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Genetic engineering cell strain which is constructed on basis of CRISPR-Cas9 systems and is capable of secreting mouse interleukin-6

A technology of interleukin and genetic engineering, applied in the field of genetically engineered cell lines that can secrete mouse interleukin-6, can solve the problems of retroviruses not having precise editing ability and affecting cell functions, etc., and achieve cost reduction, Broad application prospects and high efficiency

Pending Publication Date: 2018-10-09
湖南艾佳生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Retroviruses do not have precise editing capabilities. Viral elements are randomly integrated into the cell genome, and may be integrated into functional regions (such as integration in the exon or promoter region of a certain gene, which will affect the function of the gene), affect the function of cells

Method used

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  • Genetic engineering cell strain which is constructed on basis of CRISPR-Cas9 systems and is capable of secreting mouse interleukin-6
  • Genetic engineering cell strain which is constructed on basis of CRISPR-Cas9 systems and is capable of secreting mouse interleukin-6
  • Genetic engineering cell strain which is constructed on basis of CRISPR-Cas9 systems and is capable of secreting mouse interleukin-6

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Design and construction of a suitable site-directed cleavage vector

[0037] 1.1 Extract the original plasmid of the cutting vector

[0038] Using the plasmid mini-extraction kit (purchased from Kangwei Century, Cat. No. CW0511C), a small amount of the original plasmid of the cut vector (purchased from addgene, cat.

[0039] 1.2 Restriction cutting the vector original plasmid

[0040]Using an endonuclease (purchased from Neb, catalog number R0539L), the plasmid extracted in 1.1 was digested according to the experimental procedure of the product manual, and the digested product was recovered.

[0041] 1.3 Screening suitable sgRNA target sequence fragments

[0042] 1.3.1 Select sgRNA target sites.

[0043] According to the mouse ROSA26 genome sequence (NCBI Reference Sequence: NC_000072.6) given on the NCBI website, multiple target sites were selected for testing, and only three of the target sites were tested below. The sequence is as follows:

[0044] sg1...

Embodiment 2

[0063] Example 2 Construction of the donor vector with mouse interleukin-6 and antibiotic selection marker (puromycin)

[0064] 2.1 Extract the original plasmid of the donor vector

[0065] Using the plasmid mini-extraction kit (purchased from Kangwei Century, Cat. No. CW0511C), a small amount of the original plasmid of the donor vector (purchased from Addgene, Cat. No. plasmamid#37200) was extracted according to the experimental steps in the product manual.

[0066] 2.2 Digestion of the original plasmid of the donor vector

[0067] Using endonucleases (purchased from Neb, product numbers R3103S and R0146S), the plasmid extracted in 2.1 was double-digested according to the experimental procedure in the product manual, and the digested products were recovered.

[0068] 2.3 Cloning of mouse interleukin-6 protein coding region sequence

[0069] 2.3.1 Collect mouse SP2 / 0 cells;

[0070] 2.3.2 Use the Trizol method to extract mRNA;

[0071] 2.3.3 Using the mRNA Reverse Transcri...

Embodiment 3

[0095] Example 3 Electroporation of targeted cleavage vectors and donor vectors into mice and Sp2 / 0 myeloma cells

[0096] 3.1 Culture mouse SP2 / 0 myeloma cells

[0097] Resuscitate and culture mouse SP2 / 0 myeloma cells (purchased from ATCC, Cat. No. CRL-1581). See the table below for cell culture media.

[0098] Table 1 Components of Complete Medium for Mouse Myeloma Cells

[0099]

[0100]

[0101] 3.2 Setting the parameters of the electrorotator

[0102] The present invention adopts H1 new high-efficiency cell electrotransfection instrument (purchased from Suzhou Yida Biotechnology Co., Ltd.), and uses the following experimental parameters to conduct electrotransfection experiments.

[0103] Table 2 H1 electrorotor SP2 / 0 parameter list

[0104]

[0105] Note: The electroporation volume is 60ul, and 5 duplicate holes are set for each group.

[0106] 3.3 Electroporation of SP2 / 0 cells

[0107] 3.3.1 Cell count

[0108] Observe the cells under a microscope, col...

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Abstract

The invention provides a genetic engineering cell strain which is constructed on the basis of CRISPR-Cas9 systems and is capable of secreting mouse interleukin-6. Target sequences of sgRNA for specifically targeting mouse ROSA26 genes are shown as SEQ ID NO.1, and site-specific cleavage vectors capable of simultaneously expressing the sgRNA and cas9 proteins are further constructed; donor vectorswith mouse interleukin-6 and ROSA gene homogenous sequences are further constructed, site-specific integration is carried out on the mouse interleukin-6 at ROSA26 sites of mouse cells SP2 / 0, selectionis carried out by the aid of selection markers to obtain homozygous cell strains, and the genetic engineering cell strain with a mouse interleukin-6 high-expression function can be obtained. The genetic engineering cell strain has the advantage that the genetic engineering cell strain can be used for producing hybridoma cells with high-proportion secretion capacity and has a broad application prospect in the field of monoclonal antibody preparation.

Description

technical field [0001] The invention belongs to the field of genetic engineering and genetic modification, in particular to a genetically engineered cell strain capable of secreting mouse interleukin-6 constructed on the basis of a CRISPR-Cas9 system. Background technique [0002] CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) system is a prokaryote-specific immune system against exogenous genetic material, through sequence-specific RNA-mediated cutting and degradation of exogenous DNA, including Phage and foreign plasmids can cause the loss or partial loss of the target gene function. The CRISPR / Cas system can be used as a site-specific gene editing system. Its biggest features are simple operation, low cost, and high efficiency. It is a new gene editing technology newly discovered in the past two years and widely used in basic research. In the CRISPR / Cas9 system, crRNA (CRISPR-derived RNA) combines with tracrRNA (trans-activatin...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22C12N5/10C12N15/06C12P21/08
CPCC12N5/0693C12N9/22C12N15/02C12N15/907C12N2510/00C12N2510/02
Inventor 艾立平蒋明贵
Owner 湖南艾佳生物科技股份有限公司
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