Mammalian artificial chromosome pilot production facility: Large-scale isolation of functional satellite DNA-based artificial chromosomes

Background: A pilot production facility has been established to isolate mam malian artificial chromosomes at high purity by using flow cytometric techn iques. Dicentric chromosomes have been generated by the targeted amplificat ion of pericentric heterochromatic and centromeric DNA by activating the "m egareplicator." Breakage of these dicentric chromosomes generates satellite DNA-based artificial chromosomes (SATAC) from 60 to 400 megabases. Methods: For large-scale production, we have developed cell lines capable o f carrying one or two SATACs. A SATAC, because of a high adenine-thymine (A T) composition, is easily identified and sorted by using chromomycin A3 and Hoechst 33258 stains and a dual laser high-speed flow cytometer. A prototy pe SATAC (60 megabases) has been characterized. The prototype SATAC has bee n isolated from an original rodent/human hybrid cell line and transferred b y using modified microcell fusion into a CHO production cell Line. Results: Metaphase chromosomes from this production cell line were isolated in a modified polyamine buffer, stained, and sorted by using a modified sh eath buffer that maintains condensed chromosomes. SATACs are routinely sort ed at rates greater than 1 million per hour. Sorted SATACs have been transf erred to a variety of cells by using microcell fusion technology and were f ound to be functional. Conclusions: By developing new SATAC containing cell lines with fewer numbe rs of chromosomes in conjunction with operating a high speed flow sorter we have effectively generated an efficient production facility geared purely for the isolation of SATACs. (C) 1999 Wiley-Liss, Inc.