Stable gene expression from a mammalian artificial chromosome

Area of Science:

• Genetics
• Molecular Biology
• Cell Biology

Background:

• Mammalian artificial chromosomes (MACs) are valuable tools for gene delivery and studying chromosome biology.
• Previous work demonstrated that PAC-based vectors containing alpha-satellite DNA can generate mitotically stable MACs in human cells.

Purpose of the Study:

• To determine if a functional hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene can be assembled into a MAC.
• To assess the stability and expression of a gene delivered via a MAC.

Main Methods:

• Co-transfection of HPRT-deficient HT1080 cells with two PACs: one containing alpha-satellite DNA (PAC7c5) and another with a 140 kb human HPRT gene.
• Isolation of cell lines containing MACs hybridized with alpha-satellite and HPRT probes.
• Analysis of MAC segregation, kinetochore association, and HPRT gene expression.
• Phenotypic confirmation of HPRT deficiency complementation using HAT selection.

Main Results:

• Successfully generated MACs containing both alpha-satellite DNA and the HPRT gene.
• MACs demonstrated efficient segregation and association with kinetochore proteins.
• Stable expression of HPRT message was observed for over 60 days without selection.
• Phenotypic complementation of HPRT deficiency was confirmed by growth on HAT selection.

Conclusions:

• PAC-based vectors are a viable route for incorporating genes into MACs.
• MACs can stably deliver and express functional genes in mammalian cells.
• This approach holds potential for delivering genomic copies of genes and achieving stable transgene expression.