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

Figure 7.

Figure 7.

Use of the retrofitting vector BRV-tTS-EYFP to regulate expression of a gene loaded into the HAC by doxycycline. (a) Diagram of the retrofitting vector BRV-tTS-EYFP. The vector contains two targeting sequences (hooks) that are released by BsiWI endonuclease digestion. In vivo recombination in yeast between the vector and a YAC containing the gene of interest (isolated by TAR cloning) leads to assembly of a circular YAC/BAC molecule containing the gene of interest plus the tTS cassette all of which can be inserted into the HAC by Cre-lox recombination. (b) Inactivation of a gene expression by modulating the epigenetic status of the underlying HAC kinetochore chromatin. The BRV-tTS-EYFP vector along with a gene of interest is inserted into the single loxP site of the alphoidtetO-HAC. In the presence of doxycycline, the tTS cannot bind to tetO sequences in the HAC, and this region of the HAC is transcriptionally active. Removal of doxycycline leads to tTS-induced heterochromatinization of this region followed by gene silencing. (c) Fluorescence images of HT1080 cells with the HAC carrying the BRV-tTS-EYFP vector cultured in dox+ and dox− media followed by 10 days of culture without HAC selection (in HAT−, BS− medium). (d) Relative mean EYFP fluorescence of cells carrying the BRV-tTS-EYFP vector (FACS). (e) Mitotic stability of the HAC measured by FISH after growing the cells in the presence or absence of doxycycline. Open bars correspond to the frequency of HAC loss when the cells were grown in the presence of doxycycline. Black bars correspond to the frequency of HAC loss when doxycycline was excluded from the medium.