Efficient HaCaT cell transfection method

A cellular and efficient technology, applied in the biological field, can solve the problems of low efficiency, failure to meet experimental requirements, and troublesome experimental operation process.

Inactive Publication Date: 2015-09-16
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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AI Technical Summary

Problems solved by technology

[0004] The cell membrane of HaCaT cells is relatively thick, difficult to digest, and even more difficult to transfect. It is very difficult to transfect foreign DNA into HaCaT cells through liposomes. The transfection efficiency is very low and often fails to meet the experimental requi

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  • Efficient HaCaT cell transfection method
  • Efficient HaCaT cell transfection method

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Embodiment 1

[0014] 1. Inoculate HaCaT cells in a 12-well plate with a plating density of about 40-50%, and culture in a 37°C incubator for 12-24 hours;

[0015] 2. Cell transfection group: GFP plasmid transfection group (GFP is green fluorescent protein, transfection efficiency can be judged according to fluorescence intensity), 0.005% TX-10+GFP plasmid transfection group. The former group was set as the control group and the latter as the experimental group.

[0016] 3. Cell transfection steps: a. Dilute polyethylene glycol octylphenyl ether (TX-10) to 0.1% (0.1g / 100ml) with complete medium (DMEM+10%FBS), i.e. complete culture per 100ml Add 0.1g of polyethylene glycol octylphenyl ether to the base, miscible); b. Take a 1.5ml EP tube, add 100μl DNA concentration buffer (EC buffer), 3.2μl Enhancer and 600ng GFP plasmid, Let it stand for 5 minutes; c, add 10 μl Effectene, gently pipette evenly, and let it stand for 10 minutes; d, add 290 μl of complete medium to make a total volume of 400 ...

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Abstract

The invention discloses an efficient HaCaT cell transfection method. In the process that transfection plasmids enter a HaCaT cell through a liposome method, TX-10 is added in a transfection system of the HaCaT cell. As the TX-10 is added in the transfection process, permeability of a eukaryotic cell membrane is improved, and a liposome-DNA compound is promoted to effectively enter the cell. The method is very effective for HaCaT cells thick in cell membrane and difficult to transfect through a traditional liposome method and improves the transfection efficiency by several times. The method also has the applicability for other cells thick in cell membrane and difficult to transfect; as polyethylene glycol octylphenol ether has toxicity to cells when the use amount of polyethylene glycol octylphenol ether is high and the effect is not obvious when the use amount is low, the use amount of the polyethylene glycol octylphenol ether needs to be considered. For HaCaT cells, the best dosage of the TX-10 is 0.005%; under the best dosage, the death rate of HaCaT cells is lowest and the transfection efficiency is highest.

Description

Technical field: [0001] The invention belongs to the field of biology, and in particular relates to a method for efficiently transfecting HaCaT cells. Background technique: [0002] HaCaT cells are human skin immortalized keratinocytes derived from normal human skin keratinocytes. In view of its advantages of easy culture and multiple passages, it is used as a tool cell instead of human normal keratinocytes. , through the influence of various drugs, compounds, physical and chemical factors on HaCaT cells to study the cell proliferation, cell cycle and skin pathological mechanism of human skin keratinocytes. [0003] Cell transfection refers to the process by which eukaryotic cells acquire new genetic markers due to the incorporation of foreign DNA. There are four main methods for exogenous gene transfection into cells: electric shock method, calcium phosphate method, virus-mediated method and liposome-mediated method. Due to the strict control and difficulty of the experim...

Claims

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Application Information

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IPC IPC(8): C12N15/88
Inventor 李志远李彩月马文波
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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