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Monoclonal cell as well as preparation method and application thereof

A monoclonal, cell technology, applied in the field of cell engineering, can solve the problem of difficult and high expression of AhR

Inactive Publication Date: 2017-12-19
THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to provide a monoclonal cell and its preparation method and use in order to overcome the problem of difficult stable and high expression of AhR in the prior art

Method used

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  • Monoclonal cell as well as preparation method and application thereof
  • Monoclonal cell as well as preparation method and application thereof
  • Monoclonal cell as well as preparation method and application thereof

Examples

Experimental program
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preparation example Construction

[0029] The method for preparing monoclonal cells provided by the invention is characterized in that the method comprises:

[0030] (1) Lentiviral packaging of the plasmid carrying the AhR-encoding gene;

[0031] (2) transfect cells with successfully packaged lentivirus;

[0032] (3) culturing the transfected cells in a medium containing puromycin, thereby screening to obtain monoclonal cells.

[0033] In step (1), the plasmid carrying the gene encoding AhR is preferably a GV358 vector with the gene encoding the aryl hydrocarbon receptor inserted between the BamHI and AgeI restriction sites. The method for obtaining the plasmid can be a conventional method, for example, the AhR gene (such as NM_013464) can be amplified by PCR in a cDNA library (primers used are shown in SEQ ID NO: 1 and 2), and the amplified product The GV358 vector was digested with BamHI and AgeI respectively, purified and recovered, then connected into the GV358 vector with T4 ligase, transformed and scree...

Embodiment 1

[0040] This example is used to illustrate the obtaining of the cell line of the present invention.

[0041](1) Experimental materials: RAW264.7 cell line and HEK293T cell line were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences and preserved by the Field Surgery Research Institute of Chongqing Third Military Medical University; GV358 vector and virus infection reagent ENi.S. were purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd.; BamHI, AgeI restriction endonuclease, and T4 DNA ligase were purchased from NEB in the United States; agarose gel DNA recovery kit and plasmid mini-prep kit were purchased from Beijing Tiangen; Su was purchased from Shanghai Sangong; according to the full-length sequence of mouse AhR (NM_013464) reported in the NCBI database, primers P1 / P2 were designed, the forward primer was 5'-GAGGATCCCCGGGTACCGGTCGCCACCATGAGCAGCGGCGCCAACATC ACC-3' (SEQ ID NO: 1), the reverse The primer is 5'-...

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Abstract

The invention relates to the field of cell engineering, and discloses a monoclonal cell as well as a preparation method and application thereof. The preservation number of the monoclonal cell is CCTCC NO:C2017118. The invention also discloses a method for expressing an aromatic hydrocarbon receptor. The method comprises the step of culturing the cell. In addition, the invention also discloses the application of the cell on in-vitro study of regulating an inflammatory cell factor through the aromatic hydrocarbon receptor. According to the monoclonal cell as well as the preparation method and the application thereof provided by the invention, a monoclonal cell strain for stably expressing AhR is obtained, the cell character is uniform, and the gene expression of the cell in a passage process keeps stable, so that an obtained result is more stable and reliable through utilizing the monoclonal cell strain for stably expressing the AhR for conducting an experiment.

Description

technical field [0001] The invention relates to the field of cell engineering, in particular to a monoclonal cell and its preparation method and use. Background technique [0002] Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that not only participates in the metabolism of poisons and foreign substances, but also regulates the body's immune response and biological rhythms. In recent years, studies have shown that AhR plays a prominent role in mediating immune and inflammatory responses, and its mechanism needs to be further elucidated. AhR can regulate the functions of T cells, B cells, dendritic cells (DC), natural killer cells and neutrophils, and also plays an important role in the inflammatory response of macrophages, but due to the lack of experimental The related research of the model is not deep enough. Since macrophages are highly differentiated mature cells, the efficiency of transient transfection is extremely low and it is easy to ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12P21/02G01N33/68G01N33/569
CPCC07K14/705C12N5/0694C12N15/86C12N2510/00C12N2740/15043C12P21/02G01N33/56966G01N33/6869
Inventor 朱俊宇马晓媛田李星梁华平
Owner THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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