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. 2012 Oct 11;40(22):11477–11489. doi: 10.1093/nar/gks926

Figure 1.

Figure 1.

Linearization of the C-HAC. (A) Plasmids pSP73-TEL08-5′HPRT1-loxP, pSP73-TEL08-BLAS-loxP and the CRE-expression plasmid pOG231 were co-transfected into the hprt−/− E10B1 hamster cell line carrying the C-HAC. CRE/loxP-induced recombination resulted in a linearized HAC with a blasticidin selection marker and reconstituted the HPRT1 minigene, which was excised from the HAC. The orientation of the loxP sites in the constructs pSP73-TEL08-5′HPRT1-loxP and pSP73-TEL08-BLAS-loxP is designed in such a way that it does not allow any other recombination. (B–D) FISH analysis on E10B1 metaphase spreads with a PNA telomeric probe (green) counterstained with DAPI (blue). HACs displayed the expected four telomeric signals. Strong telomere signals were present on the hamster chromosomes due to the presence of interstitial telomeric repeats in the CHL cell line. (E) Rehybridization of the metaphases with an alphoid-20 DNA probe (red). (F–H) FISH with hamster Cot1 DNA (green) and alphoid-20 DNA probes (red). (C and G) Detailed image showing the PNA telomeric signal/hamster Cot1 signal in grayscale. (D and H) Corresponding color-inverted, grayscale DAPI images. Arrowheads point to the HACs.