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Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes

a technology of artificial chromosomes and chromosomes, which is applied in the field of methods for preparing artificial chromosomes, can solve the problems of numerous limitations of the current system, risk of disruption, and inability to meet all the requirements of the available vector, and achieve the effect of stable chromosomal vector, small stable self-replicating artificial chromosomes, and reduced macs

Inactive Publication Date: 2007-03-15
HADLACZKY GYULA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0023] Isolation of the 15 Mb (or 30 Mb amplicon containing two 15 Mb inverted repeats) or a 30 Mb or higher multimer, such as 60 Mb, thereof should provide a stable chromosomal vector that can be manipulated in vitro. Methods for reducing the size of the MACs to generate smaller stable self-replicating artificial chromosomes are also provided.

Problems solved by technology

The presently available systems, however, have numerous limitations, particularly where persistent, stable, or controlled gene expression is required.
These limitations include: (1) size limitations because there is a limit, generally on order of about ten kilobases (kB), at most, to the size of the DNA insert (gene) that can be accepted by viral vectors, whereas a number of mammalian genes of possible therapeutic importance are well above this limit, especially if all control elements are included; (2) the inability to specifically target integration so that random integration occurs which carries a risk of disrupting vital genes or cancer suppressor genes; (3) the expression of randomly integrated therapeutic genes may be affected by the functional compartmentalization in the nucleus and are affected by chromatin-based position effects; (4) the copy number and consequently the expression of a given gene to be integrated into the genome cannot be controlled.
The limitations of existing gene delivery technologies, however, argue for the development of alternative vector systems suitable for transferring large (up to Mb size or larger) genes and gene complexes together with regulatory elements that will provide a safe, controlled, and persistent expression of the therapeutic genetic material.
At the present time, none of the available vectors fulfill all these requirements.
Artificial chromosomes for expression of heterologous genes in yeast are available, but construction of defined mammalian artificial chromosomes has not been achieved.
Such construction has been hindered by the lack of an isolated, functional, mammalian centromere and uncertainty regarding the requisites for its production and stable replication.
Unlike in yeast, there are no selectable genes in close proximity to a mammalian centromere, and the presence of long runs of highly repetitive pericentric heterochromatic DNA makes the isolation of a mammalian centromere using presently available methods, such as chromosome walking, virtually impossible.
Methods provided for isolation of these chromosomes, however, provide preparations of only about 10-20% purity.

Method used

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  • Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes
  • Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes
  • Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes

Examples

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example 1

General Materials and Methods

[0249] The following materials and methods are exemplary of methods that are used in the following Examples and that can be used to prepare cell lines containing artificial chromosomes. Other suitable materials and methods known to those of skill in the art may used. Modifications of these materials and methods known to those of skill in the art may also be employed.

[0250] A. Culture of Cell Lines, Cell Fusion, and Transfection of Cells

[0251] 1. Chinese hamster K-20 cells and mouse A9 fibroblast cells were cultured in F-12 medium. EC3 / 7 (see, U.S. Pat. No. 5,288,625, and deposited at the European Collection of Animal cell Culture (ECACC) under accession no. 90051001; see, also Hadiaczky et al. (1991) Proc. Natl. Acad. Sc. U.S.A. 88:8106-8110 and U.S. application Ser. No. 08 / 375,271) and EC3 / 7C5 (see, U.S. Pat. No. 5,288,625 and Praznovszky et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:11042-11046) mouse cell lines, and the KE1-2 / 4 hybrid cell line w...

example 2

Preparation of EC3 / 7, EC3 / 7C5 and related cell lines

[0277] The EC3 / 7 cell line is an LMTK− mouse cell line that contains the neo-centromere. The EC3 / 7C5 cell line is a single-cell subclone of EC3 / 7 that contains the neo-minichromosome.

[0278] A. EC3 / 7 Cell Line

[0279] As described in U.S. Pat. No. 5,288,625 (see, also Praznovszky et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:11042-11046 and Hadlaczky et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:8106-8110) de novo centromere formation occurs in a transformed mouse LMTK− fibroblast cell line (EC3 / 7) after cointegration of λ constructs (λCM8 and λgtWESneo) carrying human and bacterial DNA.

[0280] By cotransfection of a 14 kb human DNA fragment cloned in λ (λCM8) and a dominant marker gene (λgtWESneo), a selectable centromere linked to a dominant marker gene (neo-centromere) was formed in mouse LMTK− cell line EC3 / 7 (Hadlaczky et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:8106-8110, see FIG. 1). Integration of the heterologous DNA...

example 3

Minichromosome Transfer and Production of the λ-neo-Chromosome

[0300] A. Minichromosome Transfer

[0301] The neo-minichromosome (referred to as MMCneo, FIG. 2C) has been used for gene transfer by fusion of minichromosome-containing cells (EC3 / 7C5 or EC3 / 7C6) with different mammalian cells, including hamster and human. Thirty-seven stable hybrid cell lines have been produced. All established hybrid cell lines proved to be true hybrids as evidenced by in situ hybridization using biotinylated human, and hamster genomic, or pMCPE1.51 mouse long interspersed repeated DNA probes for “chromosome painting”. The MMCneo has also been successfully transferred into mouse A9, L929 and pluripotent F9 teratocarcinoma cells by fusion of microcells derived from EC3 / 7C5 cells. Transfer was confirmed by PCR, Southern blotting and in situ hybridization with minichromosome-specific probes. The cytogenetic analysis confirmed that, as expected for microcell fusion, a few cells (1-5%) received (or retained...

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Abstract

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, methods for amplification of nucleic acids 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. Methods for use of the artificial chromosomes are also provided.

Description

RELATED APPLICATIONS [0001] This application is a continuation of and claims priority under 35 U.S.C. §120 to copending U.S. application Ser. No. 10 / 219,694, filed Aug. 14, 2002, to Gyula Hadlaczky, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHOD FOR PREPARING ARTIFICIAL CHROMOSOMES, which is a divisional of U.S. application Ser. No. 09 / 724,693, now abandoned, filed Nov. 28, 2000, to Gyula Hadlaczky, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIAL CHROMOSOMES, which is a continuation of U.S. application Ser. No. 08 / 835,682, now abandoned, filed Apr. 10, 1997 to Gyula Hadlaczky and Aladar Szalay, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIAL CHROMOSOMES, which is a continuation-in-part of U.S. application Ser. No. 08 / 695,191, filed Aug. 7, 1996, now U.S. Pat. No. 6,025,155, to GYULA HADLACZKY and ALADAR SZALAY, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIAL CHROMOSOMES...

Claims

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Application Information

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IPC IPC(8): A01H1/00C12Q1/68C07H21/04C12N15/82C12N5/04A01H5/00A01K67/027A61K38/00A61K48/00C12N5/10C12N15/09C12N15/113C12N15/62C12N15/85
CPCA01K67/0275C12N2830/002A01K2227/105A01K2227/30A01K2267/01A01K2267/03A61K38/00A61K48/00C07K2319/036C07K2319/61C12N15/113C12N15/625C12N15/82C12N15/85C12N15/8509C12N2310/111C12N2800/20C12N2800/206C12N2800/208A01K2217/05A61P43/00
Inventor HADLACZKY, GYULASZALAY, ALADAR A.
Owner HADLACZKY GYULA
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