Functional complementation of a genetic deficiency with human artificial chromosomes

We have shown functional complementation of a genetic deficiency in human c ultured cells, using artificial chromosomes derived from cloned human genom ic fragments. A 404-kb human-artificial-chromosome (HAC) vector, consisting of 220 kb of alphoid DNA from the centromere of chromosome 17, human telom eres, and the hypoxanthine guanine phosphoribosyltransferase (HPRT) genomic locus, was transferred to HPRT-deficient HT1080 fibrosarcoma cells. We gen erated several cell lines with low-copy-number, megabase-sized HACs contain ing a functional centromere and one or possibly several copies of the HPRT1 gene complementing the metabolic deficiency. The HACs consisted of alterna ting alphoid and nonalphoid DNA segments derived only from the input DNA (w ithin the sensitivity limits of FISH detection), and the largest continuous alphoid segment was 158-250 kb. The study of both the structure and mitoti c stability of these HACs offers insights into the mechanisms of centromere formation in synthetic chromosomes and will further the development of thi s human-gene-transfer technology.