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Method for enhance transport capability of gene mediated by non-viral vector

A non-viral and genetic technology, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, microorganisms, etc., can solve problems such as safety hazards and transportation capacity constraints, and achieve high safety and gene transportation capabilities. Improvement, low toxicity effect

Inactive Publication Date: 2009-01-07
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Viral vectors Viral vectors mainly include: retroviral vectors (CN1871356, CN1159210, CN1302334), lentiviral vectors (CN1688323, CN1487841, CN1668751, CN1633495, CN101186915), adenoviral vectors (CN1857738, CN1650021, CN157012 , CN1147837, CN1113390, CN1218512, CN1314948), etc., although viral vectors have higher gene delivery capacity, there are hidden dangers of safety
Non-viral carrier includes: the carrier of cationic liposome core (such as Lipofectamine2000), the carrier of liposome core (FuGene6), the carrier of cationic polymer core (CN101016550), polyamine carrier, peptide carrier (CN1821399), Composite carriers (CN101016549) and other biological material carriers (CN1786163, CN1375334), etc., although they have higher safety than virus carriers, their transport capacity has always restricted their application

Method used

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  • Method for enhance transport capability of gene mediated by non-viral vector
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  • Method for enhance transport capability of gene mediated by non-viral vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Applying a compound that promotes the level of autophagy at the same time as non-viral vector-mediated gene transport [use cationic liposome as gene transport carrier, Luciferase as reporter gene, rapamycin (rapamycin) and Trehalose is a compound that increases the level of autophagy in cells].

[0028] Cells were seeded in 24-well cell culture plates at a density of approximately 3-5×10 4 / well, cultivated overnight, and set aside.

[0029] Prepare the transfection complex, the method is as follows: 1.5 μl of Lipofectamine2000 per well, dissolved in 100 μl DMEM (with serum and no antibiotics) medium, mixed well and incubated at room temperature for 5 min, DNA was 2 μg per well, dissolved in 100 μl DMEM (with serum and no antibiotics) Antibiotics) medium, mix well and incubate at room temperature for 5 minutes, then the two dissolve and mix evenly, and incubate at room temperature for 20-25 minutes.

[0030] The transfection process method is as follows: ov...

Embodiment 2

[0036]Example 2: Applying a compound that promotes the level of autophagy at the same time as the non-viral carrier-mediated gene transport [Using polyamine transfection reagents as the gene transport carrier, GFP as the reporter gene, rapamycin (rapamycin ) and trehalose (trehalose) as compounds that increase the level of autophagy].

[0037] The cells were seeded in a 24-well cell culture plate at a density of about 3-5×10 4 / well, cultured overnight, and ready for use (same as Example 1).

[0038] The preparation of the transfection complex and the transfection process were the same as in Example 1.

[0039] The detection method is as follows:

[0040] (1) The cells were collected after 24 hours of culture, and the cells were lysed with a cell lysate (Beiyuntian Company), boiled in water for 10 minutes, and then transferred to a nylon membrane (Amersham Company) by wet transfer after 12% SDS-PAGE electrophoresis. Western blot was performed using anti-GFP (Santa Cruz Compa...

Embodiment 3

[0043] Example 3: Introducing a gene that increases the level of autophagy at the same time as the gene transport mediated by a non-viral carrier (liposome is used as a gene delivery carrier, Luciferase is used as a reporter gene, and p53 is used as a gene that improves the level of autophagy) .

[0044] Cells were seeded in 24-well cell culture plates at a density of approximately 3-5×10 4 / well, cultured overnight, ready for use (same as Example 1).

[0045] The preparation of the transfection complex was as follows: 1.5 μl of Lipofectamine2000 per well was dissolved in 100 μl of DMEM (containing serum and no antibiotics) medium, mixed well and incubated at room temperature for 5 min, the reporter gene DNA was 2 μg per well, and the p53 expression plasmid was 2 μg per well. Dissolve 1 μg of wells (the control group is p53 blank vector plasmid) in 100 μl DMEM (containing serum and no antibiotics) medium, mix well, incubate at room temperature for 5 minutes, and then the two ...

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Abstract

The invention relates to a method for improving the gene transportation capacity of a non-viral carrier medium which improves the autophagy level of a cell simultaneously or before transporting the gene of the non-viral carrier medium in 2 hours. The method for improving the autophagy level of the cell includes the physical, chemical and biological stimulations which cause the autophagy level of the cell to be improved. The invention has remarkable effects for improving the gene transportation capacity of the non-viral carrier medium by improving the autophagy level of the cell, in particular to the primary cells that are hard to be transfected [like osteoblast, myotube and MEF] in particular. Simultaneously the poison of the utilized non-viral carrier is lower and the safety is high.

Description

technical field [0001] The technology belongs to the field of gene delivery. It can improve the ability of non-viral vector-mediated gene delivery by increasing the level of autophagy, and has low cytotoxicity. Background technique [0002] Gene delivery is the technical basis of gene therapy, and the ability of gene delivery directly affects the efficiency of gene therapy. Currently, the vectors for gene delivery mainly include viral vectors and non-viral vectors. Viral vectors Viral vectors mainly include: retroviral vectors (CN1871356, CN1159210, CN1302334), lentiviral vectors (CN1688323, CN1487841, CN1668751, CN1633495, CN101186915), adenoviral vectors (CN1857738, CN1650021, CN157012 , CN1147837, CN1113390, CN1218512, CN1314948) etc., although viral vectors have higher gene delivery capacity, there are potential safety hazards. Non-viral carrier includes: the carrier of cationic liposome core (such as Lipofectamine2000), the carrier of liposome core (FuGene6), the car...

Claims

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Application Information

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IPC IPC(8): C12N5/00C12N15/63
Inventor 满娜温龙平郑芳于乐
Owner UNIV OF SCI & TECH OF CHINA
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