I BREVETTI
1) Mammalian artificial chromosomes and methods of using same
Abstract
The present invention provides a mammalian artificial chromosome (MAC), comprising a centromere and a unique cloning site, said MAC containing less than 0.1% of the DNA present in a normal haploid genome of the mammalian cell from which the centromere was obtained. The invention further provides a MAC, wherein the unique cloning site is a nucleic acid sequence encoding a selectable marker. The invention also provides methods of preparing a MAC. In addition, the invention provides methods of stably expressing a selectable marker in a cell, comprising introducing a MAC containing the selectable marker into the cell. The invention also provides a cell containing a MAC expressing an exogenous nucleic acid sequence and a transgenic mammal expressing a selectable marker.
2) Artificial mammalian chromosome
The invention relates to the field of gene therapy, gene expression, and vectors for these uses. In particular, the invention relates to the development and use of an artificial or synthetic chromosome for gene expression and gene therapy in mammals, and especially humans. The invention allows the controlled construction of stable synthetic or artificial chromosomes constructed from isolated segments of purified DNA. Functional minimal segments preferably include centromeric DNA, telomeric DNA, and genomic DNA. The artificial chromosome performs the essential chromosomal functions of naturally-occurring chromosomes so as to permit the chromosome to function as an effective vector for gene therapy.
3) Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes
Methods for preparing cell lines that contain artificial chromosomes, methods for preparation of artificial chromosomes, methods for purification of artificial chromosomes, methods for targeted insertion of heterologous DNA into artificial chromosomes, and methods for delivery of the chromosomes to selected cells and tissues are provided. Also provided are cell lines for use in the methods, and cell lines and chromosomes produced by the methods. In particular, satellite artificial chromosomes that, except for inserted heterologous DNA, are substantially composed of heterochromatin are provided. Methods for use of the artificial chromosomes, including for gene therapy, production of gene products and production of transgenic plants and animals are also provided.
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