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
Mentions: In this study, two transcriptional transactivator tTA -containing vectors, tetR-VP64-IRES-DsRed2 (Figure 1a; Supplementary Figure S1) and tetR*-VP16-IRES-DsRed2 (Supplementary Figure S2a) were constructed (see Materials and Methods for details) and then inserted into the loxP site of the alphoidtetO-HAC in HPRT-deficient human HT1080 cells (Figure 1b) in order to determine the consequences of a single copy tTA expression on HAC stability. The tetR-VP64-IRES-DsRed2 vector contains the tTAVP64 sequence (four tandem repeats of the minimal VP16 domain DALDDFDLDML fused with TetR), the color marker DsRed2 and the 3'-end HPRT sequence fragment. The tTAVP64 and DsRed2 sequences are transcribed from the same CAG promoter but are separated by an internal ribosome entry site (IRES) sequence (Figure 1a). Therefore, while the tetR-VP64-IRES-DsRed2 vector encodes a single tTAVP64/DsRed2 transcript it is translated into two proteins: tTAVP64 (which binds to the multiple tetO-sequences in the alphoid array of the HAC) and DsRed2 (which is translated from IRES at a lower efficiency). The cHS4 insulator flanks the whole cassette. The tetR*-VP16-IRES-DsRed2 vector is identical to tetR-VP64-IRES-DsRed2 but contains only one copy of the VP16 domain. The tTAVP64 or tTAVP16 binding to tetO sequences is negatively regulated by doxycycline.
Affiliation: Developmental Therapeutic Branch, National Cancer Institute, Bethesda, MD 20892, USA.