Most BAC transgene copies are transcriptionally activated with gene sequences co-linear with BAC sequences. (A–C) RNA FISH (red) shows DHFR BAC and Hsp70 gene transcripts relative to GFP-lac repressor (green) after gene induction. DNA is stained by DAPI (gray, left column). (A and B) Log phase cells with DHFR BAC transgenes showing RNA FISH using the entire BAC as probe. (A) D10 cell line with 057L22-K-8.32-C29 DHFR BAC containing lac operator repeats inserted 64 kb upstream of the DHFR gene. (B) 110–5-E4 cell line with 560 M7-K-8.32_C5 DHFR BAC containing lac operator repeats inserted into the DHFR gene fifth intron. (C) Hsp70_5 cell line 5 (top) or 10 (bottom) mins after heat shock induction using Hsp70 transcribed region as RNA FISH probe. RNA FISH signals are adjacent or partially overlapping with GFP signals in all three cell lines. (D–F) DNA FISH reveals actual localization of transcribed gene sequences relative to overall BAC sequence. Co-localization is much more extensive than that observed with RNA FISH and co-linear with the linear BAC signals. (D and E) Pooled PCR probes covering most of DHFR gene (red), total BAC sequence (green) in D10 (D) or 110–5-E4 (E) cells. (F) Hsp70_5 cell line 30 min after heat shock. Approximately 2 kb Hsp70 gene probe (red) localized used tyramide amplification method, relative to directly labeled BAC probe (green). Bar, 2 µm.