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Human artificial chromosome (hac) vector

a human and artificial chromosome technology, applied in the field of human artificial chromosomes (hac), can solve the problems of not being able to realistically modify the chromosome for every target gene, not being able to control the insertion of foreign dna, and not being able to make clear the structure, etc., and achieve the effect of stably retaining cells

Inactive Publication Date: 2006-08-17
KYOWA HAKKO KIRIN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016] It is an object of the invention to provide a human artificial chromosome vector which is retained stably in cells, allows for easy insertion of large-size foreign genes and is introduced into cells, and a method for producing the same.
[0017] To solve the above problems, the inventors have conducted intensive study to 1) establish a chromosome vector which are free from extra genes and can be retained stably in animal cells and 2) establish an expression system by providing a cloning site for the chromosome vector and inserting a target gene into the cloning site as an expression cassette. Specifically, a modified chromosome was prepared from human chromosome 21 by removing a known gene from its long arm, the stability of DT40 hybrid cells retaining the modified chromosome in long-term subculture was confirmed, a loxp sequence and an hCMV promoter were inserted into the proximal region of the long arm on the modified chromosome in a site-specific manner, the GFP gene was introduced into the modified chromosome using the Cre / loxP system, and expression of GFP was confirmed. The inventors found from the results that the problems described above might be solved by establishing a HAC vector based on fragments from human chromosome 21 and completed the present invention.

Problems solved by technology

If the vector has an origin of replication, it produces a number of copies in the cells temporarily; however, they will be omitted gradually in the absence of selection pressure due to unequal partition to daughter cells during cell divisions.
However, it is not realistic to modify chromosomes for every target gene.
These showed systems for the insertion / expression of foreign genes using chromosome fragments as a vector, but the structure was not made clear and the insertion of foreign DNA was not controlled.
Homologous recombination was used to introduce foreign genes into the HAC in a site-specific manner, though this approach had low insertion efficiency and was unsuitable for general purposes.
Although the method of inserting a target gene into an artificial chromosome is simple excepting that a natural chromosome fragment was used and loxP sites were randomly inserted, using an aberrant chromosome from a patient (mild mental retardation) is problematic in terms of safety and impractical.
It is supposed that extra genetic expression disturbs propagation of host cells retaining chromosome fragments.
However, somatic cells of most animal species have extremely low homologous recombination frequency so that a lot of effort is required to obtain recombinants.
This minichromosome was transferred into mouse ES cells by the microcell fusion method, but was unstable.
However, as described above, the establishment of HACs itself has not yet been completed, and for the introduction of foreign genes, only random insertion of drug resistant genes has been suggested; besides no detailed analysis has been done.

Method used

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  • Human artificial chromosome (hac) vector
  • Human artificial chromosome (hac) vector
  • Human artificial chromosome (hac) vector

Examples

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Effect test

example 1

Preparation of HAC Vector by Deleting Distal Region of the Long-Arm of Human Chromosome 21

(1) Construction of a Construct for Telomere Truncation

[0251] As a telomere truncation vector (targeting vector) for use in deleting a distal region of the long-arm of human chromosome 21, PBS-TEL / Puro (Kuroiwa, Nucleic Acids Res., 26:3347, 1998) was used. Based on the nucleotide sequence (Accession No. AL163204) of the long-arm distal region of human chromosome 21, which was obtained from the GenBank database, a target sequence for use in inserting the telomere truncation vector was designed. To amplify the sequence, primer oligonucleotides were used. The sequences of the primer oligonucleotides, to which a recognition sequence for restriction enzyme BamH I was added, are shown below:

(SEQ ID No.1)#21telF1: 5′-CGCGGATCCAGAGAGAGCCTGGAATGCCTGGTAGTGT(SEQ ID No.2)#21telR1: 5′-CGCGGATCCCCAGTGCCCTGAGATCTTGTGATTTCTC

[0252] DT40 hybridoma cell retaining human chromosome 21 was prepared by a microce...

example 2

Insertion of loxp Sequence into the Proximal Region of Human Chromosome 21 in HAC Vector

(1) Construction of a Construct for Inserting loxP

[0263] As a basic plasmid for inserting a loxP sequence into the human artificial chromosome (HAC) prepared in Example 1, pSF1 (Lifetech) was used. The nucleotide sequence of a loxP insertion site, that is, a proximal region of the long-arm of human chromosome 21, was obtained from the GenBank database (Accession No. AL163203). The sequences of primer oligonucleotides used in amplifying 2 target sequences for homologous recombination are shown below:

