Telomere stabilization, via activation of either telomerase or the Alternative Lengthening of Telomeres (ALT) pathway, is necessary for tumors to acquire an unlimited replicative potential. Most human ALT-positive cell lines and tumors have several distinctive characteristics in common including long and highly heterogeneous telomeres, multiprotein complexes called ALT-associated PML nuclear bodies (APBs) in which telomeric components (DNA and protein) co-localize with the PML nuclear body, and extrachromosomal circular DNA molecules that are composed of telomeric repeats. However, the relationship between extrachromosomal telomeric circles, APBs and telomere elongation has yet to be established due to the lack of a genetically tractable system in human cells to study the activation and regulation of the ALT-pathway. This also complicates and hampers the development of therapeutics targeting this telomerase- independent pathway. As an initial step towards developing a tractable genetic assay system, we have bioengineered a mammalian artificial chromosome to manufacture, on demand, extra- chromosomal telomeric circles in multiple cell types. The regulated production of extra- chromosomal telomeric circles will allow for the systematic testing of the impact of these circles on characteristics of the ALT pathway. This technology can be used in multiple cell types and/or combined with RNAi technology to test the role of specific pathways (genes) in ALT activation, a field that thus far has been refractory to controlled manipulation and exploration in human-derived model systems.