Generation of an similar to 2.4 Mb human X centromere-based minichromosomeby targeted telomere-associated chromosome fragmentation in DT40

A linear mammalian artificial chromosome (MAC) will require at least three types of functional element: a centromere, two telomeres and origins of rep lication. As yet, our understanding of these elements, as well as many othe r aspects of structure and organization which may be critical for a fully f unctional mammalian chromosome, remains poor. As a way of defining these va rious requirements, minichromosome reagents are being developed and analyse d. Approaches for minichromosome generation fall into two broad categories: de novo assembly from candidate DNA sequences, or the fragmentation of an existing chromosome to reduce it to a minimal size. Here we describe the ge neration of a human minichromosome using the latter, top-down, approach. A human X chromosome, present in a DT40-human microcell hybrid, has been mani pulated using homologous recombination and the targeted seeding of a de nov o telomere. This strategy has generated a linear similar to 2.4 Mb human X centromere-based minichromosome capped by two artificially seeded telomeres : one immediately flanking the centromeric alpha-satellite DNA and the othe r targeted to the zinc finger gene ZXDA in Xp11.21. The chromosome retains an alpha-satellite domain of similar to 1.8 Mb, a small array of gamma-sate llite repeat (similar to 40 kb) and similar to 400 kb of Xp proximal DNA se quence. The mitotic stability of this minichromosome has been examined, bot h in DT40 and following transfer into hamster and human cell lines. In all three backgrounds, the minichromosome is retained efficiently, but in the h uman and hamster microcell hybrids its copy number is poorly regulated. Thi s approach of engineering well-defined chromosome reagents will allow key q uestions in MAC development (such as whether a lower size limit exists) to be addressed. In addition, the 2.4 Mb minichromosome described here has pot ential to be developed as a vector for gene delivery.