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Method for selecting a high expression recombinant cell line

一种细胞、选择性的技术，应用在重组DNA技术、生物化学设备和方法、使用载体引入外来遗传物质等方向，能够解决耗费时间和成本、劳动密集等问题

Active Publication Date: 2012-02-29
株式会社斯特利恩
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0007] However, even when using the conventional dhfr / MTX gene amplification system that significantly increased the production of the relevant protein by introducing the gene encoding the relevant protein into dhfr-deficient CHO cells, followed by treatment of the cells by stepwise increasing the MTX concentration, selection with a high yield High-rate cell populations are also labor-intensive, time-consuming, and cost-intensive due to the prolonged method of treating cells with increasing concentrations of MTX and the number of cell populations to be screened in this method ranges from 500 to 4,000 or more

Method used

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  • Method for selecting a high expression recombinant cell line
  • Method for selecting a high expression recombinant cell line
  • Method for selecting a high expression recombinant cell line

Examples

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Effect test

example 1

[0065] Example 1: Introducing an inoperable polyA

[0066] In the first step of examining gene expression levels based on the presence or absence of polyA, in order to be operably linked to the pCT107 vector expressing an IgG antibody ( figure 1 ), the polyA at the 3' end of the three transcription units (dhfr gene, heavy chain gene, and light chain gene) in ) was inoperable, and the pCT107 vector was cleaved with each of Rsr II, PmeI, and Cla I restriction enzymes. Restriction enzymes Rsr II, Pme I, and ClaI are specific sequences that exist between the 3' end of each of the dhfr gene, heavy chain gene, and light chain gene and the corresponding polyA to enable the expression of IgG antibodies. Linearized enzymes.

[0067] Treatment with restriction enzymes was performed in the following manner. Prepare three test tubes and add (i) 30 μg pCT107 vector DNA and 10 units (U) Rsr II (R0501S, NEB); (ii) 30 μg pCT107 vector DNA and 10 units Pme I (R0560S , NEB); and (iii) 30 m...

example 2

[0068] Example 2: Transfection into CHO cell lines

[0069] The vector linearized according to Example 1 was transfected into the same amount of CHO DG44 cells. Transfection in the control and test groups shown in Table 1 below was performed.

[0070] [Table 1]

[0071] Control group (A)

[0072] Transfection was performed as follows. Using MEMα medium (1140076, Invitrogen) supplemented with 10% FBS (12105, Sigma) to culture CHO DG44 cells at 0.5×10 6 Cells / well were seeded into 6-well plates, and after 24 hours, the medium was replaced with FBS-free MEMα medium. After 30 minutes, 2.5 μg of vector DNA from each of the control group (A), test group (B), test group (C) and test group (D) was added together with 500 μl of Opti-SFM medium (12309-050 , Invitrogen) were added together to each well, after which 6.25 microliters of LTX (15338-100, Invitrogen) were added thereto and vortexed well using a vortex mixer. The resulting mixture was allowed to stand at room ...

example 3

[0073] Example 3: Determination of IgG Antibody Gene Expression Using ELISA

[0074] In order to determine whether the IgG antibody gene was expressed, ELISA was performed 3 days after transfection. First, goat anti-human immunoglobulin G (Fcγ) (109-006-098, Jackson ImmunoReserarch) was adsorbed onto a 96-well microtiter plate (449824, Nunc). Plates were blocked with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA, Bovine Serum Albumin), and serially diluted samples were added to each well. After allowing the plates to stand at room temperature for 2 hours, samples were treated with a peroxidase-labeled goat anti-human kappa antibody (I1514, Sigma) for detection. After standing at room temperature for 1 hour, the sample was reacted with tetramethyl benzidine (TMB, tetramethyl benzidine), and 1N HCl was added thereto to stop the reaction. Human IgG1κ (A7164, Sigma) purified from myeloma plasma at a concentration of 250 ng / ml was used as a standard. ...

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Abstract

The present invention relates to a method for selecting a high expression recombinant cell line using an expression vector including: (i) a gene expression cassette containing a selection marker gene to which polyA is inactively connected; and (ii) a gene expression cassette for coding target recombinant proteins to which polyA is actively connected. The method according to the present invention enables the selection of high productivity cell clones from cell populations of 10 times or more less than the cell populations of conventional cell line selection methods. Particularly, the method of the present invention uses low density MTX in selecting high productivity cell clones to shorten the time taken for developing a cell line, as compared to a conventional phased gene amplification strategy which increases the amount of MTX and includes several amplification phases. The method of the present invention achieves an improved efficiency in producing proteins in the event general selection marker genes other than MTX are used.

Description

technical field [0001] The present invention relates to a method for selecting a cell line (hereinafter referred to as "high-yielding clone") that highly expresses a related protein, and more particularly, to a method for selecting a high-yielding clone by using an expression vector comprising (i) a gene expression cassette comprising a selectable marker gene inoperably linked to the polyadenylation signal (polyA); and (ii) encoding the relevant recombinant protein and operably linked to the polyadenylation signal (polyA) a gene expression cassette for a signal; and relates to said expression vector. Background technique [0002] In the field of biology or medicine, by inserting the gene of the related protein to be produced into the expression vector, transfecting the expression vector into a cell line that produces the protein, culturing the resulting cell line in large quantities, and using a suitable method to isolate and Purification of cultured cell lines to obtain pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/65C12N15/64C12N15/52
CPCC12N15/1086C12N15/65C12N15/69C12N15/85C12N2800/24C12N2830/46C12N2830/50C12P21/02
Inventor 曺明三张珉硕金锺黙李炫周宋侑哲金满洙
Owner 株式会社斯特利恩
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