DEV032060 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. QPCR analysis of gene expression in differentiating ES cells. (A) Expression of marker genes in D3 and CCE ES cells differentiated via embryoid body (EB) formation for 14 days. For Oct4, a marker of undifferentiated cells, the value on day 0 was set to 100, whereas for Fgf5 and Gata4, which are markers of differentiated cells, the value on day 14 was set to 100. The mean of three (CCE) and two (D3) biological replicates is given with s.d. (B) Left panel shows Igf2r (grey squares) and Airn (black diamonds) expression, whereas right panel shows Slc22a2 (black diamonds) and Slc22a3 (grey squares) expression, in D3 ES cells during 5 days of RA differentiation. The value on day 5 was set to 100. The mean of two biological replicates is given with s.d. The Slc22a2 day-5 Ct value of 34 indicates very low expression, whereas that for Slc22a3 of 27 indicates higher expression in differentiated ES cells. (C) Left panel shows Igf2r (grey squares) and Airn (black diamonds) expression in CCE ES cells differentiated as EBs for 14 days. Right panel shows the same analysis in D3 ES cells differentiated as EBs for 14 days. The value on day 14 was set to 100. The mean of three (CCE) and two (D3) biological replicates is given with s.d. (D) Expression of Slc22a2 (black diamonds) and Slc22a3 (grey squares) in CCE (left panel) and D3 (right panel) cells differentiated as in C. (E) Mean Ct values of Airn, Igf2r, Slc22a2, Slc22a3 and cyclophilin A in ES cells differentiated for 5 days in RA (left panel) or 14 days as EBs (right panel). Low-level expression is indicated by high Ct values. Slc22a2 is inconsistently detected in undifferentiated ES cells (Ct values >35).

• Supplemental Figure S2 -

  Fig. S2. Expression of other imprinted genes during ES cell differentiation. (A) Expression of Kcnq1 (black squares), Ckdn1c (grey squares) and the ncRNA Kcnq1ot1 (black diamonds) in CCE (left) and D3 (right) ES cells differentiated in RA for 5 days (top panels) or differentiated as EBs for 14 days (lower panels). The highest value obtained for each differentiation series was set to 100. The mean of three biological replicates is depicted with the s.d. Kcnq1ot1 and Cdkn1c both showed an increase over the 5-day differentiation period that was delayed by 1 day compared with Igf2r and Airn (see Fig. S1), Kcnq1, which is normally expressed only in heart, showed anomalous behaviour. Primers and Taqman probes were as follows. Kcnq1 QPCR: F-TCACCGTCTTCCTCATTGTTCT, TM-TCTGCCTCATCTTCAGTGTCCTGTCCACTA, R-GGCCAGAGCGGCATACTG; Kcnq1ot1 QPCR: F-GCCCAAACCTTAGTCCTCCAT, TM-TTGTTGTTCTTGTTGGTCACATTCCCTTTTCT, R-GAAAGCACTCCTCCCCATTT; Cdkn1c QPCR: F-TGCACCCGGGACTGCT, TM-CCAATGCGAACGACTTCTTCGCCA, R-ACGCCTTGTTCTCCTGCG. (B) Expression pattern of Igf2 (black diamonds) and the ncRNA H19 (grey squares) in CCE (left) and D3 (right) ES cells differentiated in RA for 5 days (top panels) or differentiated as EBs for 14 days (lower panels). Both Igf2 and H19 showed a similar expression profile as seen for Igf2r and Airn (see Fig. S1). Details as A. Primers and Taqman probes were as follows. H19 QPCR: F-CATCCAGCCTTCTTGAACACC, TM-GGGCTGGCGCCTTGTCGTAG, R-GGGAAAAGTGAAAGAACA; Igf2 QPCR, F-GGCTTCTACTTCAGCAGGCCT, TM-AAGCCGTGCCAACCGTCGCA, R-ACTCTTCCACGATGCC.

• Supplemental Figure S3 -

  Fig. S3. Persistent low-level paternal Igf2r expression in mouse embryo fibroblast cells. (A) Non-quantitative RT-PCR using 30 cycles showing Igf2r and Airn expression from the maternal allele (+/Thp, upper panel) and from the paternal allele (Thp/+, lower panel) in 13.5 dpc MEFs. Thp is a 6 Mbp deletion of mouse chromosome 17 that includes the whole Igf2r imprinted cluster (Barlow et al., 1991). Using this standard PCR cycle number, Igf2r expression is seen in MEFs that contain only the maternal allele (+/Thp) and also as a weaker band in cells that contain only the paternal allele (Thp/+). By contrast, Airn expression is only seen in MEFs containing a paternal allele (Thp/+). Gapdh does not lie in the Thp deletion and was used as a loading control. In +/Thp MEFs, the maternal Igf2r promoter is unmethylated and the Airn promoter is methylated; the opposite pattern is seen in Thp/+ MEFs, which have a methylated paternal Igf2r promoter and an unmethylated paternal Airn promoter (Regha et al., 2007). (B) Northern blot that is overexposed to show low-level paternal Igf2r expression (Thp/+) compared with very high-level maternal Igf2r expression (+/Thp) using probe HX. The same exposure time shows that Airn is expressed from the paternal allele (Thp/+) but is not detected from the maternal allele (+/Thp) using probe Airp105. The left-hand lanes show the same samples as analyzed in A, and the right-hand lanes show a biological replicate. All lanes contained equal amounts of rRNA as assessed by Methylene Blue staining (data not shown). However, degradation of the 108 kb Airn ncRNA has clearly occurred in lane 4, whereas the 8.7 kb Igf2r transcript does not appear to be degraded in the same sample. (C) QPCR of the same RNA samples used in A and B quantifying Igf2r (assay Igf2rex4/5, upper panel) and Airn (lower panel) expression from the maternal (+/Thp) and paternal (Thp/+) chromosomes. The ratio of maternal to paternal Igf2r expression is 80:1, whereas the ratio of maternal to paternal Airn expression is 1:1000. Error bars show the s.d. of three replicates.

  Reference

  Barlow, D. P., Stoger, R., Herrmann, B. G., Saito, K. and Schweifer, N. (1991). The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tme locus. Nature 349, 84-87.