Supplementary information
Figure Legends
Supplementary Figure 1. Expression stability of the HPRT-HAC in vitro
5 x 102 cells of 3 independent D98OR (HPRT-HAC) clones were plated in condition
with HT or HAT media and colonies were counted on day 10 after May-Giemsa staining.
(a) May-Giemsa staining in 3 independent D98OR (HPRT-HAC) clones at 30PDL. (b)
The expression stability of the HPRT-HAC in D98OR after a long term-culture.
Supplementary Figure 2. PFGE and Southern blotting of the HAC
Southern blots of PFGE size-separated BamHI restriction fragments, hybridized with a
human chromosome 21-derived -satellite probe detected the integrity of the HACs
during transfer from the donor to recipient cells. (a) -satellite fingerprint of the
HPRT-HAC in the donor CHO cells and recipient D98OR cells. It was noted that the
-satellite probe was hybridized to alphoid array on chromsome 13, in addition to
chromosome 21. Because of RFLP, not all but some alphoid arrays on the HAC
(arrowheads) could be distinguished from those on the endogenous chromosome 21 in
D98OR background. Restriction fragments were compared in three independent D98OR
clones carrying the HPRT-HAC, at early (p1) and late (p30) passage. (b) -satellite
fingerprint of the p53-HAC in the donor CHO cells and recipient mouse ES cells or p53
deficient mGS cells. Mouse and hamster DNA not containing the HAC was used as
controls.
Supplementary Figure 3. In vivo and in vitro stability of HAC in mouse.
(a,d) FISH analyses for ES (HAC) cells and spleen cells of chimeric mice containing the
HAC. Digoxigenin-labeled human COT-1 DNA (red) was used to detect the HAC in
mouse ES cells (a) and chimeric spleen cells (d). Chromosomal DNA was
counterstained with DAPI. The inset shows an enlarged image of the HAC. (b) Mitotic
stability of the HAC in mouse ES cells. Mitotic stability was determined by FISH using
human COT-1 DNA after a long period of culture without selection. (c)
Immunochemical staining of ES (HAC) cells on day 7 after induction via the SDIA
method using Tuj-1 antibody.
Supplementary Figure 4. FISH analyses for mGS (p53-HAC) cells and real time
RT-PCR analyses for p53-/-mGS (p53-HAC) cells after X-ray irradiation.
(a) FISH analyses of normal mGS cells containing the p53-HAC. Digoxigenin-labeled
human COT-1 DNA (red) and biotin-labeled RP6-6J15 (green) were used to detect the
HAC and the p53 gene on the HAC, respectively, in mGS cells. The inset shows an
enlarged image of the p53-HAC. (b) Real-time RT-PCR analyses using mouse p21
specific primer for the p53-/-mGS (p53-HAC) cells after X-ray irradiation (8 Gy). The
irradiated cells were collected after 0, 1, 2, 4, and 6 hours and used for real time
RT-PCR.  -actin was used as an internal control.

Supplementary Figure 5. In vitro differentiation of p53-/-mGS (p53-HAC) cells.
(a) The differentiation of p53-/-mGS (p53-HAC) cells into dopaminergic neurons via
the SDIA method. Bright field and fluorescence field images after immunochemical
staining using Tuj-1 antibody are shown in the upper and lower panels, respectively. (b)
Proportion of Tuj-1-positive p53-/-mGS (p53-HAC) cells after differentiation.
Tuj-1-positive colonies were counted on day 7 after induction. ES and p53-/-mGS cells
were used as positive and negative controls, respectively. Each measurement represents
the mean ± standard deviation of three independent experiments. A statistical analysis
was performed using the two-tailed Student’s t-test.