LM3 cell line with Midkine stable low expression and construction method of LM3 cell line

A construction method and low-expression technology, applied in the field of biomedicine, can solve the problems of less Midkine protein, unclear regulation mechanism of Midkine and liver cancer energy metabolism, etc., and achieve high transfection efficiency

Inactive Publication Date: 2017-06-20
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on the function of Midkine mainly occurs in the aspects of tumor proliferation, migration and epithelial-to-mesenchymal transition (EMT), but there are few reports on the biologica

Method used

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  • LM3 cell line with Midkine stable low expression and construction method of LM3 cell line
  • LM3 cell line with Midkine stable low expression and construction method of LM3 cell line
  • LM3 cell line with Midkine stable low expression and construction method of LM3 cell line

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Embodiment 1

[0027] Example 1. Construction of recombinant expression vector pLenti-MDKshRNA

[0028] (1) Purchase the commercially available pSM2 vector with Midkine shRNA sequence from Open Biosystems. The Midkine shRNA gene was amplified by PCR, and the primer sequences were as follows:

[0029] MDKshRNA Forward Primer: 5′-AAGCCCTTTGTACACCCCTAAGCCT-3′

[0030] MDKshRNA Reverse primer: 5′-ACCTGGTGAAACTCACCAGGGATT-3′

[0031] The PCR amplification reaction system is as follows:

[0032]

[0033] PCR reaction conditions:

[0034]

[0035] (2) The above PCR product was cleaned and recovered with TaKaRa MiniBEST DNA Fragment Purification Kit from Treasure Biotech Co., Ltd., and dissolved in 110 μl sterile water. Refer to the instruction manual for the operation steps.

[0036] (3) Perform Mlu I and Xho I double enzyme digestion on the recovered PCR products. The enzyme digestion reaction system is as follows: 50 μl enzyme digestion system, add 5 μl PCR product, 10U Mlu I, 10U Xho ...

Embodiment 2

[0043] Embodiment 2, the establishment of low expression Midkine stable cell line, the specific implementation steps are as follows:

[0044] (1) After extracting a large amount of pLenti-MDKshRNA recombinant plasmid, measure the OD value.

[0045] (2) Inoculate 1×106 HEK293T cells in a 100mm culture dish, add 10ml of DMEM medium containing 10% fetal bovine serum, 1×P / S, and place in 37°C, 5% CO 2 Cultivate in an incubator, and the cell confluence is about 50-70% the next day.

[0046] (3) Cell transfection experiment. Mix the recombinant plasmid pLenti-MDKshRNA and viral packaging plasmids psPAX2 and pVSVG into the Opti-MEM basic culture medium at a mass ratio of 10:9:1. The specific operation is as follows: 3 μg of pLenti-MDKshRNA recombinant plasmid, 2.7 μg of Add the pVSVG packaging plasmid to 580 μl Opti-MEM basic culture medium, mix well, then add 12 μl Transfect Reagent (ThermoFisher) transfection reagent, mix well, let stand at room temperature for 20 minutes; take o...

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Abstract

The invention relates to the field of biomedical research, and in particular to a construction method of an LM3 stable cell line with Midkine low expression. The LM3 cell line with Midkine stable low expression is constructed by virtue of an RNA interference technology. The construction method comprises the following steps: conducting primer design and target fragment amplification, and constructing Midkine shRNA recombinant plasmid; then, collecting a liquid medium containing viruses, filtering the liquid medium, adding the filtered liquid medium to a culture dish inoculated with LM3 cells in advance and conducting co-culture; and finally, conducting Puromycin resistance screening and sub-culturing on the LM3 cells accepting transfection, so that the LM3 stable cell line with Midkine low expression is finally obtained. With the establishment of the cell line, a new experimental material is provided for researching a molecular action mechanism of Midkine in tumors and a regulatory effect of the Midkine in tumor energy metabolism.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an LM3 stable cell line with stable and low expression of Midkine protein and a construction method thereof. Background technique [0002] Midkine protein is an important cytokine with a molecular weight of 13kDa, which can be combined with heparin and is a member of the heparin-binding growth factor family. In recent years, studies have found that Midkine protein is not expressed or lowly expressed in normal human tissues, but overexpressed in a variety of malignant tumors, including liver cancer, pancreatic cancer, breast cancer, and intestinal cancer. Studies have found that Midkine protein has a variety of biological functions, such as promoting cell mitosis, cell proliferation, angiogenesis, inducing cell malignant transformation, anti-apoptosis and other activities, thereby affecting the proliferation, differentiation and apoptosis of tumor cells, and has become a tumor biology ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/12
CPCC12N5/0693C12N15/867
Inventor 朴海龙刘秀梅夏天
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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