A method for improving the efficiency of chitosan transporting siRNA in vitro

A technology of chitosan and chitosan solution, which is applied to other methods of inserting foreign genetic materials, recombinant DNA technology, etc., can solve the problems of increasing economic costs and poor efficiency, and achieves strong practicability, simple operation, and low cost. cost effect

Active Publication Date: 2021-09-21
FOURTH MILITARY MEDICAL UNIVERSITY
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Problems solved by technology

[0003] However, although chitosan / siRNA has been proven to have a certain gene silencing effect, the overall efficiency is not good
Therefore, most scholars have abandoned chitosan, and some scholars have tried to chemically modify chitosan to improve its transfection efficiency, but these modifications have virtually increased the economic cost.

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  • A method for improving the efficiency of chitosan transporting siRNA in vitro
  • A method for improving the efficiency of chitosan transporting siRNA in vitro
  • A method for improving the efficiency of chitosan transporting siRNA in vitro

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Embodiment Construction

[0030] The present invention will be further described in detail below in conjunction with specific embodiments, which are explanations of the present invention rather than limitations.

[0031] The method for improving chitosan in vitro transport siRNA efficiency disclosed by the invention comprises the following steps:

[0032] (1) prepare chitosan and siRNA solution

[0033] ①. Prepare 0.2M acetic acid and 0.2M sodium acetate respectively, and mix the two according to the ratio of 1:10 to form a buffer solution with a pH of 5.5; weigh an appropriate amount of chitosan and dissolve it in the buffer solution so that the concentration is 1mg / ml, and the shell The molecular weight (kDa) / deacetylation degree (%) of the polysaccharide is 30 / 85, 30 / 95, 150 / 85, 150 / 95 respectively; use a disposable needle filter with a pore size of 0.22 μm to filter and sterilize;

[0034] ②. The siRNA (siGFP) sequence targeting green fluorescent protein GFP used in the experiment

[0035] Justic...

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Abstract

The invention discloses a method for improving the efficiency of chitosan transporting siRNA in vitro, and belongs to the technical field of in vitro siRNA transport. Include the following steps: 1) adopt the method for directly mixing to prepare chitosan / siRNA complex; 2) form the chitosan / siRNA complex in step 1) in Hank's buffered saline solution (HBSS) containing calcium and magnesium transfected cells. At the same time, the method of the present invention also proposes a "one-step method" transfection, that is, directly adding chitosan solution and siRNA in the same proportional amount to HBSS containing calcium and magnesium to transfect cells without premixing. The method is simple in operation and strong in practicability. The method can realize the high-efficiency transfer of siRNA by chitosan in various cell lines, and greatly reduces the cost of the siRNA transfer carrier in vitro.

Description

technical field [0001] The invention belongs to the technical field of in vitro siRNA transfer, relates to a method for optimizing chitosan in vitro transfer, in particular to a method for improving the efficiency of chitosan in vitro transfer of siRNA. Background technique [0002] RNA interference technology is currently the most widely used gene negative intervention method, which can not only be used as a laboratory gene silencing tool, but also widely used in the research and development of siRNA-related therapeutic drugs. In the research of RNA interference technology, the development of efficient, safe and economical siRNA delivery carrier is the main direction of research. At present, there are many commercial siRNA transfer carriers in vitro, including lipofectamine 2000, RNAiMAX, N-TER Nanoparticle siRNA TransfectionSystem, Magic TM siRNA Transfection Reagent, jetPEI, etc., but they have the disadvantages of high price and cumbersome operation. Therefore, a lot o...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/87
CPCC12N15/87
Inventor 宋文张玉梅雒静何奕德
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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