Edinburgh Research Explorer

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

Research output: Contribution to journalArticlepeer-review

  • Artem V. Kononenko
  • Nicholas C O Lee
  • Mikhail Liskovykh
  • Hiroshi Masumoto
  • William C. Earnshaw
  • Vladimir Larionov
  • Natalay Kouprina

Related Edinburgh Organisations

Open Access permissions

Open

Documents

  • Download as Adobe PDF

    Rights statement: This is a pre-copyedited, author-produced version of an article accepted for publication in Nucleic Acids Research following peer review. The version of record Artem V. Kononenko, Nicholas C.O. Lee, Mikhail Liskovykh, Hiroshi Masumoto, William C. Earnshaw, Vladimir Larionov, Natalay Kouprina, Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette, Nucleic Acids Research, Volume 43, Issue 9, 19 May 2015, Page e57, https://doi.org/10.1093/nar/gkv124.

    Final published version, 1.59 MB, PDF document

Original languageEnglish
Article numbere57
JournalNucleic Acids Research
Volume43
Issue number9
DOIs
Publication statusPublished - 20 May 2015

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 alphoid(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(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(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.

    Research areas

  • DNA-Mediated Cell Transformation and Nucleic Acids Transfer, Recombinant DNA expression

Download statistics

No data available

ID: 30212646