Supplementary Methods
Chromosome elimination cassette. The insert of loxP2 cassette vector, pBS246
(Gibco-BRL) was PCR-amplified and subcloned into pGEM-T Easy (Promega). A
loxP site was re-cloned in the opposite orientation (pGEM-CEC). The CAG-gfp/IRES
(internal ribosome entry site).puro-pA fragment was integrated into the pGEM-CEC
to make pCEC-CAG-gfp/IRES.puro-pA for random insertion (Fig. 1a). For Ch6
elimination, the pGEM-CEC was subcloned into pBSIISK(+) (Strategene) (pBS-CEC).
The Pgk-neo/IRES.gfp-pA fragment was then integrated into the pBS-CEC to generate
pCEC-Pgk-neo/IRES.gfp-pA. For homologous recombination in the Gt(ROSA)26Sor
locus, the CEC-Pgk-neo/IRES.gfp-pA was integrated into pROSA26-pA 1 (Fig. 3a).
ESC transfection. To obtain stable transformants, ScaI linearized pCEC-CAGgfp/IRES.puro-pA DNA was electroporated into HM1 ESCs using a Gene Pulser II
(BioRad). The Cre expression vector, pBS185 (pCMV-Cre) (Gibco-BRL) was
transfected into hybrid cells with lipofectamine 2000 (Invitrogen) for transient
expression. For integration of CEC-Pgk-neo/IRES.gfp-pA into the Gt(ROSA)26Sor
locus by homologous recombination, PvuI linearized plasmid DNA was
electroporated into HM1 ESCs.
Cell fusion. HM1 ESCs (129/Ola: Mus musculus domesticus) deficient for the Hprt
gene were cultured in ESC medium on inactivated primary embryonic fibroblast
feeder cells 2. CEC-transgenic HM1 ESCs were electro-fused with thymocytes
collected from female 129ROSA26 (M. m. domesticus) or JF1 (M. m. molossinus)
mice 3. Hybrid cell clones were selected with HAT medium.
FACS sorting. Hybrid cells were suspended in PBS with 2% fetal bovine serum and
2 g/ml Propidium Iodide (Sigma). Approximately 10,000-500,000 cells were applied
for qualitative analysis and 100-1,000 GFP-negative hybrid cells were collected with
FACS Vantage (Becton Dickinson).
FISH analysis. Chromosomal analysis was performed following standard G-banding
procedures. For mapping, chromosome preparations were hybridized and detected
with a CEC vector specific probe as described 4. For chromosome painting,
chromosome preparations were hybridized and detected with STAR FISH probes
(Cambio) specific to Ch6, Ch11, Ch12 and Ch17 according to the chromosome
painting protocol.
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Genomic PCR and RT-PCR. Genomic PCR products were amplified for 35 cycles
with an annealing temperature of 60oC. D1Mit234, D2Mit493, D17Mit133, D6Mit183,
D6Mit102 and D6Mit14 were used as mouse chromosome-specific markers for
detecting DNA sequence polymorphisms between 129 and JF1; D1Mit234 (129; 143
bp and JF1; 173 bp),
primer 1; 5’-TCCATTATTCCCAGAACCCA -3’ and
primer 2; 5’-TCAATGTTATTTTTTGCATCTGTG-3’,
D2Mit493 (129; 127 bp and JF1; 97 bp),
primer 1; 5’-GTCTCTACCTGAGTTTCCATCACA-3’ and
primer 2; 5’-TCCCGAGTTGTCCCTCTATG-3’,
D17Mit133 (129; 158 bp and JF1; 142 bp),
primer 1; 5’-TCTGCTGTGTTCACAGGTGA-3’ and
primer 2; 5’-GCCCCTGCTAGATCTGACAG-3’,
D6Mit183 (129; 94 bp and JF1; 190 bp),
primer 1; 5’-TTCTCAATGAACACTAGAACATTCG-3’ and
primer 2; 5’-AAAACACAGGTAGAAAACATACATACA-3’,
D6Mit102 (129; 177 bp and JF1; 125 bp),
primer 1; 5’-CCATGTGGATATCTTCCCTTG-3’and
primer 2; 5’-GTATACCCAGTTGTAAATCTTGTGTG-3’,
D6Mit14 (129; 155 bp and JF1; 147 bp),
primer 1; 5’-ATGCAGAAACATGAGTGGGG-3’ and
primer 2; 5’-CACAAGGCCTGATGACCTCT-3’.
GFP DNA was PCR-amplified with the primer set,
GFPF; 5’-CGTAAACGGCCACAAGTTCA-3’ and
GFPR; 5’-CGCTTTACTTGTACAGCTCGT-3’.
