Skip to main content
. 2013 Apr 3;41(10):e107. doi: 10.1093/nar/gkt205

Figure 6.

Figure 6.

Doxycycline regulated expression of the tTS-EYFP transgene loaded into the HAC. (a) A diagram illustrating time-course analysis of tTS-EYFP expression in the cells grown in different media. Samples 1–11 correspond to cultures used for FACS analysis. The cultures marked by red stars were analyzed by ChIP (see below). (b) Relative mean EYFP fluorescence determined by FACS of cells carrying the tTS-EYFP cassette (construct #4) grown in different media. Sample 1 corresponds to control HT1080 cells without the EYFP transgene (fluorescence background). Sample 2 corresponds to the cells with the HAC carrying the tTS-EYFP cassette grown in HAT and dox+ medium for 30 days. Sample 3 corresponds to the cells grown for 22 days in dox+ medium without selection. Sample 4 corresponds to the cells grown for 22 days without doxycycline. Sample 5 corresponds to the cells grown for a month in dox− medium with BS selection. Sample 6 corresponds to the cells grown in bsr−dox− medium. Samples 7, 8 and 9 correspond to the cells grown in dox+ BS+ medium for 24 h, 6 and 12 days, correspondingly. Samples 10 and 11 correspond to the cells treated either TSA or SAHA in dox+ medium. Error bars, SD (n = 3). (c) Transcription of Hygro, TK, EGFP and HPRT genes from the HAC. The level of the transgene transcripts from cells cultured in dox+ or dox− medium was analyzed by RT–PCR. The housekeeping gene, BRCA1, was used as an internal control. Lane 1 (dox−) and lane 2 (dox+) correspond to transcripts for BRCA1. Lane 3 (dox−) and lane 4 (dox+) correspond to transcripts for Hygro. Lane 5 (dox−) and lane 7 (dox+) correspond to transcripts for TK. Lane 6 (dox−) and lane 8 (dox+) correspond to transcripts for HPRT. Lane 9 (dox−) and lane 10 (dox+) correspond to transcripts for EGFP. Lane M- GeneRuler™ 1-kb DNA ladder. (d) ChIP analysis of H3K4me3 chromatin in the transgene cassette of the HAC in the presence and absence of doxycycline. The cell samples used for ChIP correspond to samples 2 and 6 in Figure 6a and b. Enrichment is shown relative to the 5S rRNA control locus. Satellite 2 sequence, Sat2, corresponding endogenous pericentromeric repeats was included as a negative control. (e) ChIP analysis of CENP-A chromatin in the HAC in the presence and absence of doxycycline. Enrichment is shown relative to the chromosome 21 centromere.