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Method of introducing target-specific foreign gene

An exogenous, genetic technology, applied to other methods of inserting foreign genetic material, cells modified by introducing foreign genetic material, using a vector to introduce foreign genetic material, etc.

Pending Publication Date: 2021-11-16
CHUGAI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, it is clear that even with the introduction of exogenous DNA into the genome, it is not always possible to obtain transformed cells expressing high

Method used

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  • Method of introducing target-specific foreign gene
  • Method of introducing target-specific foreign gene
  • Method of introducing target-specific foreign gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0142] [Example 1] Preparation of Landing Pad Plasmid

[0143] Created a "landing rack" containing a DNA cassette that was used as a target site for the introduction of the gene of interest during the cassette exchange reaction and integrated it into the plasmid ( figure 1 ). The landing rack plasmid carries the green fluorescent protein (GFP) gene as a marker gene, which is used to identify gene high expression regions on the genome of CHO cells. It also has the dihydrofolate reductase (DHFR) gene as a selectable marker after the introduction of the landing shelf plasmid. DNA sequences (loxP1 and loxP2) recognized by recombinase Cre are inserted into both ends of the two genes, DHFR gene and GFP gene. The DHFR gene and the GFP gene present between these two loxPs were removed during the cassette exchange reaction and replaced with genes encoding the polypeptide to be produced loaded on the recombinant plasmid. In the following examples, an attempt was made to generate anti...

Embodiment 2

[0144] [Example 2] Preparation of recombinant plasmid

[0145] A "recombinant plasmid" was prepared for a cassette exchange reaction with the DNA cassette inserted into the landing pad on the CHO cell genome ( figure 2 ). The recombinant plasmid carries the DHFR gene and the antibody gene consisting of heavy and light chains, and loxP is inserted at both ends of these genes. As evaluation antibodies, one type of IgG1 antibody (mAb-A) and two types of IgG2 antibodies (mAb-B, C) were used.

[0146] Each antibody recognizes a different antigen as follows:

[0147] mAb-A: GYM329 / anti-latent myostatin antibody / IgG1;

[0148] mAb-B: CIM331 / anti-IL-31 receptor antibody / IgG2 (WO 2010 / 064697);

[0149] mAb-C: SA237 / anti-IL-6 receptor antibody / IgG2 (WO 2016 / 027859).

[0150] Although figure 2 A recombinant plasmid carrying antibody genes consisting of one set of heavy chain / light chains was used, but the configuration of the recombinant plasmid can be appropriately modified acco...

Embodiment 3

[0151] [Example 3] Preparation of host cells for site-specific integration (TI)

[0152] The landing pad plasmid was transfected into host cells (CHO-DXB11 ) using Nucleofector 2b from LONZA (Nucleofector is a registered trademark of Lonza Cologne GmbH). The landing pad plasmid used for transfection was linearized with the restriction enzymes EcoRV and SalI. Four hours after transfection, the medium was changed to a hypoxanthine / thymidine-free medium, and shaking culture of the cells was started. After about two weeks, single cell sorting was performed using Sony's Cell Sorter SH800. When sorting, sort the cell population within the top 2% with high GFP fluorescence intensity. A 488nm semiconductor laser was used to excite GFP. Single-cell sorted cells are expanded and cultured, and genomic DNA is extracted from each cell clone. Using the recovered genomic DNA, the copy number of the GFP gene introduced into each cell clone was measured using Bio-Rad's QX200 Droplet Digita...

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Abstract

A hotspot useful for transformation by animal cell TI is provided. This hotspot was discovered near LOC103164262 in the CHO cell genome. Additionally, the present invention relates to transformed cells into which foreign DNA has been introduced into said hotspot, and a method for culturing said cells to produce polypeptides coded by said DNA.

Description

technical field [0001] The present disclosure relates to methods for introducing exogenous DNA into the genome of animal cells in a target-specific manner, transformed cells obtained by using these methods, and methods for producing polypeptides encoded by exogenous DNA. Background technique [0002] A method of expressing a polypeptide such as a cytokine or an antibody in cultured cells by gene recombination technology and mass-producing the polypeptide is well known. Such polypeptide production techniques generally include the steps of introducing a polynucleotide encoding a polypeptide of interest into cells in an expressible form to produce transformed cells and recovering the accumulated polypeptide of interest from culture thereof. Widely used as cells to be transformed are cells of microorganisms, insects, plants or animals. Among them, animal cells are widely used as suitable host cells for obtaining animal-derived polypeptides. By expressing a polypeptide in anima...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/90C12P21/00C12P21/08C12N15/11C12N15/12C12N15/13
CPCC07K16/2866C07K2317/52C07K2317/14C12N15/907C12N15/90C07K16/00C12N15/85
Inventor 小松将大小村健太若原裕二
Owner CHUGAI PHARMA CO LTD
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