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Reconstruction and application of targeted site-specific integrated CHO cell line

A fixed-point integration and cell line technology, applied to cells modified by introducing foreign genetic material, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve problems such as limited utilization rate, limited wide and long-term industrial application

Active Publication Date: 2015-08-05
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

Due to the randomness of chromosome 1 integration, it needs to be obtained through a large number of screenings, and the utilization rate is very limited, which is very limited for extensive and long-term industrial applications

Method used

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  • Reconstruction and application of targeted site-specific integrated CHO cell line
  • Reconstruction and application of targeted site-specific integrated CHO cell line
  • Reconstruction and application of targeted site-specific integrated CHO cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 FRT Location Screening in Chromosome 1 of CHO Cells

[0019] 1. Cell culture

[0020] Suspension-cultured CHO cells are preferred, and glutamine, etc. are added to the suspension medium, in 5% CO 2 , 37°C, cultured in a cell shaker at 130rpm / min.

[0021] 2. Plasmid transfection and preliminary screening of cells

[0022] The pBAT-FRT plasmid was linearized with ScaI restriction endonuclease, and after the digested product was recovered, the Lonza Cell Line was used to Kit V kit and refer to the instructions for pBAT-FRT plasmid electrotransfection. The cells transformed with the plasmid were cultured in shake flasks (5% CO 2 , 37°C, 130rpm / min) to 48h, the cell viability and cell number were detected by a cell counter.

[0023] Pressurized screening was carried out 48 hours after transfection: the concentration of bleomycin was 50 μg / ml, 100 μg / ml, 250 μg / ml, 500 μg / ml, 750 μg / ml and 1000 μg / ml, and the pressure was gradually increased to obtain the pri...

Embodiment 2

[0036] Example 2 Homologous recombination of exogenous gene and protein expression

[0037] 1. Expression plasmid construction

[0038] a) Construction of pBAT-dhfr expression vector

[0039] The pBAT-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 Pre-denaturation at ℃ 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 1 (tota...

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Abstract

The invention relates to an FRT-sCHO cell line with an integrated exogenous gene at a specific site and application of the FRT-sCHO cell line. The cell line is prepared by using a Chinese hamster ovary cell (CHO) as a host cell and inserting an identification tag into the terminal (1q12 and 1q13) regions of the long arm of chromosome No. 1 of the host cell. The FRT-sCHO cell line provided by the invention supports integration of exogenous genes at specific sites and maintains high-efficiency and stable expression of exogenous proteins for a long time.

Description

technical field [0001] The invention relates to the field of cell genetic engineering, in particular to a FRT-sCHO cell line for site-specific integration of foreign genes and its use in the field of biopharmaceuticals. Background technique [0002] Improving the expression level and stability of exogenous genes in mammalian cells has always been an ever-growing pursuit 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 th...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85
Inventor 高小平李生伟罗弟祥付伟代燕平程琳张晟
Owner 成都金洛克锶生物技术有限公司
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