Nano-particles capable of improving gene transfection efficiency and preparation method of gene transfection reagent based on particles

A technology of nanoparticle and gene transfection, which is applied in the direction of introducing foreign genetic material using vectors, recombinant DNA technology, etc., and can solve the problem of low transfection rate

Inactive Publication Date: 2014-06-18
常州碳宇纳米科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the invention is to overcome the low deficiencies in the transfection rate of existing artificially synthesized transfection reagents, provide a...

Method used

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  • Nano-particles capable of improving gene transfection efficiency and preparation method of gene transfection reagent based on particles
  • Nano-particles capable of improving gene transfection efficiency and preparation method of gene transfection reagent based on particles
  • Nano-particles capable of improving gene transfection efficiency and preparation method of gene transfection reagent based on particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1. Transfection experiments on H293T cells based on polyethyleneimine (PEI) mixed NONPs gene transfection reagent

[0021] (1) Preparation of polyethyleneimine (PEI) mixed NONPs gene carrier and gene transfection reagent

[0022] Select pEGFP as the reporter gene (DNA), and polyethyleneimine (PEI) as the gene transfection carrier. The atomic force results show that the structure of the PEI-DNA complex is very compact, ranging in size from tens to hundreds of nanometers such as image 3 (a); while the PEI / NONP / DNA complex is relatively loose, independent DNA strands can be seen in the atomic force microscope such as image 3 (b). The illustration shows that NONPs may slightly interfere with PEI-DNA recombination.

[0023] 0.5 μg of pEGFP DNA plasmid, 4 μL of PEI and NONPs were mixed and dissolved in phosphate-buffered saline (PBS) to make a 100 μL solution. Incubate for 30 minutes at room temperature. 1% agarose gum in TAE buffer (containing 40 ml tris, 1 ml...

Embodiment 2

[0033] Embodiment 2. Based on the transfection experiment of the gene transfection reagent of polyethyleneimine (PEI) mixed NONPs to Hela cell

[0034] (1) Preparation of polyethyleneimine (PEI) mixed NONPs gene carrier and gene transfection reagent

[0035] Except that the cultured cells were Hela cells, other raw materials and experimental procedures were the same as in Example 1.

[0036] (2) Evaluation of gene transfection efficiency

[0037] The experimental procedure is the same as in Example 1. After incubation for 48 hours, the transfection efficiency of the Hela cells was detected by flow cytometry. When the nitrogen-to-phosphorus (N / P) ratio (N / P) was 32, the transfection efficiency exceeded 50%. Under the same conditions, the transfection efficiency of the PEI system without NONPs was only 40%. It can be proved that the use of NONPs transfection reagents has increased the transfection efficiency of Hela cells by more than 25%.

[0038] (3) Cytotoxicity evaluatio...

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Abstract

The invention belongs to the technical field of biological medicines and aims at improving the gene transfection efficiency by adopting nano-particles NoNPs released through cells. The nano-particles exist extensively in a biosystem and an extracellular circulatory system, such as bronchoalveolar lavage fluid, body fluid, blood, saliva, cow milk or urine. A non-virus gene transfection vector is doped with the nano-particles so that the DNA (Desoxvribose Nucleic Acid) carrying capacity of the gene transfection vector can be improved remarkably without increase of cytotoxicity. Experimental results indicate that the NONPs, which are extracted from the cow milk and added to the PEI gene transfection vector, are capable of remarkably improving the gene transfection efficiency under the circumstance of hardly increasing the cytotoxicity. The major use of the nano-particles is to serve as the gene transfection reagent which can be used for cell biology research, gene transfection preparation production, gene therapy and the like.

Description

Technical field: [0001] The invention relates to a kind of nano particles (NONPs) naturally produced by organisms, which can be used to prepare high-efficiency gene transfection reagents by means of doping. Background technique: [0002] Gene transfection is a process in which eukaryotic cells obtain new genetic markers due to the incorporation of exogenous DNA, which can be used to produce genetic products such as proteins, RNA, antibodies, etc.; or introduce control genes into cells to regulate the functions of internal genes / enzymes For cell biology research; or to repair lost / damaged genes for gene therapy. Gene transfection technology is not only an important tool for studying transgene and gene expression, but also a key step in gene therapy. An ideal gene transfection reagent should have the following characteristics: efficient transfection; safety; low cytotoxicity; simple method; time-saving and economical. [0003] At present, the main gene transfection reagents ...

Claims

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Application Information

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IPC IPC(8): C12N15/63
Inventor 刘遵峰贾凤美
Owner 常州碳宇纳米科技有限公司
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