Use of a human minichromosome as a cloning and expression vector for mammalian cells.
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.
Guiducci
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Istituto Pasteur-Fondazione Cenci Bolognetti, c/o Dipartimento di Biologia Cellulare e dello Sviluppo, Università 'La Sapienza', Via degli Apuli 1, 00185 Roma, Italy.
Ascenzioni
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Auriche
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Piccolella
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Guerrini
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Donini
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D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D016466:CHO Cells; D002503:Centromere / Q000235:genetics; D002878:Chromosomes, Human, Pair 1 / Q000235:genetics*; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D018390:Gene Targeting; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D006822:Hybrid Cells; D017404:In Situ Hybridization, Fluorescence; D007376:Interleukin-2 / Q000235:genetics; D051379:Mice; D014162:Transfection
D018076:DNA, Complementary; D005819:Genetic Markers; D007376:Interleukin-2; D004247:DNA