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. 2007 Jan 24;2(1):e175. doi: 10.1371/journal.pone.0000175

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Yamagishi et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Analysis of the insulator fragment using targeted transgene experiments. (A) Scheme of experimental design. The Hoxd9/lacZ transgene, along with an insulator candidate fragment, was inserted into the region just downstream of Evx2 by a gene-targeting technique using ES cells. The resulting ES cells were injected into blastocysts to establish transgenic mice. (B) The XhoI-NsiI (XNs) fragment (red) was separated into three fragments—BamHI-BamHI (BB), EcoRI-BglII (EBg), and XhoI-BamHI (XB)—each of which were translocated together with the Hoxd9/lacZ reporter transgene. (C–H) LacZ-stained 11 dpc embryos. XNs (C) and XB (F) blocked lacZ gene expression in brain, while BB (D) and EBg (E) failed to do so. Based on these results, we divided the XB fragment into two fragments—XhoI-EcoRI (XE) and EcoRI-EcoRI (EE)—which we used to make targeted transgenic mice having a similar configuration to that shown in panel (A). Embryos harboring XE (G) and EE (H) displayed lacZ gene expression in brain but did not display expression patterns indicative of insulation activity.