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: Next, we examined the kinetics of HAC loss in one of HT1080 clones after 3, 4, 7 and 14 days of culture in the medium with and without doxycycline. As seen, the fraction of cells carrying the HAC decreased dramatically in doxâˆ’ medium after 4 days of culture while remaining relatively stable in dox+ medium (Figure 3).
Affiliation: Developmental Therapeutic Branch, National Cancer Institute, Bethesda, MD 20892, USA.