Journal Article

Use of a Human Minichromosome as a Cloning and Expression Vector for Mammalian Cells

Cristiana Guiducci, Fiorentina Ascenzioni, Cristina Auriche, Enza Piccolella, Anna Maria Guerrini and Pierluigi Donini

in Human Molecular Genetics

Volume 8, issue 8, pages 1417-1424
Published in print August 1999 | ISSN: 0964-6906
Published online August 1999 | e-ISSN: 1460-2083 | DOI:
Use of a Human Minichromosome as a Cloning and Expression Vector for Mammalian Cells

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A natural human minichromosome (MC1) derived from human chromosome 1 was shown to be linear and to haveasizeof 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 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.

Journal Article.  6451 words.  Illustrated.

Subjects: Genetics and Genomics

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