SUPPLEMENTARY INFORMATION

Transcription

1 doi:.38/nature08267 Figure S: Karyotype analysis of ips cell line One hundred individual chromosomal spreads were counted for each of the ips samples. Shown here is an spread at passage 5, predominantly diploid. More than 75% of the cells showed normal mouse karyotype of 40 chromosomes. 2

2 Oct3/4 Nanog Figure S2. Methylation analysis of and Nanog promoter regions. Genomic DNA from ips cell lines (, IP36D-3, ) at passage as well as from MEFs and ES cells were isolated and bisulfite treated. and Nanog promoter regions were amplified with nested primers (Supplementary Table S2). Ten randomly selected clones were sequenced and analyzed. Open and filled circles represents unmethylated and methylated CpG dinucleotides, respectively. The three ips cell lines are very different in methylation patterns compared to the parental -GFP MEFs, but are very similar to those from normal ES cells, reflecting the epigenetic remodeling that occurred in concert with the reprogramming events. 2

3 Sox GFP-ES IP36D-3 IP4D-6 0 -GFP-MEF D6 transfected MEF 0.0 -GFP-ES IP36D-3 IP4D-6 0 -GFP-MEF D6 transfected MEF Klf4 c-myc GFP-ES IP36D-3 IP4D-6 0 -GFP-MEF D6 transfected MEF 0.0 -GFP-ES IP36D-3 IP4D-6 0 -GFP-MEF D6 transfected MEF Figure S3. Quantitative RT-PCR to detect transgene expression Real-time RT-PCR was used to detect total, endogenous and transgenic expression of the four factors, Sox2, c-myc and Klf4 in -GFP ES (an ES line with integrated -GFP expression), IP36D3, IP20D3, IP4D, IP4D-6, 0, -GFP MEF and a positive control, day 6 transfected MEF. The assays included primers specifically designed to discriminate endogenous (black bars) from transgenic (open bars) RNA expression, and total expression is shown with grey bars. Relative expression levels (Yaxis) of these genes in each sample were first normalized to their endogenous GAPDH level, and then displayed as their relative ratio to expression of these genes in -GFP ES. Primer sequences are listed in Supplementary Table S3. 3

4 c-myc Figure S4. Validation of genomic integration of transgenes by Southern blot analysis Total genomic DNA was extracted from -GFP ES, -GFP MEF, IP36D3, IP20D3, IP4D and the corresponding tetraploid mice. DNA digested with Bam H and BglII were hybridized with and c-myc cdna probes, respectively. Arrowheads indicate endogenous or c-myc bands found in all the samples. Asterisk indicates extra bands in the ips samples corresponding to viral integration into the genome. Note that the different ips lines have different integration patterns, but the three ips cell lines and their corresponding 4N-comp animals showed the same patterns. 4

5 Figure S5: Identification of integration patterns in ips cell lines and 4N-comp mice by reverse PCR. Genomic DNA was extracted from the ips cell lines, IP4D-6, 0 as well as the tails of IP4D 4N-comp, IP4D6 4N-comp, IP4D 4N-comp and a normal C57/DBA (B6D2F) mice. The integration patterns of these samples were identified by reverse PCR procedures of neighboring genomic regions by nested primers specific for. Bands above the red line or not present in the C57/DBA control were considered positive patterns for integration. Note that the three 4N-comp mice displayed different integration patterns to each other, but similar to the ips lines (IP4D, P4D6 and IP4D) they originated from. 5

6 2 8 Relative expression Pou5f Sox2 Nanog Klf4 Myc Lin28 Fgf4 Gdf3 Catnb Cfc Dppa5 Slc2a3 Zfp296 MEF CL 0-2 Selected genes Figure S6. Expression profiles of selected pluripotent marker genes from microarray analysis. 6

