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. 2009 Apr 6;185(1):87–100. doi: 10.1083/jcb.200809196

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© 2009 Hu et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 1. — Generation of stable cell lines with multi-copy, inducible BAC transgenes. (A) Transposable element with 19-bp Tn5 mosaic ends (red) used to insert 256mer lac operator repeat (green) and selectable marker (yellow) into BACs. (B) BACs used in this study (top to bottom): DHFR 057L22-K-8.32-C-29, DHFR 560M7-K-8.32-C-5, Hsp70 92G8-K/N-8.32-C-2, and Metallothionein 134B2-K/N-8.32-C-8. Inducible genes whose expression we monitored (red), other genes (gray), transposable element (green), and vector backbone (blue) are shown in linear form. (C and D) DNA counterstaining is with DAPI (blue). (C) Transgenes localized in mitotic spreads of the DHFR D10 cell line. Left two panels: lac repressor staining (green); right two panels: FISH (BAC probe, red). (D) Fiber FISH (red) provides copy number estimates with lac operator probe (left) and shows contiguous, tandem BAC insertions with BAC probe (right) for the Hsp70_5 cell line. Bar (C and D), 2 µm.