Construction of a mammalian artificial chromosome (MAC) will develop our understanding of the requirements for normal chromosome maintenance, replication and segregation while offering the capacity for introducing genes into cells. Construction of MACs with telomere, centromere and replication function has been approached by two methods. The 'top down' strategy uses artificially induced chromosome truncations as a means to define a minimal chromosome that retains the mitotic properties of a normal chromosome. The 'build up' approach has focused on attempts to assemble MAC vectors containing functionally defined telomere repeats together with candidate centromere and replication origin sequences. Here we report on significant advances in both areas, with particular emphasis on two reports showing that stable, low copy number MACs containing a functional centromere can be produced following transfection of naked DNA into the human HT1080 cell line. One approach used a transfection mixture of cloned synthetic α-satellite arrays up to 1 Mb in length and unlinked telomeric DNA, in either the presence or absence of random human genomic DNA fragments. In the second approach, MACs were formed from a defined yeast artificial chromosome (YAC) DNA molecule containing 100 kb of highly homogeneous alphoid DNA retrofitted with human telomere repeats. These results demonstrate for the first time that α-satellite DNA can seed de novo centromeres in human cells, indicating that this repetitive sequence family plays an important role in centromere function. The stability of these MACs suggests that they have potential to be developed as gene delivery vectors.
ASJC Scopus subject areas
- Molecular Biology