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Establishment and use of red fluorescent specific labeled mitochondria cell lines

A red fluorescence, mitochondrial technology, applied in fluorescence/phosphorescence, cells modified by introducing foreign genetic material, and determination/inspection of microorganisms, etc., can solve the problems of being limited to 440-529nm, high background, short excitation and emission wavelength , to achieve the effect of low cost, convenient use, and no influence on the biological activity of cells

Inactive Publication Date: 2009-08-05
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first fluorescent protein is green fluorescent protein (GFP) cloned from jellyfish (Aequorea victoria). Green fluorescent protein has been widely used in protein molecular labeling and intracellular molecular tracking, which has promoted biological research. The disadvantage is that The emission spectrum of these fluorescent proteins is limited to 440-529nm, and the excitation and emission wavelengths are relatively short, which can excite certain substances in cells to produce fluorescence, and the background of intracellular imaging is relatively high

Method used

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  • Establishment and use of red fluorescent specific labeled mitochondria cell lines
  • Establishment and use of red fluorescent specific labeled mitochondria cell lines
  • Establishment and use of red fluorescent specific labeled mitochondria cell lines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] This example describes the screening method of the Vero cell line that specifically labels mitochondria with red fluorescence provided by the present invention.

[0018] (1) Determination of G418 screening concentration for Vero cells

[0019] Inoculate Vero cells in 6-well plates, remove the medium when the cells grow to 80% monolayer, and screen with 200 μg / ml, 400 μg / ml, 600 μg / ml, 800 μg / ml and 1000 μg / ml of G418 medium respectively kill. The medium was changed every 3 days (the concentration of G418 in the medium of each group was not changed), and the culture was continued for 2 weeks. The concentration of G418 for screening and killing Vero cells was determined according to the cell death situation.

[0020] The results showed that the Vero cells were screened with different concentrations of G418-containing medium, the medium was replaced every 3 days, and the culture continued for 2 weeks. According to the cell death, the optimal concentration of G418 for scre...

Embodiment 2

[0025] This example describes the identification of the cell line V-Mito, which specifically labels mitochondria with red fluorescence, provided by the present invention.

[0026] (1) Laser confocal microscope observation

[0027] After the islands of resistant clones were picked and expanded for culture, the cells were planted in a 35mm culture dish with a glass bottom and observed under a confocal laser microscope. The scanning parameters were set as excitation wavelength 488nm, detection excitation wavelength 585nm, resolution 512×512, and 8bit. Fluorescence images were processed by software to obtain the fluorescence intensity of the delineated cell area.

[0028] The result shows: the cell line V-Mito that the red fluorescence specific labeling mitochondria provided by the present invention scans under the laser confocal microscope, visible cell mitochondria are specifically marked by red fluorescent protein (see figure 2 ).

[0029] (2) Identification of mitochondria...

Embodiment 3

[0033] This example describes the stability of the cell line V-Mito specifically labeled with red fluorescence provided by the present invention.

[0034] Culture the above-mentioned cell line V-Mito with red fluorescence-specific labeling of mitochondria in normal medium without G418. After the cells grow to a single layer, carry out subculture, and repeat passage for 30 times in normal medium without G418. , to observe the stability of the cell line. The above-mentioned mitochondria-specific marker cell line V-Mito was passaged for 30 times, and then frozen in a liquid nitrogen tank. After one week, the cells were revived, cultured with normal medium, frozen again in a liquid nitrogen tank, and repeated for 3 times. Second, the expression of fluorescent protein in mitochondrial-labeled Vero cells was observed.

[0035] The results show that: the established mitochondrial marker Vero cell line V-Mito is cultured in a normal medium without G418, repeated passage 30 times and ...

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Abstract

The invention provides Vero clone V-Mito of red fluorescence specificity marking mitochondria. Ultrapure plasmid pDsRed2-Mito transfects Vero cells through lipidosome LipofectamineTM2000 mediate, and is carried out with masculine clone screening through a culture medium with G418 so as to select masculine clones. The masculine clones are enlarged, cultured and built with an organ named V-Mito. The cellular organ mitochondria of the cells can steadily give out red fluorescence under laser light, and the specificity shows the shape of the mitochondria. The cellular organ V-Mito of red fluorescence specificity marking mitochondria has no damage to the cells, does not affect the biology activity of the cells, has steady heritability, can reproduce for many times, can proliferate without limitation, and has the advantages of convenient use and low cost. The cellular organ V-Mito has wide application prospects under normal and abnormal physiological status of cells, and under pathogenicbacteria or virus infection state on research aspects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the establishment and application of a cell line V-Mito that specifically marks mitochondria with red fluorescence. Background technique [0002] Mitochondria are energy conversion organelles in cells, widely present in various eukaryotic cells, and convert proton concentration gradients into ATP through oxidative phosphorylation to provide energy for various life activities of cells. The main function of mitochondria is to carry out oxidative phosphorylation and store calcium ions. In animal cells, more than 80% of ATP is synthesized in mitochondria. It contains its own DNA and is a semi-autonomous organelle of the cell. Mitochondria are generally linear or punctate in shape, and are evenly distributed in the cytoplasm. When the metabolism and energy demand state of the cell changes, the mitochondria can move freely in the cytoplasm. Mitochondria are involved in various biological a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10G01N21/64C12Q1/02
Inventor 于涟魏永伟章晓栋侯贤宏
Owner ZHEJIANG UNIV
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