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NDUFS3 gene knock-down and/or overexpression stable-transfected strain and construction method thereof

A technology of gene overexpression and construction method, which is applied in the field of NDUFS3 gene knockdown and/or overexpression stable transfection and its construction, to achieve the effect of reducing experimental time, optimizing experimental steps, and shortening screening time

Active Publication Date: 2019-11-12
KUNMING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In addition, the traditional method of constructing stable cell lines can only achieve a maximum transfection rate of 90%, unless complex and difficult experimental methods such as limiting dilution and monoclonal screening can be used to achieve 100% transfection rate

Method used

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  • NDUFS3 gene knock-down and/or overexpression stable-transfected strain and construction method thereof
  • NDUFS3 gene knock-down and/or overexpression stable-transfected strain and construction method thereof
  • NDUFS3 gene knock-down and/or overexpression stable-transfected strain and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The present invention provides a method for constructing a stably transfected strain with NDUFS3 gene overexpression and a stably transfected strain with NDUFS3 gene knockdown or overexpression, which comprises the following steps:

[0052] seed board

[0053] Human melanoma cell lines in logarithmic growth phase, including cell lines SK-MEL-110, A875, A375, digested with trypsin, made into 5×10 with complete medium 3 cells / mL of cell suspension and seeded in 6-well plates. Wait for the cells to attach and begin to divide and proliferate.

[0054] High titer virus transfection

[0055] Introduce the target gene into the cell line:

[0056] a) Overexpression of the lentiviral packaging vector: the primer sequence for expressing the target gene includes an upstream primer as shown in SEQ ID No.1 and a downstream primer as shown in SEQ ID No.2. The target gene is amplified by PCR using the upstream primer shown in SEQ ID No.1 and the downstream primer shown in SEQ ID N...

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Abstract

The invention discloses a NDUFS3 gene knock-down and / or overexpression stable-transfected strain and a construction method thereof, and relates to the technical field of genetic engineering. The construction method particularly comprises the steps of inserting a target gene into a cell strain, and screening the transfected cell strain through 8-10<mu>g / mL of puromycin to obtain the NDUFS3 gene knock-down and / or overexpression stable-transfected strain. Compared with a conventional stable-transfected cell strain construction method, the construction method provided by the embodiment of the invention can reduce and optimize test steps, the transfection efficiency is improved, viruses being high in titer can be selected in a disposable manner for transfection, and through high-concentration drug screening, the test time can be reduced, the screening time can be shortened to 5 days, and low-concentration puromycin is not needed for maintaining.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular, to a stably transfected strain of NDUFS3 gene knockdown and / or overexpression and a construction method thereof. Background technique [0002] The traditional method of stably transfecting cell line construction usually requires preliminary experiments to detect the MOI value of each cell line and the half-lethal concentration of puromycin, and then calculate the virus concentration at the time of cell transfection according to the MOI value, according to the half-lethal concentration of puromycin For drug screening, the MOI value and the half-lethal concentration of puromycin are different for each cell line, so that every time a new cell line is transfected, tedious pre-experiments are required. [0003] In the traditional method of constructing stable transfected cell lines, 72 hours after virus transfection, the half-lethal concentration of puromycin obtained in the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/113C12N15/867C12R1/91
CPCC07K14/47C12N5/0686C12N15/113C12N15/86C12N2310/14C12N2510/00C12N2740/15043
Inventor 朱月春易子寒蒋露熊国行赵雷况应敏张巧
Owner KUNMING MEDICAL UNIVERSITY