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An improved non-liposome transfection kit and its application method

A technology of liposome transfection and transfection reagent, which is applied in the biological field, can solve the problems such as the need to improve the transfection efficiency and the low transfection efficiency, and achieve the effects of wide application range, improved transfection efficiency and strong stability.

Active Publication Date: 2021-06-25
广州晶欣生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Chinese patent CN102604114B discloses a star-shaped cationic polymer containing a dendritic polylysine base unit and a preparation method thereof. The star-shaped cationic polymer containing a dendritic polylysine base unit has good biocompatibility and can Biodegradable and its metabolites are less toxic, but when used for gene transfection, the transfection efficiency is low, and the transfection efficiency needs to be improved

Method used

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  • An improved non-liposome transfection kit and its application method
  • An improved non-liposome transfection kit and its application method

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1 Improved non-liposome transfection kit of the present invention and using method thereof

[0036] The improved non-liposome transfection kit of the present invention comprises: star-shaped cationic polymers, sodium chloride, 4-hydroxyethylpiperazineethanesulfonic acid and citric acid-sodium citrate buffer which are packaged independently. Among them, the packaging specifications of star-shaped cationic polymer, sodium chloride, 4-hydroxyethylpiperazineethanesulfonic acid and citric acid-sodium citrate buffer can be set as required, such as: star-shaped cationic polymers can be included in the kit Polymer 10mg, sodium chloride 1.5g, 4-hydroxyethylpiperazineethanesulfonic acid 0.5g and citric acid-sodium citrate buffer 100mL, wherein the concentration of citric acid-sodium citrate buffer is not less than 10mmol / L .

[0037] The method for applying the improved non-liposome transfection kit of the present invention to carry out gene transfection comprises the...

Embodiment 2

[0043] Example 2 Application of the improved non-liposome transfection kit of the present invention to carry out gene transfection to adherent cells

[0044] The method for applying the improved non-liposome transfection kit of the present invention to carry out gene transfection to adherent cells comprises the following steps:

[0045](1) After inoculating cells in a single well of a 6-well plate, culture for 18-24 hours, and transfect when the cell fusion rate reaches 60-80%. Before transfection, replace with 3 mL of fresh medium containing 2% serum Medium, cultured for 1-2 hours; high concentration of serum will affect the transfection efficiency, the use of low serum fresh medium with a serum content of no more than 5% can improve the transfection efficiency;

[0046] (2) dissolving the star-shaped cationic polymer in sterile water and adjusting the pH to 7.0 to obtain the transfection reagent; wherein the concentration of the star-shaped cationic polymer is 1 mg / mL;

[0...

Embodiment 3

[0052] Example 3 Application of the improved non-liposome transfection kit of the present invention to carry out gene transfection to suspension cells

[0053] The method for applying the improved non-liposome transfection kit of the present invention to carry out gene transfection to suspension cells comprises the following steps:

[0054] (1) Add 1.0×10 6 Cells at a concentration of / mL were cultured with 25 mL of fresh medium containing 2% serum for 2 to 3 hours;

[0055] (2) dissolving the star-shaped cationic polymer in sterile water and adjusting the pH to 7.0 to obtain the transfection reagent; wherein the concentration of the star-shaped cationic polymer is 1 mg / mL;

[0056] (3) Sodium chloride and 4-hydroxyethylpiperazineethanesulfonic acid are dissolved in citric acid-sodium citrate buffer, and the pH value is adjusted to 7.2 to obtain the enhancing reagent; wherein, citric acid-sodium citrate buffer The concentration of liquid is 10mmol / L, and the final concentrat...

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Abstract

The invention discloses an improved non-liposome transfection kit and an application method thereof, wherein the improved non-liposome transfection kit of the present invention comprises: a star-shaped cationic polymer used to prepare a transfection reagent; and Sodium Chloride, 4‑Hydroxyethylpiperazineethanesulfonic Acid, and Citric Acid‑Sodium Citrate Buffers for the Enhancement Reagent. The components in the improved non-liposome transfection kit provided by the present invention have good biocompatibility, low immunogenicity, low toxicity and good safety, and are easy to operate and highly reproducible when used for gene transfection. Moreover, the transfection efficiency and cell survival rate are high, and the application range is wide, and it can be applied to gene transfection of various adherent cell lines and suspension cell lines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an improved non-liposome transfection kit and an application method thereof. Background technique [0002] Gene therapy refers to the introduction of exogenous normal genes into target cells to correct or compensate diseases caused by gene defects and abnormalities, and achieve therapeutic purposes. Gene therapy is currently an effective method for the treatment of cancer and congenital immune system diseases. The key to its implementation is to select the appropriate gene carrier and gene introduction method, so that the gene can be safely, efficiently, controllable and stable expression. [0003] Gene vectors widely used at present are mainly divided into two categories: viral vectors and non-viral vectors. [0004] Viruses are the earliest vectors for gene therapy, and they are also the gene vectors that have been studied the most in clinical trials and are closest t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90
CPCC12N15/90
Inventor 魏军李莉好
Owner 广州晶欣生物科技有限公司