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The generation in vitro of mammalian artificial chromosomes, in view of the possibility of developing new technologies for gene therapy, is still an ambitious goal. Mammalian artificial chromosomes, to be used as cloning and expression vectors, have been constructed either by de novo synthesis or by reduction of pre-existing chromosomes. In the work here reported, we introduced a loxP sequence into the pericentromeric region of a chromosome 9-derived X-ray-reduced minichromosome, with the purpose of generating a human chromosome vector (HCV). The modified accessory chromosome is linear and mitotically stable, has lost at least 1400 kb of alpha satellite DNA and normally binds CENP-B, CENP-C and CENP-E. The efficiency of gene targeting via loxP mediated homologous recombination was tested using the histone H2B-Green Fluorescent Protein chimaeric gene as a reporter. The frequency of site-specific insertion of the exogenous sequence was found to be about 50% and to occur in a controlled way with regard to the number of copies. The expression level of the fusion protein was stable over prolonged time in culture.

Original publication

DOI

10.1159/000048801

Type

Journal article

Journal

Cytogenetics and cell genetics

Publication Date

01/2001

Volume

94

Pages

113 - 120

Addresses

Department of Genetics and Microbiology A. Buzzati-Traverso, University of Pavia, Italy.

Keywords

Chromosomes, Artificial, Human, Centromere, Animals, Humans, Chromosomal Proteins, Non-Histone, Recombinant Fusion Proteins, DNA, Satellite, RNA, Messenger, Gene Targeting, Mutagenesis, Insertional, Recombination, Genetic, Sequence Homology, Nucleic Acid, Genes, Reporter, X-Rays, Attachment Sites, Microbiological, Cricetinae