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Gene transporter and its preparation method and application

A transporter and gene technology, applied in the field of medicine, can solve the problems of inapplicability to embryonic cells, and achieve the effects of low toxicity, good dispersibility, and small particle size

Active Publication Date: 2022-03-22
FUYANG NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, based on the specificity of embryonic cells, the vectors provided by these traditional techniques are not suitable for embryonic cells

Method used

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  • Gene transporter and its preparation method and application
  • Gene transporter and its preparation method and application
  • Gene transporter and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0036] The preparation method of the above-mentioned gene transporter, the preparation method comprises: adding the nano-metal particle solution to the solution of the polyethyleneimine, stirring, and the self-assembly of the polyethyleneimine and the nano-metal particle to form a The gene transporter of the nucleocapsid structure.

[0037] Preferably, the mass percent content of polyethyleneimine in the polyethyleneimine solution is 0.01-1%, and the mass percent content of nano-metal particles in the nano-metal particle solution is 1-3%.

[0038] Preferably, the stirring temperature is 22-27°C, the stirring speed is 700-900r / min, and the stirring time is 45-50h.

[0039] Preferably, the preparation method is further subjected to membrane filtration after stirring, including but not limited to using a 220nm filter membrane. The obtained gene transporter can be stored at low temperature, including but not limited to 4°C.

[0040] The embodiment of the present invention also p...

Embodiment 1

[0050] 1 Materials, equipment and methods

[0051] 1.1 Materials

[0052] Polyethyleneimine (PEI, Sigma-Aldrich company, relative molecular mass: 25000, 1800), chloroauric acid (HAuCl 4 ·3H 2 O, Sigma-Aldrich Company, relative molecular mass 394), green fluorescent protein reporter gene plasmid EGFP (self-constructed), TUNEL cell apoptosis detection kit (Shanghai Beyond Biotechnology Co., Ltd.), Anti-phospho-H2A.X ( Merck Chemical Technology (Shanghai) Co., Ltd.), the rest of the reagents were of domestic analytical grade, and the water used in all experiments was ultrapure water.

[0053] 1.2 Equipment

[0054] Intelligent CNC temperature-controlled magnetic heating stirrer (model ZNCL-BS, Chongqing Dongyue Instrument Co., Ltd.), ultraviolet spectrophotometer (Thermo, the United States), agarose gel electrophoresis instrument (Bio-Rad Company, the United States), laser particle size analysis Instrument (Nano90, Malwern Company, UK), transmission electron microscope (JEOL-...

Embodiment 2

[0123] This example is a modification example of Example 1. The change relative to Example 1 is that in the step "1.3.1 Preparation of Gene Carrier AuNP-PEI", PEI with a molecular weight of 25,000 and PEI with a molecular weight of 1,800 were used and the two The mass ratio of the latter is 1:1.

[0124] See results Figure 12 ,according to Figure 12 , when the mass ratio of the first PEI and the second PEI is 1:1, the proportion of blastocysts showing fluorescent signals is also higher.

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Abstract

The invention relates to a gene transporter and its preparation method and application. The gene transporter includes nano-metal particles and polyethyleneimine, the gene transporter has a core-shell structure, wherein the nano-metal particles form the core of the gene transporter, and the polyethyleneimine contains molecular weight The first polyethyleneimine with a molecular weight of 20000-30000 and the second polyethyleneimine with a molecular weight of 1500-2000, the first polyethyleneimine and the second polyethyleneimine are interwoven and covered on the surface of the core to form Cover the case. After the gene transporter combines with the plasmid inserted into the exogenous target gene fragment through electrostatic adsorption, it can break through the zona pellucida barrier on the outer layer of the plasma membrane of the embryonic cell and enter the embryonic cell, and promote the breakage of the double-strand DNA of the embryonic cell to generate the exogenous target gene fragment conditions for integration. At the same time, the inventors also found that the gene transporter also has the advantages of small particle size, good dispersibility, high stability, and low toxicity.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to a gene transporter and its preparation method and application. Background technique [0002] In biological research-related experiments, it is an important experimental method to introduce specific exogenous genes into mouse early embryo cells. There are various technical methods currently used, but there are some disadvantages, such as: the required instruments are precise and expensive, the technical threshold is high, physical and chemical damage to the embryo is caused, commercial transfection reagents are not applicable and expensive, etc. In addition, embryonic cells are different from somatic cells. There is a layer of zona pellucida on the outer layer of the cytoplasmic membrane. The double-layer biological barrier makes it difficult for foreign genes to enter embryonic cells. [0003] Metal nanomaterials have been widely used in gene therapy and biological research due...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/87
CPCC12N15/87
Inventor 张迪王旗李文雍王荣程小翠李欢欢
Owner FUYANG NORMAL UNIVERSITY
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