Figure 1

Physical map of the telomeric end of the imprinted domain in mouse distal chromosome 7 in relation to the position of the BAC transgene and expression analysis of transgenic lines. A) Top line shows a genomic map of the IC2 region on distal mouse chromosome 7. Hatched region marks the KvDMR1 region that is methylated only on the maternal allele. The black arrowhead marks the position of the IC2 control region for the domain. Arrows indicate direction of transcription. Below is the map of the 85 kb Cdkn1c transgene (BAC144D14). Filled boxes show the positions of the intact genes. In addition to Phlda2, Slc22a18 and Cdkn1c, the transgene includes the 3' UTR of Napl14 (white box) but not the 5' UTR. The modified BAC used to generate line 10–15 has a β-galactosidase-neomycin (β-geo) fusion gene inserted into Cdkn1c indicated by the white arrowhead and black filled box. B) Localisation of the Cdkn1c protein expressed from the BAC transgene. Sagittal section of E12.5 wild type and Cdkn1c-null/BAC transgene (KO/Tg) embryos stained with a Cdkn1c-specific antibody. Endogenous Cdkn1c is most strongly expressed in skeletal and cardiac muscle, cartilage and the developing pituitary with lower expression in neuronal tissues. The transgenic expression is predominantly only neural, indicated by the black arrows, and in a subset of cells in the pituitary, kidney, lung and adrenal gland. C) Quantitative RT-PCR data for Cdkn1c at E13.5 for 5D3 (single copy line) and 5A4 (two copy line) transgenic embryos and 10–15 (Cdkn1c-βgeo) at E12.5. Cdkn1c levels were normalised against three reference genes: Gapdh, Actin and 18S RNA. Cdkn1c was at 1.36-fold wild type levels in the single copy line and 1.75-fold endogenous in the two copy line. Cdkn1c levels were not significantly raised in the control line, 10–15 (Cdkn1c-βgeo). D) Quantitative RT-PCR data for Cdkn1c at E12.5 for 5D3 (single copy line) showing the difference in expression between head, where the transgene is predominantly expressed, and the body, where the transgene is only expressed in a small subset of tissues. This is consistent with copy number dependent expression of Cdkn1c from the transgene in a subset of tissues.