Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette

Abstract

Human artificial chromosome (HAC)-based vectors represent an alternative technology for gene delivery and expression with a potential to overcome the problems caused by virus-based vectors. The recently developed alphoidtetO-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of the HAC kinetochore via binding of chromatin modifiers, tTA or tTS, to its centromeric tetO sequences. This provides a unique control for phenotypes induced by genes loaded into the HAC. The alphoidtetO-HAC elimination is highly efficient when a high level of chromatin modifiers as tetR fusion proteins is achieved following transfection of cells by a retrovirus vector. However, such vectors are potentially mutagenic and might want to be avoided under some circumstances. 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 tTAVP64 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 alphoidtetO-HAC for routine gene function studies, we constructed a new TAR-BRV- tTAVP64 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 alphoidtetO-HAC in hamster CHO cells from where the HAC may be MMCT-transferred to the recipient human cells.

Moving toward a higher efficiency of microcell-mediated chromosome transfer

“… 32 , 33 , 40 In hamster, mouse, and human cells, this HAC is stably maintained as an autonomous chromosome ( Figure 2b ). 33 , 34 , 38 , 39 , 41–43 We transferred the alphoid tetO -HAC from donor hamster CHO cells to different recipient cell types ( Figure 2c ) to gauge if a modification to the MMCT protocol was an improvement. In our experiments, we compared the material derived from six flasks.…”

Stable maintenance of de novo assembled human artificial chromosomes in embryonic stem cells and their differentiated progeny in mice

“…The construct containing the alphoid-tetO array p3.5 20 was labeled with Alexa546, using the ULYSIS Ò Nucleic Acid Labeling Kit (Life Technologies) as per manufacturer recommendations. This labeled probe was denatured at 75 C for 5 min, then incubated in hybridization solution containing 50% formamide at 37 C for 30-60 min, and finally incubated overnight with denatured and dehydrated metaphase spreads under the cover slip in a humidified chamber.…”

Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

“…HACs made using synthetic centromeric DNA have been extensively characterized and improved over the last decade [2][3][4][5] . They now represent an important tool to study epigenetic regulation of centromere structure and function 2,[6][7][8][9][10] , to study full-length gene functions in mutant animal or human cells 5,[11][12][13][14][15][16] , to measure chromosome instability (CIN) and identify new targets for cancer therapy [17][18][19] . Also, synthetic HACs do not interfere with embryogenesis in mouse, making them a promising tool for future gene therapeutic studies 15 .…”

“…Synthetic HACs were originally designed using a "bottom up" approach to contain only pre-defined DNA arrays 1,20,21 . Their design allows the HAC centromeres to be easily modified and inactivated/removed by targeting with chimeric proteins specifically directed to the synthetic DNA 2,4,11,14 . However, they also present a number of challenges that must be overcome to enable them to be exploited fully.…”