Human artificial chromosome with a conditional centromere for gene delivery and gene expression

Area of Science:

• Genetics
• Molecular Biology
• Epigenetics

Background:

• Human artificial chromosomes (HACs) are episomal, single-copy vectors offering controlled gene expression and avoiding random mutagenesis.
• A conditional centromere was previously developed for HACs, enabling inactivation and loss of the chromosome via tetracycline-regulated systems.

Purpose of the Study:

• To adapt the conditional centromere (tet-O) HAC vector for efficient gene delivery and expression in human cells.
• To demonstrate the stable integration and expression of a transgene within the tet-O HAC system.

Main Methods:

• Homologous recombination in DT40 cells to insert a loxP cassette into the tet-O HAC.
• Microcell-mediated chromosome transfer (MMCT) to introduce the modified HAC into Chinese hamster ovary cells.
• EGFP transgene incorporation and expression analysis in human cells.

Main Results:

• Efficient and accurate incorporation of an EGFP transgene into the tet-O HAC vector.
• Stable expression of the EGFP transgene in human cells following MMCT.
• Demonstration of transgene elimination via HAC loss upon centromere inactivation.

Conclusions:

• The tet-O HAC vector system is suitable for gene delivery and expression in human cells.
• This HAC vector offers controllable gene expression and elimination, with potential applications in gene therapy and research.