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Method for transfecting cells by utilizing bacterial magnetic particles-polyethyleneimine/deoxyribonucleic acid (BMPs-PEI/DNA)transfer complex

A technology for transfecting cells and complexes, which is applied in the field of transfecting cells with BMPs-PEI/DNA delivery complexes, can solve problems such as low transfection efficiency, and achieve the advantages of reducing cytotoxicity, reliable technical assurance, and improving transfection efficiency. Effect

Active Publication Date: 2013-07-24
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, the technology of using magnetosomes (BMPs) to connect with polyethyleneimine (PEI) to form a complex carrying target DNA to transfect cells in vitro has only been established in recent years. At present, this technology still faces many problems that need to be solved urgently. The important one is the low transfection efficiency
In addition, when BMPs are used to assist PEI to improve the transfection efficiency, the influence of the formed complex on cell morphology and the transport mechanism and distribution of BMPs-PEI in cells have not been reported in depth.

Method used

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  • Method for transfecting cells by utilizing bacterial magnetic particles-polyethyleneimine/deoxyribonucleic acid (BMPs-PEI/DNA)transfer complex
  • Method for transfecting cells by utilizing bacterial magnetic particles-polyethyleneimine/deoxyribonucleic acid (BMPs-PEI/DNA)transfer complex
  • Method for transfecting cells by utilizing bacterial magnetic particles-polyethyleneimine/deoxyribonucleic acid (BMPs-PEI/DNA)transfer complex

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Optimization of conditions for transfecting cells using BMPs-PEI / DNA transfer complex

[0051] 1. Effect of connecting and incubating BMPs-PEI / DNA ternary gene transfer complex on cell transfection efficiency by vortex method and ultrasonic dispersion method

[0052] BMPs (magnetosomes), PEI (polyethyleneimine) and DNA (pEGFP-N1, purchased from Addgene, USA) should be quantified before connection, and their concentrations should be adjusted to PEI: 2 μg / μl, BMPs: 1 μg / μl, respectively. μl, DNA: 1μg / μl; absorbance OD of DNA 260 / OD 230 ≧1.80.

[0053] Take 0.8 μl of plasmid DNA with a concentration of 1 μg / μl and 0.8 μl of PEI with a concentration of 2 μg / μl and dilute them in 48.7 μl of PBS buffer, and gently vortex to mix. QL-901, produced by Jiangmen Qilinbeier Instrument Factory. Slowly add 49.5 μl of PEI-containing PBS buffer solution to 49.5 μl of DNA-containing PBS buffer solution drop by drop, mix the two according to the volume ratio of 1:1, mix ge...

Embodiment 2

[0071] Example 2 The method of using BMPs-PEI / DNA transfer complex to transfect cells

[0072] Take 0.8 μl of plasmid DNA with a concentration of 1 μg / μl and 0.8 μl of PEI with a concentration of 2 μg / μl and dilute them in 48.7 μl of PBS buffer (0.01M, pH7.26), and gently vortex to mix. Slowly add 49.5 μl of PEI-containing PBS buffer solution to 49.5 μl of DNA-containing PBS buffer solution drop by drop, mix the two according to the volume ratio of 1:1, mix gently, and then incubate at room temperature for 10 minutes to make PEI and DNA Self-assembly begins with the formation of PEI / DNA binary nanocomposites. Take BMPs and add them to the PEI / DNA complex solution that has been connected and assembled, that is, add 1 μl of 1 μg / μl BMPs to 99 μl of PEI / DNA mixture, and finally form a 100 μl mixture to match the quality of BMPs, PEI and DNA. The ratio is 1:1.6:0.8. Use ultrasonic vibration for 45s, mix well, and incubate at room temperature for 20 minutes to fully connect BMPs ...

Embodiment 3

[0074] Embodiment 3 Morphological observation of Hela cells, Hep-G2 cells, Cos-7 cells transfected by the method of the present invention

[0075] This example comprehensively compares the changes in cell morphology and structure caused by the method in Example 2 and other conventional transfection methods. Morphological observation of cells transfected for 48 hours was performed using confocal laser microscopy to analyze the BMPs-PEI / DNA complexes used in cell transfection with other transfection reagents (including PEI and Lipofectamine TM 2000) compared to the difference in damage to cells.

[0076] Morphological observation of the transfected cells by laser confocal microscopy to prove that compared with other transfection reagents, the BMPs-PEI / DNA complex has the least damage to the cells when it is used for cell transfection, cell shrinkage, apoptosis The ratio of the transfected cells is relatively small, and the shape of the cells after transfection is basically int...

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Abstract

The invention provides a method for transfecting cells by utilizing a bacterial magnetic particles-polyethyleneimine / deoxyribonucleic acid (BMPs-PEI / DNA)transfer complex, and belongs to the technical field of the transfection of in-vitro cell genes. The method comprises following steps of: diluting DNA and PEI in a phosphate buffer solution (PBS) respectively, and mixing the DNA and the PEI uniformly to form a PEI / DNA binary nano complex; adding BMPs into a buffer system of the PEI / DNA binary nano complex, performing ultrasound oscillation, and mixing uniformly to form a BMPs-PEI / DNA ternary gene transfer complex; and adding the BMPs-PEI / DNA ternary gene transfer complex into cells to be transfected, and transfecting under the action of a magnetic field. The method is easy to operate and readily available in materials and reagent; and the using amount of the DNA can be reduced and the toxicity of the PEI on the cells can be reduced while the transfection efficiency is improved by the PEI under the assistance of the BMPs, so the method provides a reliable technical assurance for the mature application and large-range popularization of a BMPs-PEI gene delivery system.

Description

technical field [0001] The invention relates to the technical field of in vitro cell gene transfection, in particular to a method for transfecting cells by using BMPs-PEI / DNA transfer complex. Background technique [0002] Magnetosomes (BMPs, Bacterial Magnetic Particles) are novel superparamagnetic nanoparticles synthesized and secreted by magnetotactic bacteria, mainly composed of Fe 3 o 4 Constituted and surrounded by a lipid membrane that resembles a cell membrane. As a real biomineralization synthetic material, since its discovery, BMPs are being favored by more and more researchers due to their great potential in gene therapy. The use of BMPs combined with cationic polymers (such as polyethyleneimine, etc.) to assist in improving transfection efficiency belongs to the category of magnetic transfection. The so-called magnetic transfection refers to a transfection method in which nucleic acid carriers are combined with magnetic (nano) particles to complete gene delive...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63
Inventor 方美英杨万杰康晓龙董新星白莹
Owner CHINA AGRI UNIV
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