For RT-PCR of Nanog and Stella, cDNA was synthesized with oligo dT primers
from DNaseI-treated RNA extracted from ESCs, pre-Cre and post-Cre hybrid cells.
RT-PCR products were amplified for 30 cycles with an annealing temperature of
60oC with the specific primers;
Nanog F1; 5’-GCGCATTTTAGCACCCCACA-3’ and
R1; 5’-GTTCTAAGTCCTAGGTTTGC-3’,
Stella F; 5’-ACAGACTGACTGCTAATTGG-3’ and
R; 5’-GGAAATTAGAACGTACATACTCC-3’.
G3pdh was amplified with primers,
2
F; 5’-TGAAGGTCGGTGTGAACGGATTTGGC-3’ and
R; 5’-CATGTAGGCCATGAGGTCCAC-3’.
For RT-PCR of Nurr1, Tyrosine hydroxylase (TH), Neurofilament-M (NF-M), Desmin,
Thrombomodulin (TM), Gata4, Insulin, - fetoprotein (-FP), Albumin and G3pdh
with RNA extracted from pre-Cre and post-Cre CEC6tg/tg hybrid cell-derived
teratomas and undifferentiated pre-Cre CEC6tg/tg hybrid cells, cDNA was amplified
for 35 cycles with an annealing temperature of 60oC with the specific primers;
Insulin I F; 5'-TAGTGACCAGCTATAATCAGA G-3’ and
R; 5'-ACGCCAAGGTCTGAAGGT CC-3’.
Nurr1, TH, NF-M, Desmin, TM, Gata4, -FP, Albumin and G3pdh-specific primers
were described previously 3,5.
Southern blot hybridization. To detect homologous recombination events genomic
DNA was extracted from GFP-positive clones, digested with EcoRV, separated
through 1.0% agarose and transferred to a Hybond N+ membrane by alkali blotting.
The membrane was pre-hybridized, then hybridized with pROSA26-5’ plasmid1 (Fig.
3a) labeled with 32P-dCTP using Megaprime DNA labeling system (Amersham)
overnight at 42oC followed by washes in 2xSSPE/0.1% SDS and 0.1xSSPE/0.1%
SDS at 65oC.
Western blot hybridization. Whole-cell extracts were separated by electrophoresis
on 12% SDS-polyacrylamide gels. The proteins were transferred onto a PVDF
membrane (Millipore). The membrane was pre-hybridized with 3% skimmed milk
(Difco) in PBS for 1 h at room temperature and then incubated with anti-Nanog
(1:1000 dilution) (Abcam) and anti-histone H3 (1:3000) (Abcam) antibodies
overnight at 4oC. Bands were detected with the ECL Western blotting detection kit
(Amersham).
Fluorescence immunocytochemistry. Cre-treated hybrid cells and teratomas
generated by subcutaneous injection of the hybrid cells to immunodeficient SCID
mice were fixed with 4% formaldehyde for 10 min and overnight, respectively.
Following pre-treatment with blocking solution (2% skimmed milk in PBS) for 1 h,
the cells were incubated with anti-Nanog polyclonal antibody (rabbit IgG) (1:500)
(Abcam) and anti-Oct4 monoclonal antibody (mouse IgG) (1:100) (Santa Cruz)
overnight at 4oC. After rinsing, cell samples were incubated with Alexa 546conjugated goat anti-rabbit IgG antibody (1:500) (Molecular Probes) and FITC-
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conjugated anti-mouse IgG (1:1000) (Zymed) for 1 h. Paraffin sections of 6-week–old
teratomas were stained with anti-Tublin (TuJ), anti-Desmin or anti-Albumin
antibody as described previously 3.
References
1.
Srinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP
and ECFP into the ROSA26 locus. BMC Dev Biol 1, 4 (2001).
2.
Tada, M., Tada, T., Lefebvre, L., Barton, S. C. & Surani, M. A. Embryonic
germ cells induce epigenetic reprogramming of somatic nucleus in hybrid
cells. EMBO J. 16, 6510-20 (1997).
3.
Tada, M. et al. Pluripotency of reprogrammed somatic genomes in embryonic
stem hybrid cells. Dev Dyn 227, 504-10 (2003).
4.
Lawrence, J. B., Singer, R. H. & Marselle, L. M. Highly localized tracks of
specific transcripts within interphase nuceli visualized by in situ hybridization.
Cell 57, 493-502 (1989).
5.
Hatano, S. et al. Pluripotential competence of cells associated with Nanog
activity. Mech Dev 122, 67-79 (2005).
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