Gene engineering cell line receiving site-directed integration of exogenous genes

A technology of site-specific integration and exogenous genes, applied to cells modified by introducing foreign genetic material, using vectors to introduce foreign genetic material, biochemical equipment and methods, etc.

Inactive Publication Date: 2018-08-24
成都金洛克锶生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cell lines are all bottle-attached, and as an industrial application, especially in the

Method used

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  • Gene engineering cell line receiving site-directed integration of exogenous genes
  • Gene engineering cell line receiving site-directed integration of exogenous genes
  • Gene engineering cell line receiving site-directed integration of exogenous genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 FRT recognition site location screening in CHO cell chromosomes

[0021] 1. Cell culture

[0022] CHO-S cell line, in CD FortiCHO TM Add glutamine and anti-clumping agent (Anti-Clumping Agent) to Medium medium, and culture in a cell shaker at 5% CO2, 37° C., 130 rpm / min.

[0023] 2. Plasmid transfection and preliminary screening of cells

[0024] The plasmid pFRT / lacZeo was extracted according to the endotoxin-free plasmid kit manual (DP117, Tiangen Biochemical Technology Co., Ltd.), and the pFRT / lacZeo plasmid was linearized with ScaI restriction endonuclease. line Kit V kit and refer to the manual (VCA-1003, LONZA) for pFRT / lacZeo plasmid electrotransfection. The cells transfected with the plasmid were cultured in shake flasks for 48 hours, and the cell viability and cell number were detected by a cell counter. Step-by-step pressure screening after transfection: Bleomycin (Zeocin) concentration is 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 750 μg / mL a...

Embodiment 2

[0037] Example 2 Homologous recombination of exogenous gene and protein expression

[0038] 1. Expression plasmid construction

[0039] a) UCOE-dhfr expression vector construction

[0040] The UCOE-dhfr vector is obtained through artificial transformation, and it can be used for site-directed integration of foreign genes and protein expression. The gene fragment was amplified by PCR with specific primers P1 and P2. PCR reaction system 1 (total volume 50 μl): 5×Buffer 10 μl, dNTP 2 μl, primers 1 μl (P1 and P2), template UCOE vector 1 μl, Phusion enzyme 0.5 μl, and finally make up to 50 μl with double distilled water; reaction conditions: 98°C Pre-denaturation for 60s, 1 cycle, 98°C for 10s, 55°C for 30s, 72°C for 50s, 30 cycles, and finally 72°C for 7min. The gene product was detected by agarose gel electrophoresis, and the obtained gene fragment was named gene1. The gene fragment dhfr was amplified by PCR using specific primers P3 and P4. PCR reaction system 2 (total volu...

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Abstract

The present invention relates to an engineering cell line for site-directed integration of exogenous genes, and uses thereof, wherein an identification tag is inserted into the Z4q3 region of Chinesehamster ovary cells (CHO) as host cells to obtain the cell line. According to the present invention, the engineering cell line supports the site-directed integration of exogenous genes, and can maintain the efficient and stable expression of exogenous genes for a long time.

Description

technical field [0001] The invention relates to the field of cell genetic engineering, in particular to a CF cell line for site-specific integration of exogenous genes and its application in protein expression. Background technique [0002] Improving the expression level and stability of exogenous genes in mammalian cells has always been a growing demand in the field of biopharmaceuticals or genetic engineering. Although most commercial expression systems, especially expression vectors, have targeted optimized designs, such as selection of strong promoters, with pressurized screening systems, etc., it still takes a lot of effort and cost to obtain cells with high expression of foreign proteins. The integration of exogenous genes introduced by conventional techniques is random, and the insertion position does not support strong transcription, resulting in low protein expression levels. Even short-term high-level expression cannot maintain its stability, and increases with the...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85
CPCC12N15/85C12N2800/30
Inventor 李生伟高小平张晟付伟王雯茜代燕平
Owner 成都金洛克锶生物技术有限公司
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