7 Table S. Summary of ips lines derived to date. Genetic background B6D2F (GFP + ) C57x29S2 (GFP - ) NT = Not Tested Experiment number Exp4D- Exp4D-2 Exp4D-5 Exp20D- Exp20D-2 Exp20D- Exp36D-2 Exp4D-C ips cell lines Teratoma formation 2N chimera mice (Number) % Chimerism Germline transmission (Number) 4N complementation ability Developed to No. of alive 4Ncomp animals YES YES(6) YES (4) D IP4D-2 NT YES <D.5 IP4D-3 NT YES (4) -90 <.5 IP4D-4 NT NT <.5 IP4D-5 NT NT NT IP4D-6 NT YES () YES (3) D9.5 IP20D- YES YES (5) 5-80 D3.5 IP20D-2 YES YES () 30 NT YES YES (4) YES () D5.5 IP20D-4 NT NT NT IP20D-5 NT NT NT IP20D-6 NT NT NT IP20D-7 NT NT NT IP20D-8 NT NT NT IP20D-9 NT NT NT IP20D- NT NT NT IP20D- NT NT NT IP20D-2 NT NT NT NT NT NT IP20D-4 NT NT NT IP20D-5 YES NT NT IP20D-6 YES NT NT IP20D-7 YES NT NT IP20D-8 YES NT NT IP20D-9 YES YES (4) -70 YES () D3.5 IP20D-20 YES NT NT IP20D-2 YES NT NT IP20D-22 NT NT NT IP20D-23 NT NT NT IP36D-2 NT NT NT IP36D-3 YES YES (6) 5-70 D.5 0 NT YES(8) -90 YES() D NT NT D NT NT NT 04 NT NT NT 05 NT NT NT 06 NT NT NT 7

8 Table S2. PCR primer sequences for methylation studies Gene Accession number Primer sequence Nested PCR NM_03633 F: GAGGATTGGAGGTGTAATGGTTGTT R: CTACTAACCCATCACCCCCACCTA st Round F: CAAGCTTTGGGTTGAAATATTGGGTTTATTT R: CGGATCCCTAAAACCAAATATCCAACCATA 2 nd Round Nanog NM_02806 F: AAGTATGGATTAATTTATTAAGGTAGTT R: AAAAAACCCACACTCATATCAATATA st Round F: AAGTATGGATTAATTTATTAAGGTAGTT R: CAACCAAATCAACCTATCTAAAAA 2 nd Round 8

9 Table S3. Primer sequence for real-time RT-PCR Target gene expression level Endogenous Exogenous / transgene Name endo- endo- Sox2 endo-myc endo-klf4 ex- ex-sox2 ex-c-myc ex-klf4 Primer sequence F: TCTTTCCACCAGGCCCCCGGCTC R: TGCGGGCGGACATGGGGAGATCC F: TAGAGCTAGACTCCGGGCGATGA R: TTGCCTTAAACAAGACCACGAAA F: TGACCTAACTCGAGGAGGAGCTGGAATC R: AGTTTGAGGCAGTTAAAATTATGGCTGAAGC F: CCATCGGACCTACTTATCTGC R: AAAACCTCAAACCAAAACCC F: CCCAGTGTGGTGGTACGGGAAATC R: AGTTGCTTTCCACTCGTGCT F: CCCAGTGTGGTGGTACGGGAAATC R: TCTCGGTCTCGGACAAAAGT F: CCCAGTGTGGTGGTACGGGAAATC R: GCTCGCTCTGCTGTTGCTGGTGATAG F: CCCAGTGTGGTGGTACGGGAAATC R: GTCGTTGAACTCCTCGGTCT T- F: GGCTTCAGACTTCGCCTCC R: AACCTGAGGTCCACAGTATGC Total T-Sox2 T-c-Myc F: GCGGAGTGGAAACTTTTGTCC R: CGGGAAGCGTGTACTTATCCTT F: ATGCCCCTCAACGTGAACTTC R: CGCAACATAGGATGGAGAGCA T-Klf4 F: GTGCCCCGACTAACCGTTG R: GTCGTTGAACTCCTCGGTCT 9