Reverse non-viral vector gene transfection method

A non-viral vector and gene transfection technology, which is applied in the field of reverse non-viral vector gene transfection, can solve the problems of poor controllability and short time, and achieve the effects of high efficiency, simple operation and good transfection efficiency

Inactive Publication Date: 2012-08-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the expression time of gene transfection using non-viral vectors is short, and the release of gene drugs by conventional transfection methods is poorly regulated. In order to achieve the sustained and high-efficiency gene expression required in some gene therapy, repeated administration is often required, while the opposite The transfection method can realize the control of the release of the gene complex to meet the needs of long-term drug delivery or smart drug delivery
But there is no report about the reverse non-viral vector gene transfection method in the prior art

Method used

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  • Reverse non-viral vector gene transfection method
  • Reverse non-viral vector gene transfection method
  • Reverse non-viral vector gene transfection method

Examples

Experimental program
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Effect test

Embodiment 1

[0035] (1) Apply an aqueous polymer compound solution with a concentration of PEG (Mw=3400) of 2 mg / ml and a concentration of fibronectin of 20 μg / ml to a 24-well cell culture plate (manufactured by Corning, USA), and remove after incubation for 4 hours. The liquid was washed twice with PBS buffer solution (that is, phosphate buffer solution with a Tween-20 concentration of 0.05% by mass and a pH of 7.4) to prepare a surface-modified cell culture plate.

[0036] (2) Using chitosan as a non-viral carrier, mix it with plasmid DNA containing a gene encoding luciferase (ie, pGL3 plasmid DNA, Institute of Pharmacology, Zhejiang University) to prepare a complex of chitosan and DNA.

[0037](3) The complex of chitosan and DNA was added dropwise to the above-mentioned surface-modified cell culture plate, and after incubation for 2 hours, human cervical cancer HeLa cells were added for transfection.

Embodiment 2

[0039] (1) Soak the hyaluronic acid scaffold in a polymer with a concentration of 60 μg / ml of PLL (Mw=100,000) and a polypeptide containing RGD sequence (GRGDSP, synthesized by Shanghai Sangon Bioengineering Co., Ltd.) In the compound aqueous solution, the liquid was removed after incubation for 1 h, and washed twice with PBS buffer solution to prepare the surface-modified hyaluronic acid scaffold.

[0040] (2) Lipofectamine2000 (Invitrogen Company) was used as a non-viral vector and mixed with pGL3 plasmid DNA to prepare a complex of Lipofectamine2000 and DNA.

[0041] (3) The complex of Lipofectamine2000 and DNA was added dropwise onto the surface-modified hyaluronic acid scaffold, incubated for 0.5 h, and then added to human liver cancer HepG2 cells for transfection.

Embodiment 3

[0043] (1) The concentration of anionized gelatin (Mw=50,000) is 500 μg / ml, and the concentration of protamine (Sigma company) is 200 μg / ml of polymer compound aqueous solution coated on the cell culture dish (produced by Hangzhou Shengyou Company) ), after incubation for 12 h, the liquid was removed, washed twice with PBS buffer solution, and the surface-modified cell culture dish was prepared.

[0044] (2) to HD (produced by Roche) as a non-viral vector, prepared by mixing with pGL3 plasmid DNA Complexes of HD and DNA.

[0045] (3) Will The complex of HD and DNA was added dropwise onto the above-mentioned surface-modified cell culture dish, and after incubation for 5 hours, leukemia K562 cells were added for transfection.

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Abstract

The invention discloses a reverse non-viral vector gene transfection method which comprises the following steps: (1) leading water solution of a polymer compound to be in contact with a cell culture vessel or a biological material cell culture support, removing liquid after incubation and preparing the surface modified cell culture vessel or the surface modified biological material; (2) mixing a non-viral vector with genes, and preparing a compound of the non-viral vector and the genes; and (3) dripping the compound of the non-viral vector and the genes into the surface modified cell culture vessel or the surface modified biological material, further adding cells to be transfected after the incubation and carrying out the gene transfection. The method has the advantages of simple operation and low cost, and can significantly improve the gene transfection efficiency of the non-viral vector under the situation that serum exists and control the release of the gene compound.

Description

technical field [0001] The invention relates to the technical field of gene transfection, in particular to a method for reverse non-viral vector gene transfection. Background technique [0002] Since gene therapy first entered clinical trials in 1989, it has experienced nearly two decades of development, and its application has also expanded from the initial treatment of genetic disorders to the prevention and treatment of acquired diseases. So far, thousands of gene therapy cases have been reported. Treatments are approved to enter clinical trials. Gene recombination and transfection technologies have also entered more and more research fields, such as tissue engineering and regenerative medicine. For gene recombination and gene transfection, the construction of a vector that is safe and can produce genes with high efficiency and localized expression is the key. As we all know, there are two types of gene therapy vectors: viral vectors and non-viral vectors. Although vira...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/87C12N15/85
Inventor 高建青何彩霞胡瑜兰
Owner ZHEJIANG UNIV
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