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Method for improving high-molecular polymer carrier gene transfection efficiency based on cell cycle control

A high-molecular polymer and gene transfection technology, applied in the field of biomedicine, can solve the problems of fast tumor cell division and short cell cycle

Active Publication Date: 2016-01-13
宁波宁瑞生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tumor cells also have cell cycle characteristics as described above, but tumor cells divide rapidly and have a shorter cell cycle

Method used

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  • Method for improving high-molecular polymer carrier gene transfection efficiency based on cell cycle control
  • Method for improving high-molecular polymer carrier gene transfection efficiency based on cell cycle control
  • Method for improving high-molecular polymer carrier gene transfection efficiency based on cell cycle control

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Cell culture, cell cycle arrest

[0025] HeLa cells were cultured in RPMI-1640 medium containing 10% FBS in a constant temperature incubator, and the temperature of the incubator was set at 37°C, CO 2 Concentration 5%.

[0026] RO-3306 was purchased from Sigma, and was dissolved in DMSO into a 10 mM stock solution, which was aliquoted and frozen at -20°C. When in use, use RPMI-1640 cell culture medium containing 10% FBS to dilute it to a working concentration of 9uM (that is, to obtain a serum-containing medium containing 9uM RO-3306).

[0027] Cell cycle synchronization method: HeLa cells are plated in a six-well plate, and the number of cells in each well is 1×10 3 Each, volume 2mL. Incubate the cells overnight in a 37°C incubator.

[0028] The synchronization steps are:

[0029] (1) Absorb the original culture medium,

[0030] (2) Add 2mL serum-containing medium containing 9uMRO-3306 to wells that need to be synchronized, add 2mL serum-containing medi...

Embodiment 2

[0032] Embodiment 2EGFP, Luciferase gene transfection

[0033] (1) HeLa six-well plate, the number of cells is 200,000 / well;

[0034] (2) After the cells adhere to the wall, add the synchronization reagent RO-3306 prepared in the embodiment to synchronize for 16 hours;

[0035] (3) After the synchronization is completed, the control group is replaced with a serum-free medium (), and the synchronized group is replaced with a serum-free medium (RPMI-1640) containing RO-3306 at a concentration of 9uM;

[0036] (4) Add the prepared polyplex (PEI-DNA complex) to a six-well plate for transfection,

[0037] DNA-PEI complex configuration:

[0038] Dilute the DNA to 40 μg / mL with pH 7.4 HEPES buffer, weigh an appropriate amount of PEI and dissolve it with HEPES and dilute it to 38.4 μg / mL, mix the two in equal volume, vortex for 10 seconds, and let stand for 30 minutes to form nanoparticles .

[0039] (5) After 3 hours, all serum-free medium (RPMI-1640) was replaced with serum-cont...

Embodiment 3

[0046] Example 3 DNA / PEI complex enters the nucleus process

[0047] In order to further confirm whether the entry of PEI-DNA complex into the nucleus depends on the cell cycle. We simultaneously labeled DNA and PEI for gene transfection. Observe the normal growing cells with confocal, 3 hours after adding the DNA / PEI complex, it is found that most of the complexes are located in the periphery of the nucleus, and only 1%-5% of the cells are in the division phase, and the chromosomes of the cells in the early and middle division phase are shrunk , the cell is raised, and the complex is in the center of the cell, occupying the position of the protocellular nucleus; 5 hours after adding the DNA / PEI complex, only a few complexes enter the nucleus, and it can be observed that DNA-PEI appears in a state of aggregation in the division formation In the nuclei of the two daughter cells (results such as Figure 6A and Figure 6B shown).

[0048] The particle size of the dendritic PE...

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Abstract

The invention discloses a method for improving high-molecular polymer carrier gene transfection efficiency based on cell cycle control. The method includes the steps of synchronizing a target cell to a G2 / M boundary by an RO-3306 synchronization reagent, and then adding a high-molecular polymer carrier-exogenous target gene composite for gene transfection. The method has the advantage that since the RO-3306 is added to synchronize a cancer cell to the G2 / M during gene transfection, the gene transfection efficiency can be improved to a great extent.

Description

technical field [0001] The invention belongs to the field of biomedicine, and particularly relates to a specific enzyme activity inhibitor R0-3306 of cell cycle-dependent kinase 1. Efficiency of gene transfection with polymeric vectors. Background technique [0002] Vectors used for gene transfection can be divided into two categories: viral vectors and synthetic vectors. The earliest vectors used were recombinant viruses. The advantage of using recombinant virus as a vector is high transfection efficiency, but the disadvantage is that there are certain safety hazards. Recombinant viruses may mutate into wild-type pathogenic virions. In addition, the virus itself is immunogenic and may trigger an immune response. Synthetic carriers include liposomes and polymers. Among polymer carriers, cationic polymers are flexible in structure and are expected to provide multiple functions required for efficient gene delivery while maintaining good biocompatibility, low production cos...

Claims

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Application Information

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IPC IPC(8): C12N15/85
Inventor 刘祥瑞周雪飞刘欣祝鼎成周珠贤唐建斌申有青
Owner 宁波宁瑞生物科技有限公司
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