Human artificial chromosome vector

a technology of artificial chromosomes and vectors, applied in the field of human artificial chromosome vectors, can solve the problems of limited development of the use of them, and achieve the effects of high efficiency, stably produced, and high efficiency

Inactive Publication Date: 2012-09-13
SANFORD APPLIED BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0044]Human antibodies may be produced with high efficiency by introducing the HAC vector comprising the human antibody heavy chain gene, the human antibody light chain gene, and the human antibody surrogate light chain gene of the present invention into an animal as compared to the vector produced by the conventional HAC technology. In addition, an animal which is capable of producing a human antibody with high efficiency may be stably produced by introducing the human artificial chromosome vector of the present invention into an animal.
[0045]Furthermore, as a preferred embodiment, when a human artificial chromosome vector which further comprises a gene of a non-human animal-derived IgM heavy chain constant region in addition to the human antibody heavy chain gene, the human antibody light chain gene, and the human antibody surrogate light chain gene is introduced into an animal, a human antibody may be produced with a further higher efficiency, and an animal which is capable of producing a human antibody with such high efficiency may be stably produced.

Problems solved by technology

In addition, due to factors such as the kind of antigen, the number of exposure to an immunogen, and the amount of donor serum which may be collected, in preparing a polyclonal antibody; the development of the use thereof is limited.

Method used

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Examples

Experimental program
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example

Example 1

Construction of a Targeting Vector

[0167](1) Construction of a Targeting Vector pTEL'hisDpurolox2272F9R9

[0168]Methods described in the publication (Kuroiwa et al., Nat Biotechnol. 18: 1086-1090, 2000, Kuroiwa et al., Nat Biotechnol. 20: 889-894, 2002 and Kuroiwa et al., Nat Biotechnol. 27: 173-181, 2009) were basically used for construction of a targeting vector.

[0169]Specifically, a genomic DNA fragment Dk-F9R9 used as a homology arm was amplified by PCR consisting of 40 cycles of 98° C. for 10 seconds and 68° C. for 9 minutes by using two primer DNAs of kD-F9 (5′-tcgaggatccgccagggagacagatgccaagtacggtttag-3′) (SEQ ID NO:1) and kD-R9 (5′-tcgaggatccaggatctttgggggactgaatggggtgtgct-3′) (SEQ ID NO:2) and a genomic DNA of a chicken DT40 cell line KTL1 (Kuroiwa et al., Nat. Biotechnol. 27: 173-181, 2009) having the human chromosome 2 as a template.

[0170]Subsequently, the plasmid pTEL'hisDpurolox2272 was constructed in the following order.

[0171]First, the plasmid pPURlox2272 was co...

example 2

Construction of KSL-HAC

[0210](1) Modification of the Human Chromosome 2 in a Chicken DT40 Cell

[0211]In order to generate a deletion at the AP104134 site of the human chromosome 2 and insert a lox2272 sequence and a promoterless puror cassette, the targeting vector pTEL'hisDpurolox2272F9R9 was linearized with SrfI (Stratagene) and introduced into KTL1 (Kuroiwa et al., Nat. Biotechnol. 27: 173-181, 2009) which was a chicken DT40 cell line having the human chromosome 2 fragment digested at the CD8A gene locus by electroporation (550 V, 25 μF). The electroporation of the DT40 cell was carried out by a method described in the publication (Kuroiwa et al., Nat. Biotechnol. 18: 1086-1090, 2000).

[0212]Colonies were subjected to selection by histidinol (0.5 mg / ml, Sigma) for two weeks and the sensitivity to puromycin (1 μg / ml, Sigma) was measured as an index of deletion of the puror cassette on the CD8A gene locus.

[0213]The chromosome DNA was extracted from a colony having the puromycin sensi...

example 3

Construction of KSL-HACΔ

[0259](1) Modification of the Human Chromosome 14 in a DT40 Cell

[0260]In order to integrate a lox511 sequence and a CAG promoter on the AL512355 site which is positioned on the centromere side about 300 kb from the IgH gene locus, the targeting vector pSC355CAGlox511hisDDT linearized with SrfI (Stratagene) was introduced into the DT40 cell maintaining an intact human chromosome 14 labeled with pSTneo [Katoh et al., Cell Structure and Function, 12, 575-580, 1987; Japanese Collection of Research Biologicals (JCRB) Bank, Deposit Number VE039] by electroporation (550 V, 25 μF). The electroporation method into the DT40 cell is described in the publication (Kuroiwa et al., Nat. Biotechnol. 18: 1086-1090, 2000).

[0261]The colony was subjected to selection by histidinol (0.5 mg / ml, Sigma) for two weeks, and thus a genomic DNA extracted from the resistant colony was subjected to PCR screening.

[0262]PCR was carried out with 40 cycles of 98° C. for 10 seconds and 68° C. ...

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Abstract

A human artificial chromosome vector comprising a human antibody heavy chain gene, a human antibody light chain gene, and a human antibody surrogate light chain gene.

Description

TECHNICAL FIELD[0001]The present invention relates to a human artificial chromosome vector comprising a human antibody heavy chain gene, a human antibody light chain gene, and a human antibody surrogate light chain gene, an animal having the human artificial chromosome vector, and a method for producing a human antibody.BACKGROUND ART[0002]The development of a technology of producing a chimeric animal by fusing micronuclei comprising human chromosome fragments with cells having pluripotent differentiation to obtain hybrid cells allows to prepare a non-human animal maintaining large exogenous genes to be produced (Non-Patent Document 1 and Patent Document 1).[0003]Subsequently, a method for constructing a desired human artificial chromosome (hereinafter abbreviated to HAC) vector to produce the non-human animal has been proposed, by which the HAC including a wide range of human antibody gene loci has been established.[0004]First, as a method for modifying a chromosome fragment to be ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027C12N15/85
CPCC07K16/00C07K16/1278C12N15/85C12N2800/208A01K2267/01A01K2207/15A01K2217/072A01K2227/101C12N15/8509A01K67/0278A01K2217/052
Inventor KUROIWA, YOSHIMIMATSUSHITA, HIROAKISANO, AKIKO
Owner SANFORD APPLIED BIOSCI
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