(SEQ ID No. 9)#21qEcoF:5′-CCGGAATTCCTCTGGGTTTCTGGTGAAGC;(SEQ ID No. 10)#21qEcoR:5′-CCGGAATTCTGTAGATCCTGCCATTGTGG;(SEQ ID No. 11)#21qBaF:5′-CGCGGATCCTTGGCTCCAAAAGGTACCAC;(SEQ ID No. 12)#21qBaR:5′-CGCGGATCCCTATCCTCGCCACTGTGTCC.

[0264] Using the genomic DNA extracted from a DT40 hybridoma cell retaining human chromosome 21, as a template, two target sequences were amplified by PCR. Each of them was di...

example 3

Transfer of HAC Vector Derived from Human Chromosome 21 into Hamster Cell Line

(1) Microcell Fusion and Isolation of Drug Resistant Clone

[0272] As a chromosome donor cell, DT40 cell (DT40(#21) bsd-79) retaining a HAC vector derived from human chromosome 21 obtained in Examples 1 and 2 by deleting a long-arm distal region and inserting a loxp sequence was used. As a chromosome recipient cell, Chinese hamster ovary derived cell line, CHO-K1 (available from ATCC, Accession No. JCRB9018) was used.

[0273] First, microcells were prepared from about 109 DT40 (#21) bsd-79 cells. The DT40 (#21) bsd-79 cells were cultured up to a cell density corresponding to about 60 to 70% saturation in a culture solution (10% FBS, 1% ChS, 50 μM 2-mercaptoethanol, DMEM) containing colcemid (0.075 μg / ml, Demecolcine, Wako Pure Chemical Industries, Ltd.) for 12 to 15 hours to induce micronuclei. The cells were centrifugally collected, suspended in serum-free DMEM, and seeded in twelve 25 cm2-centrifugation ...

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Abstract

The present invention relates to a human artificial chromosome (HAC) vector and a method for producing the same. The present invention further relates to a method for introducing foreign DNA using a human artificial chromosome vector and a method for producing a cell which expresses foreign DNA. Furthermore, the present invention relates to a method for producing a protein.

Description

TECHNICAL FIELD [0001] The present invention relates to a human artificial chromosome (HAC) vector and a method for producing the same. The present invention further relates to a method for introducing foreign DNA using a human artificial chromosome vector and a method for producing a cell which expresses foreign DNA. Furthermore, the present invention relates to a method for producing a protein. BACKGROUND ART [0002] A vector for introducing and expressing an foreign gene in mammalian cells is not only an essential tool for the study of basic life science, but it has also played an important role in applying the results to practical use in industry (for example, large-scale production of drugs) and clinical practice (for example, gene therapy). Progress in genetic engineering technology after the later half of the 1970's facilitated the isolation and amplification of particular gene DNA fragments (gene cloning) using Escherichia coli and yeast. Cloned DNA has been used conventional...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C12N5/08C12N15/63A61K35/12A61K35/28A61K35/545A61K35/57A61K38/18A61K38/20A61K38/22A61K38/36A61P3/06A61P3/10A61P7/04A61P7/06A61P9/00A61P9/10A61P13/12A61P19/02A61P21/04A61P25/16A61P25/28A61P29/00A61P35/00A61P37/02A61P37/04A61P43/00C12N1/15C12N1/19C12N1/21C12N5/10C12N15/09C12N15/85C12P21/02
CPCC12N2800/208C12N2800/30C12N15/85A61P1/04A61P3/00A61P3/06A61P3/10A61P7/00A61P7/02A61P7/04A61P7/06A61P9/00A61P9/08A61P9/10A61P13/12A61P19/02A61P19/08A61P21/04A61P25/16A61P25/28A61P29/00A61P35/00A61P35/02A61P37/02A61P37/04A61P43/00C12N5/10C12N15/09C12N15/63
Inventor OSHIMURA, MITSUOKATOH, MOTONOBUTOMIZUKA, KAZUMAKUROIWA, YOSHIMIKAKEDA, MINORU
Owner KYOWA HAKKO KIRIN CO LTD
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