Reagent composition for improving cell transfection efficiency

A technology of transfection efficiency and composition, which is applied in the direction of introducing foreign genetic material, microorganisms, and using microcapsules using vectors, can solve problems such as lack of means, achieve high transfection efficiency, simple operation, and improve transfection and expression effects. Effect

Active Publication Date: 2018-10-30
FUJIAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the popularity of this method is limited by many factors such as electroporation efficiency, cell viability, and differences in experimental instruments.
In addition, primary cells are highly resistant to various transfection methods, making efficient gene editing methods for cells relatively scarce

Method used

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  • Reagent composition for improving cell transfection efficiency
  • Reagent composition for improving cell transfection efficiency
  • Reagent composition for improving cell transfection efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Verifying the Effect of Mixture A on Cell Transfection Efficiency in L929 Cells

[0048] The test method is as follows:

[0049] In this experiment, liposome Lipofectamine 2000 transfection method was used to transfect L929 cells. Seed L929 cells in a 6-well plate according to the appropriate concentration, in the presence of 5% CO 2 Cultivate in a 37°C incubator for 12 hours. When the cells are completely attached to the wall and grow to 60-90%, the transfection can begin; change the medium 1 hour before transfection, and use 1 ml of fresh DMEM complete medium containing 10% FBS Replace the old medium; add mixed solution A to the DMEM complete medium, the ratio of mixed solution to medium is 1:1000; the final concentration of mixed solution A in the medium is 0.25 μM BX795, 5 μM Ruxolitinib, 5 μM Tofacitinib Citrate, 5 nMactinomycin D. Prepare the transfection mixture: in a 1.5 ml round-bottomed sterile tube, mix 40 μl of Opti-MEM solution and 2 μl of Lipo...

Embodiment 2

[0051] Example 2 In L929 cells, the effect of mixing drugs in pairs on transfection efficiency was verified

[0052] The test method is as follows:

[0053] In this experiment, two mixtures of BX795 (final concentration 0.25 μM), Ruxolitinib (final concentration 5 μM), TofacitinibCitrate (final concentration 2.5 μM) and Actinomycin D (final concentration 5 nM) were prepared in proportion to 6 groups, and the control group was The solvent DMSO was added to the complete DMEM medium (1 μl DMSO / ml DMEM medium); the experimental group was L929 cells in which two mixed drugs were added to the complete DMEM medium: BX795+Ruxolitinib, BX795+Tofacitinib Citrate, BX795+Actinomycin D, Ruxolitinib+ Tofacitinib Citrate, Ruxolitinib+Actinomycin D, Tofacitinib Citrate+Actinomycin D.

[0054] In this experiment, liposome Lipofectamine 2000 transfection method was still used to transfect L929 cells. L929 cells were seeded in 6-well plates in 5% CO 2 Cultivate in a 37°C incubator for 12 hour...

Embodiment 3

[0056] Example 3 In L929 cells, verify the influence of three drug mixtures on transfection efficiency

[0057] The test method is as follows:

[0058] In this experiment, any three drug mixtures of BX795 (final concentration 0.25 μM), Ruxolitinib (final concentration 5 μM), TofacitinibCitrate (final concentration 2.5 μM), and Actinomycin D (final concentration 5 nM) were divided into 4 groups in proportion , the systems are: 1 control group, the solvent DMSO (1μl DMSO / ml DMEM) was added to the complete DMEM medium; 2-4 experimental groups were L929 cells mixed with three drugs added to the complete DMEM medium: BX795+Ruxolitinib+ Tofacitinib Citrate, BX795+Ruxolitinib+Actinomycin D, BX795+Tofacitinib Citrate+Actinomycin D, Ruxolitinib+Tofacitinib Citrate+Actinomycin D.

[0059] In this experiment, liposome Lipofectamine 2000 transfection method was still used to transfect L929 cells. Seed L929 cells in a 6-well plate according to the appropriate concentration, in the presen...

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Abstract

The invention relates to a reagent composition for improving the cell transfection efficiency, and belongs to the technical field of biology. The reagent composition comprises two or more of a compound BX975 which is 0.01 to 10 micrometers, a compound Ruxolitinib which is 0.1 to 100 micrometers, a compound Tofacitinib which is 0.1 to 100 micrometers, and a compound Actinomycin D which is 0.1 to 100 nM. Reagent mixed liquor and compounds containing NDA and transfection reagents or lentivirus packaged with destination vector are respectively added into cell culture fluid, so that the cell transfection efficiency is improved. The reagent composition is simple to operate, high in repeatability and large in application range, and is successfully applied to cell lines, such as L929 and BI, and primary cell, such as mice primary lung fibrocyte and T lymphocyte.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a reagent composition for improving cell transfection efficiency. Background technique [0002] Gene therapy is a new research field in contemporary medicine and biology. It introduces normal genes or therapeutic DNA sequences into target cells in a certain way to correct gene defects or exert therapeutic effects, so as to achieve the purpose of treating diseases. In order to achieve the desired therapeutic effect, scientists have been studying effective ways to deliver plasmid DNA. At present, conventional exogenous expression systems are mainly divided into two categories: one is the use of viral vectors, and the mainstream three viral vectors include lentiviral vectors, retroviral viral vectors and adenoviral (AAV) vectors , these approaches are mainstream options for clinical trials such as gene therapy. Viral vectors such as AAV vectors have the characteristics of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N15/88
CPCC12N15/86C12N15/88C12N2740/15043
Inventor 陈骐傅雅娟郑立群
Owner FUJIAN NORMAL UNIV
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