Use of a Human Minichromosome as a Cloning and Expression Vector for Mammalian Cells
| Autor: | Cristina Auriche, Cristiana Guiducci, Anna Maria Guerrini, Enza Piccolella, Pierluigi Donini, Fiorentina Ascenzioni |
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| Rok vydání: | 1999 |
| Předmět: |
Genetic Markers
DNA Complementary Centromere Genetic Vectors Population Gene Expression Apoptosis CHO Cells Hybrid Cells Biology Molecular cloning Transfection Mice Minichromosome Aldesleukin Cricetinae Genetics Animals Humans education Molecular Biology In Situ Hybridization Fluorescence Genetics (clinical) education.field_of_study Expression vector Gene targeting DNA General Medicine Molecular biology Chromosomes Human Pair 1 Cell culture Gene Targeting Interleukin-2 |
| Zdroj: | Human Molecular Genetics. 8:1417-1424 |
| ISSN: | 1460-2083 0964-6906 |
| Popis: | 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 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. |
| Databáze: | OpenAIRE |
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