Use of a Human Minichromosome as a Cloning and Expression Vector for Mammalian Cells
Human Molecular Genetics
A natural human minichromosome (MC1) derived from human chromosome 1 was shown to be linear and to have a size of 5.5 Mb. Human IL-2 cDNA and the neo gene were co-transfected into a MC1-containing human-CHO hybrid cell line. Integration of the foreign genes was directed to the pericentromeric region of MC1 by co-transfection of chromosome 1-specific satellite 2 DNA. A number of G418-resistant transfectants were obtained and expression of IL-2 was determined. FISH analysis demonstrated
... onstrated co-localization in the minichromosome of the IL-2 gene and of the satellite 2 DNA. An IL-2-producing clone was used in cell fusion experiments with IL-2-dependent murine CTLL cells to generate CTLL-human hybrids containing the modified minichromosome (MC1-IL2). The hybrids were able to grow in medium lacking IL-2 for 17 mean population doublings (MPD), indicating that expression of the cytokine was sufficient to relieve the IL-2 dependence of CTLL proliferation. Endogenous IL-2 production delayed the onset of apoptosis in the IL-2-dependent CTLL cells. Mitotic stability was shown to be 100% in the human-CHO hybrids and 97% per MPD in CTLL cells. These results demonstrate that a natural human minichromosome can be utilized as a cloning and expression vector for mammalian cells and that the MC1 minichromosome can be engineered to deliver IL-2 to two types of cells, fibroblasts and lymphocytes.