GENE TARGETING TO THE CENTROMERIC DNA OF A HUMAN MINICHROMOSOME

A human supernumerary minichromosome (MC), previously identified as a derivative of chromosome 9, has been introduced into Chinese hamster o vary (CHO) cells by means of cell fusion. A hybrid clone containing th e MC as the only free human chromosome was isolated. A selectable mark er gene (nea) inserted into a yeast artificial chromosome (YAC) has be en successfully targeted to the MC centromeric DNA via co-transfection with chromosome-9-specific alpha satellite DNA. In situ hybridization and Southern blotting experiments demonstrated that the intact neo ge ne was integrated into the MC centromeric DNA. Studies on the clonal d istribution and on the stability of the MC either in the presence or i n the absence of the selective agent have been carried out. The MC is susceptible to further manipulations and may thus represent a model fo r the construction of a large-capacity vector for somatic gene therapy .