Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette.
Bottom Line: Here, we describe a novel system that allows verification of phenotypic changes attributed to expression of genes from the HAC without a transfection step.We demonstrated that a single copy of tTA(VP64) carrying four tandem repeats of the VP16 domain constitutively expressed from the HAC is capable to generate chromatin changes in the HAC kinetochore that are not compatible with its function.To adopt the alphoid(tetO)-HAC for routine gene function studies, we constructed a new TAR-BRV- tTA(VP64) cloning vector that allows a selective isolation of a gene of interest from genomic DNA in yeast followed by its direct transfer to bacterial cells and subsequent loading into the loxP site of the alphoid(tetO)-HAC in hamster CHO cells from where the HAC may be MMCT-transferred to the recipient human cells.
Affiliation: Developmental Therapeutic Branch, National Cancer Institute, Bethesda, MD 20892, USA.Show MeSH
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Mentions: In our first experiments, the transcription of satellite DNA in alphoidtetO array was found to be significantly increased (3-fold) after 3 days of growth in the medium lacking doxycycline in clone #1 [t-test, t(4) = 12.3554, P = 0.0002]. In clone #2, there was a significant increase (6-fold) after 2 days [t-test, t(4) = 53.9038, P < 0.0001] that further increased by 9-fold after 3 days [t-test, t(4) = 146.6633, P < 0.0001] (Figure 5a and b). These experiments demonstrate that targeting of HAC kinetochore chromatin by a single expressed copy of the tTAVP64 leads to a significant increase of transcription of non-coding alphoidtetO array after 3 days of cell growth (Figure 5a and b). These results correlate with ChIP data on enrichment of H3K4me3, a marker for the 5' end of actively transcribed genes (28,29,50,51), on alphoidtetO array in the HAC. In two independent clones, the HAC chromatin following tTAVP64 expression failed to reveal significant structural differences after one day of culture in dox− medium but was significantly (3–4 fold) increased on the alphoidtetO array of the HAC after 3 days of culture in the medium lacking doxycycline [t(4) = 31.2421, P < 0.0001 for clone #1 and t(4) = 49.8532, P < 0.0001 for clone #2] (Figure 5c and d). Thus, elevation of satellite transcription of the alphoidtetO array after 3 days was accompanied by enrichment in H3K4me3 chromatin. No significant difference in the level of CENP-A on the alphoidtetO array was observed when cells were grown in the presence or absence of doxycycline after 3 days of culture [t(4) = 2.1625, P = 0.0966] (Figure 5e). However, a longer culturing of the cells in dox− medium (4, 8 and 12 days) resulted in a dramatic decrease of the level of CENP-A (Figure 5e) followed by kinetochore inactivation and HAC loss (Figure 3) that is in agreement with our previous results on inactivation of the HAC kinetochore by tTA using viral vectors (29,31,32).
Affiliation: Developmental Therapeutic Branch, National Cancer Institute, Bethesda, MD 20892, USA.