Method for real-time monitoring on gene transfection

A real-time monitoring and gene transfection technology, applied in the field of genetic engineering, can solve the problems of high cost and complicated process, and achieve the effect of low cost, mild conditions, and easy operation and promotion

Inactive Publication Date: 2013-05-29
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the traditional methods for visually tracing the distribution and localization of exogenous transfected genes in cells mainly focus on the application of nanomaterials with optical characteristics for labeling, the process is complicated and the cost is relatively high

Method used

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  • Method for real-time monitoring on gene transfection
  • Method for real-time monitoring on gene transfection
  • Method for real-time monitoring on gene transfection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Synthesis of tetramethylrhodamine isothiocyanate-labeled polyethyleneimine:

[0036] ① Configure a dimethyl sulfoxide solution of polyethyleneimine with a concentration of 17.86mM;

[0037] ②A solution of tetramethylrhodamine tetramethylisothiocyanate in dimethyl sulfoxide with a concentration of 10 mM is prepared;

[0038] ③ According to the molar ratio of polyethyleneimine and tetramethylrhodamine tetramethylisothiocyanate, the dimethyl sulfoxide solution of polyethyleneimine and tetramethylrhodamine tetramethylisothiocyanate were mixed in a ratio of 1:10. The sulfoxide solution was mixed and stirred for 6 hours. After 6 hours, the mixed solution was transferred to a dialysis bag with a molecular weight cut-off of 3500. First, it was dialyzed three times with dimethyl sulfoxide for one day, and then continued to dialyze with ultrapure water for two days. sky.

Embodiment 2

[0039] Embodiment 2: the synthesis of the polyethylenimine of tetramethyl rhodamine isothiocyanate label:

[0040] ① Configure a dimethyl sulfoxide solution of polyethyleneimine with a concentration of 17.86mM;

[0041] ②A solution of tetramethylrhodamine tetramethylisothiocyanate in dimethyl sulfoxide with a concentration of 10 mM is prepared;

[0042] ③ According to the molar ratio of polyethyleneimine and tetramethylrhodamine tetramethylisothiocyanate, the dimethyl sulfoxide solution of polyethyleneimine and tetramethylrhodamine tetramethylisothiocyanate were mixed in a ratio of 1:20 The sulfoxide solution was mixed and stirred for 6 hours. After 6 hours, the mixed solution was transferred to a dialysis bag with a molecular weight cut-off of 3500. It was dialyzed 5 times with dimethyl sulfoxide for 2 days, and then continued with ultrapure water for 3 days. sky.

Embodiment 3

[0043] Embodiment 3: the synthesis of the polyethylenimine of tetramethylrhodamine isothiocyanate label:

[0044]Embodiment 2: the synthesis of the polyethylenimine of tetramethyl rhodamine isothiocyanate label:

[0045] ① Configure a dimethyl sulfoxide solution of polyethyleneimine with a concentration of 17.86mM;

[0046] ②A solution of tetramethylrhodamine tetramethylisothiocyanate in dimethyl sulfoxide with a concentration of 10 mM is prepared;

[0047] ③ According to the molar ratio of polyethyleneimine and tetramethylrhodamine tetramethylisothiocyanate, the dimethyl sulfoxide solution of polyethyleneimine and tetramethylrhodamine tetramethylisothiocyanate were mixed in a ratio of 1:40. The sulfoxide solution was mixed and stirred for 6 hours. After 6 hours, the mixed solution was transferred to a dialysis bag with a molecular weight cut-off of 3500. First, it was dialyzed 4 times with dimethyl sulfoxide for 1.5 days, and then continued to dialyze with ultrapure water fo...

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Abstract

The invention relates to a method for real-time monitoring on gene transfection. Based on polyethyleneimine marked by tetramethyl isothiocyanic acid rhodamine, polyethyleneimine is subjected to tetramethyl isothiocyanic acid rhodamine marking at a room temperature. The method has the advantages of simplicity of synthesis, moderate conditions, low cost and convenience for operation. Plasmid DNA (Deoxyribonucleic Acid) of a fluorescent protein is expressed by electrostatic adsorption of the polyethyleneimine marked by the tetramethyl isothiocyanic acid rhodamine, and the plasmid DNA is transfected into cells; a fluorescence imaging method is used for monitoring the distribution of the polyethyleneimine which is used as a carrier and a plasmid in the cells and the expression of the plasmid in the cells in real time; and the method is simple and easy to realize, is suitable for most of laboratories and is convenient for operation and popularization.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for real-time monitoring of gene transfection. Background technique [0002] With the completion of the Human Genome Project and the advancement of cell biology technology, gene therapy will occupy an important position in the process of overcoming the stubborn disease of human beings, and will become a common treatment method. Gene therapy refers to a new biomedical technology that introduces human normal genes or genes with therapeutic effects into human target cells in a certain way to correct gene defects or exert therapeutic effects, so as to achieve the purpose of treating diseases. Gene transfection technology has been widely used in gene therapy research. Gene transfection refers to the transfer or transport of nucleic acid molecules (genes) with biological functions into cells and the maintenance of biological functions of nucleic acid molecules (genes) in ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/02C08G73/04
Inventor 蔡林涛石碧华龚萍刘朋王碧王亚楠
Owner SHENZHEN INST OF ADVANCED TECH
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