Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTA super(VP64) expression cassette

• Published in: Nucleic acids research 2015-05, Vol.43 (9), p.e57-e57
• Main Authors: Kononenko, Artem V, Lee, Nicholas CO, Liskovykh, Mikhail, Masumoto, Hiroshi, Earnshaw, William C, Larionov, Vladimir, Kouprina, Natalay
• Format: Article
• Language: English
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkv124

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 alphoid super(tetO)-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 alphoid super(tetO)-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 tTA super(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 super(tetO)-HAC for routine gene function studies, we constructed a new TAR-BRV- tTA super(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 super(tetO)-HAC in hamster CHO cells from where the HAC may be MMCT-transferred to the recipient human cells.