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Method for constructing cell line capable of highly expressing PRMT7 and cell line

A cell line, high-expression technology, applied in the field of breast cancer cell line construction, can solve the problem of failing to significantly reduce the mortality of breast cancer patients

Inactive Publication Date: 2020-06-05
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although early diagnosis and treatment measures have improved, the mortality rate of breast cancer patients has not been significantly reduced

Method used

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  • Method for constructing cell line capable of highly expressing PRMT7 and cell line
  • Method for constructing cell line capable of highly expressing PRMT7 and cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1 pCDH-puro-PRMT7 virus packaging

[0014] (1) The desired eukaryotic expression vector for PRMT7 overexpression such as figure 1 , the PRMT7 CDS sequence is described above, and the pCDH- puro-PRMT7 plasmid construction.

[0015] (2) will 3x10 6 293T cells were planted in a 10cm dish, the original medium for cultivating 293T cells was discarded, 4ml of DMEM serum-free medium was added to it, and then temporarily placed at 37°C, 5% CO 2 (CO 2 5% air by volume) in an incubator.

[0016] (3) Take out a 1.5ml Dorf tube, first add 0.5ml DMEM serum-free medium, then mix the transfection system, add 12μg of pCDH-puro-PRMT7, add 9μg of packaging plasmid psPAX2 (Tiandz 60908-6310y), and add packaging plasmid pMD2G (Tiandz 60908-4950y) was added with 3 μg, transfection reagent polyethyleneimine (polyethyleneimine, PEI, Polyscienceas, 23966) was added with 9 μg, all were added, mixed well, and left at room temperature for 15 minutes.

[0017] (4) Add the transfectio...

Embodiment 2

[0022] Example 2 pCDH-puro-PRMT7 virus infects MCF7 cells

[0023] (1) Spread MCF7 cells on a 3.5cm plate one day in advance.

[0024] (2) The next day, when the cell coverage rate was 30%, add 1 ml of virus stock solution to the MCF7 cell culture dish.

[0025] (3) After 8 hours of infection, replace with 2 ml of DMEM medium containing 10% fetal bovine serum in volume concentration.

[0026] (4) After 48 hours, the selection of puromycin was started, the final concentration was 2ug / ml, and the MCF7 cell line stably expressing PRMT7 could be obtained after continuous selection for 3 days. According to the literature "Targeting KDM1A attenuates Wnt / β-cateninsignaling pathway to eliminate sorafenib-resistant stem-like cells inhepatocellular carcinoma" conditions to extract protein, the protein expression of PRMT7 was detected by western blot experiment (such as figure 2 ).

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Abstract

The invention discloses a method for constructing a breast cancer MCF7 cell line capable of stably over-expressing exogenous recombinant plasmid pCDH-puro-PRMT7. The full length of a PRMT7 gene is obtained; the pCDH-puro-PRMT7 plasmid is constructed; a virus is packaged, and cells are infected by the virus; puromycin is screened; and the MCF7 cell line capable of stably over-expressing the PRMT7 is obtained after amplification. The construction of the cell line is good for researching the cell biological functions of the PRMT7, such as the influences of the PRMT7 on the tumorigenic abilities of mice and the influences on the pulmonary metastasis abilities of the mice.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction of a breast cancer cell line with high PRMT7 expression. [0002] technical background [0003] Breast cancer is one of the most common malignant tumors in women, which seriously threatens women's lives. In my country, the annual incidence of breast cancer is increasing year by year. Although early diagnosis and treatment measures have been improved, the mortality rate of breast cancer patients has not been significantly reduced. Therefore, the exploration of the mechanism of breast cancer tumorigenesis has always been the focus of scientific research, which is of great significance for improving the diagnosis and treatment of tumors. [0004] Protein Arginine Methyltransferases (PRMTs) are methyl donor-dependent arginine methyltransferases that catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) onto the nitrogen atom of the arginine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12R1/91
CPCC12N15/85C12N5/0693C12N2800/107C12N2510/00
Inventor 刘扬耿鹏宇
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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