
Viral producer cell lines and infection of target cells. Retroviruses A1, A2, A3, A5, S1, S2
and S3 were generated with the GP+E-86 ecotropic packaging cell line5 and maintained as
described6, excepted that the linearized constructs (DraI) were transfected directly into the
producers using lipofectamine. Hygromycin (Roche, 200 ug ml-1) or puromycin (Sigma, 1.8 ug ml1)
selection was started on day 2, and maintained throughout the expansion in HXM media5. One
passage prior to the infection, selection was suspended and cells were maintained in the
presence of 10% newborn calf serum (NCS, Invitrogen) in DMEM (Invitrogen).
24h before
infection, producers’ media were changed for complete ES cells medium. 24h infections were
carried out using 4ug ml-1 of polybren (Sigma). Fresh media were added the next day and
selection started 48h after infection (1.5 ug ml-1 puromycin and 150 ug ml-1 hygromycin for ES
cells). Numbers of ES cell colonies surviving selection were estimated and compared to the one
inferred for the replating, in order to calculate the percentage of infection. Viral titers were kept
low to ensure low infection rate and to minimize chances of multiple integrations. For ES cells
infected with S1, the total number of colonies surviving hygromycin selection corresponded to the
estimated integration diversity since the cells were not split.
A4, S4 and S5 were VSV-G
pseudotyped retroviruses produced by the amphotropic packaging cell line 293GPG 7, as
previously described6.
Cre-induced recombination in ES cells. 107 ES cells were transfected with a cre plasmid.
Cells were resuspended in 800 ul of ES cell media and electroporated with 25 ug of supercoiled
pCX-cre or pOG2318, using 225 V and 950 uF or 230 V and 500 uF parameters, respectively.
After an incubation of 20 minutes at room temperature, cells were distributed on 3 dishes (100
mm) covered with irradiated MEFs. For every batch of electroporation, at least one population
similarly received the supercoiled pCX-EYFP1 (225 V, 950 uF) accounting for both a control of
electroporation and a negative control for G418 selection. 2 x 106 cells of the population no.9A,
no.10A and no.10B (≥ 9 x104 colonies for each) were also used as negative control for selection.
48h after electroporation, the replating was estimated and G418 selection started at 300 ug ml -1.
At that time, electroporation controls showed ≥ 50% of YFP positivity under the UV microscope.
7-9 days later, colonies were counted (to determine the frequency of G418 R colonies), isolated in
96 wells plates and expanded in 24 wells dishes before freezing. G418 selection was maintained
all through the expansion. Fractions of these clones were put aside on gelatin coated plates and
tested functionally for the losses of puromycin and hygromycin, or used to extract DNA or RNA.
DNA and RNA analysis. Genomic DNA was isolated using DNAzol and total cellular RNA with
Trizol, according to the manufacturer instructions (Invitrogen). Southern Blot and RNA analyses
were performed as previously described9.
aCGH.
aCGH was performed at the Microarray and Genomics Facility of the Roswell Park
Cancer Institute (Buffalo, NY). Because the R1 ES cells have a XY genotype, the hybridizations
were mainly carried with XX genomic DNA since the sex-mismatches provided internal controls.
Occasionally, XY control genomic DNA was employed (samples: R1, 1-3, 9-18, 9-37, 9-90, 1416). Deletions or amplifications were detected when adjacent BACs in the array presented a
Log2 ratio ~ ≤ -0.5 or ≥ 0.5, respectively. For the viewing and comparison analyses of aCGH
data from multiple clones, we used a modified version of the CGHAnalyzer 10. This version was
adapted to support mouse data and the use of local instances of publicly available database
(kindly modified by Jean-Philippe Laverdure, BioneQ-Réseau québécois de bio-informatique,
Université de Montréal, CA).
aCGH data were submitted to the GEO database
(www.ncbi.nlm.nih.gov/geo/).
Spectral karyotyping and FISH. Spectral karyotyping and FISH analyses were conducted by
The Quebec Leukemia Cell Bank under the supervision of J.H. (http://www.bclq.gouv.qc.ca/,
Maisonneuve-Rosemont Hospital, Montreal, CA).
Chimeras production. Mouse chimeras were generated by the transgenic facility of IRIC and
manipulated under the recommendation of Université de Montréal CDEA animal committee. ES
cells (129/Sv x 129/Sv-CP_F1)2 corresponding to primary clone no.9, tertiary clones 9-35 (no in
vitro phenotype) and 9-18 (with in vitro phenotype) were injected into C57BL/6 blastocysts. ES
cells from primary clone no.9 were also aggregated with CD1 morulas. Chimeric mice derived
from primary clone no.9 and 9-35 were bred with C57BL/6 mice to assess germ-line transmission
of the engineered alleles (tested by PCR specific to the junction of virus A1 and the anchoring
locus, or by PCR specific to the neomycin gene).
Equipment and settings. We scanned blots and gels using a Duoscan T1200 AGFA scanner
and the AGFA FotoLook 3.60.00 software (200 or 300 d.p.i.).
We adjusted brightness and
contrast and assembled the panels in Adobe Photoshop CS version 8.0 (Adobe Systems
Incorporated). We reduced the images in Adobe Illustrator CS (Adobe Systems Incorporated).
For spectral karyotyping (SKY), we used a microscope (Axioplan 2 Imaging, Zeiss) equipped with
a 63X / 1.40 immersion objective (Plan-Apochromat, Zeiss) and filters (Sky filter #1, DAPI and
Cube filter, Zeiss).
Images were acquired with a multi format CCD camera (C4880-80,
Hamamatsu) and a capture software (version 2.3, Spectral Imaging). Analyses were done with a
complementary software (Sky View version 1.61, Spectral Imaging).
For embryonic bodies visualization (Fig. 2b), we used an inverted microscope (Axiovert 25, Zeiss)
equipped with a 5X / 0.12 (A-plan, Zeiss) and 2.5X / 0.075 (Plan-Neofluar, Zeiss) objectives, for
R1 and primary 9 samples, respectively. We set the camera to 4X magnification (G5, Canon).
These pictures were 8-bit depth (RGB).
For tertiary 9-35, we acquired the image with a
microscope (DMIRB, Leica) equipped with a 10X / 0.30 objective (HC PL Fluotar, Leica), under
darkfiled illumination. The camera (Retiga EXi, QImaging) was equipped with a color module and
link to an acquisition software (Northern Elite version 6, EMPIX Imaging Inc.). This image was
16-bit depth (RGB).
For FISH (Fig. 2c), we employed a microscope (Axioskop 2 plus, Zeiss) with a 100X / 1.30
immersion objective (Iris Plan-Neofluar, Zeiss) and different wavelength filters (DAPI, FITC,
TRITC, Cy3, Zeiss). Images were taken with a camera (CCD-CE-4912-5010, Applied Imaging),
captured and analyzed with the same software (Cytovision version 3.6, Applied Imaging).
For foetus images (Fig. 2d), we used a stereomicroscope (MZFLIII, Leica) coupled to a 0.63X
lens (Planapo, Leica) with 1X magnification. The camera (Micropublisher 3.5, QImaging) was
linked to the acquisition software (Northern Elite version 6, EMPIX Imaging Inc.). These images
were 8-bit depth (RGB).
Calibration of each microscope was performed using a ruler or a micrometer microscope slide.
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