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Site for stably expressing protein in CHO cell gene NW_003614092.1 and application of site

A technology for stable expression and cellular genes, applied in the field of genes, can solve the problems of long time, stability affecting the time to market of products, and the level of target gene expression decline, and achieve the effect of reducing costs and shortening research and development time.

Active Publication Date: 2022-02-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Random integration is the most mature traditional method for constructing protein expression systems, but obtaining stable high-expression cell lines through random integration requires multiple screenings, which is time-consuming and expensive
At the same time, for the cell lines obtained by random integration, the loss of expression stability of the cells in the later stage of culture is completely unpredictable: this instability may not occur at all; it may also gradually appear after numerous cell divisions; It is also possible that after only a few generations of cell division, apparent instability emerges
The stability problem will not only affect the time to market of the product, but will also have certain conflicts with the management of drug regulations
In addition, the information of random integration sites is unclear, and the site effect of foreign gene integration will also lead to a significant decrease in the expression level of the target gene
According to existing literature reports, the instability of this recombinant CHO cell line appears in all recombinant CHO cell lines, making the problem of unstable expression an extremely common problem

Method used

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  • Site for stably expressing protein in CHO cell gene NW_003614092.1 and application of site
  • Site for stably expressing protein in CHO cell gene NW_003614092.1 and application of site
  • Site for stably expressing protein in CHO cell gene NW_003614092.1 and application of site

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Screening of stable expression sites

[0038] The CHO-K1-1d2 cells expressing the Zsgreen1 reporter gene screened by high-throughput flow cytometry were cultured in an adherent culture until they were in good condition, and the CHO cells at this time were regarded as the 0th generation, and then cultured continuously for 20 The expression of Zsgreen1 protein in cells at passages 0, 10, and 20 was observed under an inverted fluorescence microscope, and the average fluorescence intensity of cells at passages 0, 10, and 20 was detected by BD flow cytometry.

[0039] Observation by inverted fluorescence microscope and detection by flow cytometry showed that after 20 passages, CHO-K1-1d2 cells could still express 100% Zsgreen1 protein in the adherent culture state, and Zsgreen1 protein between different passages The expression levels are basically the same, and there is a strong green fluorescent signal.

[0040] The CHO-K1-1d2 cells that passed the verification ...

Embodiment 2

[0042] Example 2: Analysis of Lentiviral Integration Sites

[0043] Use the Lenti-X Integration Site Analysis Kit (Clontech: 631263) related to chromosome walking technology to analyze the integration site of the lentiviral vector in CHO-K1-1d2 cells. The specific steps are as follows:

[0044] (1), construction of lentiviral integration library

[0045] Collect CHO-K1-1d2 cells, use the DNA extraction kit to extract the genome, use DraI, SspI, HpaI three restriction endonucleases to digest the genome at 37°C for 16-18h, and the digestion system is as follows:

[0046]

[0047] The digested product was purified and recovered using a PCR purification kit, and the chromosome walking adapter GenomeWalker Adapter was connected to both ends of the purified digested fragment. The connection system was as follows:

[0048]

[0049] Incubate overnight at 16°C and incubate at 70°C for 5 minutes. After the reaction is terminated, 32 μl TE (10 / 1, pH 7.5) is added to the system to ...

Embodiment 3

[0064] Example 3: Target Sequence Selection

[0065] 根据就近原则,使用CCTOP CRISPR / Cas9在线预测系统对位点NW_003614092.1第1159467碱基处上下游各100bp的序列:CCCTCTCCAAACTCTTCCTGATAAGGGTTCCAGGTATGGCTTCATCTATGGAGTAGGAGTCATATTCAATCAGAAAGTGTCTGTGTTCTTCCATGGGGTTCATACAAGTATTAGACTTGGGAGCATGTCTTTCCAGCCCAGTCTTTGTAGGTCATGGGATTCCATGCTGGGTATGACTTGTGATCATTTTCCCCTCTGGAA(SEQ ID NO.1)进行预测,并挑选出编辑效率较高的靶 sequence.

[0066] The relevant parameters are set as follows:

[0067] 1) The maximum number of base mismatches allowed in the first 13 bp within the 20 bp sequence after NGG is 1;

[0068] 2) The number of all 20bp mismatched bases after NGG is 4.

[0069] The CCTOP CRISPR / Cas9 online prediction system will score the editing efficiency of the identified 5'NNNNNNNNNNNNNNNNNNNNNGG 3' target sequence, LOW efficiency(score0.74 ).

[0070] Sequences in which the predicted editing efficiency was higher than 0.56 were selected as target sequences.

[0071] Target sequence 5'-TAGGTCATGGGATTCCATGCTGG-3'(SEQ ID NO.2), score=0.75 ...

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Abstract

The invention discloses a site for stably expressing protein in the CHO cell gene NW_003614092.1 and application of the site. The obtained stable expression site is positioned on a base in a 200-bp base range with the 1159467 base of the CHO cell gene NW_003614092.1 as a central point, namely, the 1159367 to 1159567 base, and can integrate an exogenous protein gene and perform stable expression. According to the invention, a target gene is subjected to site-specific integration to a stable expression region in a site-specific integration manner, so the problem of unclear integration sites caused by random integration is solved; and through site-specific integration of an exogenous gene in the range of 1159367 to 1159567bases, relative to the stable expression site at the 1159467 base in the CHO cell gene NW_003614092.1, expression instability and a repeated and tedious cell strain screening process caused by a position effect are overcome.

Description

technical field [0001] The invention relates to a site for stably expressing protein in CHO cell gene NW_003614092.1 and its application, belonging to the field of gene technology. Background technique [0002] Chinese hamster ovary (CHO) cells were established in Dr. Theodore T. Puck's laboratory in 1957. They are immortalized non-secreting cells that rarely secrete endogenous proteins; The advantages of closer post-translational modification of human natural proteins, less susceptibility to human virus infection, large-scale culture in serum-free medium with defined chemical composition, etc., are widely used in the field of biopharmaceuticals, and more than 70% of protein drugs are produced . However, as mammalian cells, CHO cells have a long culture cycle and high culture costs. At the same time, the demand for recombinant products such as monoclonal antibodies continues to increase. The continuous growth of demand means that the specific productivity still needs to be ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/90C12N15/85C12N15/55C12N5/10
CPCC07K14/47C12N15/907C12N15/85C12N9/22C12N5/0682C12N2800/107C12N2830/008C12N2510/02
Inventor 陈蕴金坚丁学峰瞿丽丽李华钟蔡燕飞杨兆琪朱景宇鲁晨俞琪
Owner JIANGNAN UNIV
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