Novel generation of human satellite DNA-based artificial chromosomes in mammalian cells

An in vivo approach has been developed for generation of artificial chromos omes, based on the induction of intrinsic, large-scale amplification mechan isms of mammalian cells. Here, we describe the successful generation of pro totype human satellite DNA-based artificial chromosomes via amplification-d ependent de novo chromosome formations induced by integration of exogenous DNA sequences into the centromeric/rDNA regions of human acrocentric chromo somes. Subclones with mitotically stable de novo chromosomes were establish ed, which allowed the initial characterization and purification of these ar tificial chromosomes. Because of the low complexity of their DNA content, t hey may serve as a useful tool to study the structure and function of highe r eukaryotic chromosomes. Human satellite DNA-based artificial chromosomes containing amplified satellite DNA, rDNA, and exogenous DNA sequences were heterochromatic, however, they provided a suitable chromosomal environment for the expression of the integrated exogenous genetic material, We demonst rate that induced de novo chromosome formation is a reproducible and effect ive methodology in generating artificial chromosomes from predictable seque nces of different mammalian species, Satellite DNA-based artificial chromos omes formed by induced large-scale amplifications on the short arm of human acrocentric chromosomes may become safe or low risk vectors in gene therap y.