Supplementary Figure 1. Expression Stability of the HPRT-HAC in Vitro

Supplementary information

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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 neuronsvia 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.