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DNA Transmethylase defective CHO (Chinese hamster ovary) cell line and preparation method and application thereof

A methyltransferase and cell line technology, applied in the field of genetic engineering, can solve the problems of unstable expression, inability to meet vaccine requirements, and low expression level.

Inactive Publication Date: 2018-03-23
XINXIANG MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current CHO cell expression system has problems such as low expression, especially unstable expression, and cannot meet the growing needs for vaccine development, clinical treatment, and gene therapy.

Method used

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  • DNA Transmethylase defective CHO (Chinese hamster ovary) cell line and preparation method and application thereof
  • DNA Transmethylase defective CHO (Chinese hamster ovary) cell line and preparation method and application thereof
  • DNA Transmethylase defective CHO (Chinese hamster ovary) cell line and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Knockout of CHO cell Dnmt3a gene

[0030] 1.1 Determine the target site for the candidate gene

[0031]Primers (D3a-Ex1seq-L:5′-GACCACAAGAATTCCGGCTC-3′, D3a-Ex1seq-R: 5'-CGTGTGTGAATCTGTGTGGG-3') PCR amplification of the corresponding gene fragment was carried out, and the accuracy of the sequence was verified by cloning and sequencing of the PCR amplified fragment. The chimeric single-stranded guide RNA targeting site of the Dnmt3a gene assisted by the online tool (http: / / crispr.mit.edu / ) is as follows:

[0032] D3a-Ex1-98rev: 5'-ATCATCCTCCCGCTCCAAAGTGG-3';

[0033] D3a-Ex1-308fw:5′-TTTGAGGGGTCATCCTTGCAGGG3′

[0034] Two pairs of primers were designed according to the target sequence to construct the targeting sgRNA vector expressing CRISPR / Cas9.

[0035] 1.2 Construction of sgRNA vector

[0036] Use Bbs I restriction endonuclease to linearize the px330 vector plasmid and recover the linear fragment. The sgRNA oligonucleotide single strand anneals to for...

Embodiment 2

[0039] Example 2 Verification of biological characteristics of Dnmt3a-deficient CHO cells

[0040] Using wild-type CHO-K1 cells as a control, the cell growth characteristics of the obtained Dnmt3a-deficient CHO cell clones were verified, including observation of cell morphology and growth status, detection of cell proliferation by CCK-8 method, flow cytometry (FCM) Cell apoptosis was detected to verify whether the Dnmt3a-deficient CHO cell line could undergo normal growth and passage. CCK-8 kit (Cell Counting Kit-8 kit, Beyotime Biological Company) detects cell proliferation (results see figure 2 ) and the results of cell apoptosis detected by flow cytometry (results in image 3 ) suggest that the obtained Dnmt3a-deficient CHO cell line can grow and pass on normally, and the biological characteristics such as cell growth state, morphology, cell proliferation, and cell apoptosis have no significant difference from normal CHO cells.

Embodiment 3

[0041] Example 3 Construction of recombinant expression vectors driven by different promoters

[0042] The present invention uses the eukaryotic expression vector pIRESneo2 (Clontech company) as the parent carrier to construct the eukaryotic expression vector pWTY-02 (see Figure 4 ).

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Abstract

The invention relates to a DNA transmethylase defective CHO (Chinese hamster ovary) cell line and a preparation method and application thereof and belongs to the technical field of gene engineering. DNA Transmethylase Dnmt3a gene of CHO cells is knocked off by means of CRISPR / Cas9 gene editing technique, and screening and identifying are performed to obtain DNA transmethylase Dnmt3a defective CHOcells; the cells are transfected with a eukaryotic expression vector, stably expressed recombinant CHO cell strains are screened, and accordingly a novel CHO cell expression system based on DNA transmethylase deficiency is established. The recombinant gene CHO cell expression system is established via host CHO cell genetic modifications, expression level of recombinant proteins can be significantly increased, the problem of recombinant protein expression instability is solved, and recombinant protein expression stability is improved.

Description

technical field [0001] The invention relates to a preparation method and application of a DNA methyltransferase-deficient Chinese hamster ovary (CHO) cell line, belonging to the technical field of genetic engineering. Background technique [0002] In the 1980s, tissue plasminogen activator (tissue type plasminogen activator, t-PA) was successfully expressed in recombinant Chinese hamster ovary (CHO) cells for the first time and was approved by the US FDA for clinical use. The era of biopharmaceuticals based on CHO cells is coming. At present, the recombinant drug protein produced by CHO cells has accounted for nearly 70% of the production of mammalian cells. However, the current CHO cell expression system has problems such as low expression, especially unstable expression, and cannot meet the growing demand for vaccine development, clinical treatment, and gene therapy. Therefore, using gene and cell engineering methods to study the recombinant expression of CHO cells is of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/113C12N15/90C12N15/85C12N15/11C12R1/91
CPCC12N9/1007C12N15/113C12N15/85C12N15/907C12N2310/10C12Y201/01072
Inventor 贾岩龙郭潇王天云安文琪马超援路江涛邱乐乐
Owner XINXIANG MEDICAL UNIV
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