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Lentiviral expression vector for permanently labeling nuclei and labeling method

An expression vector and lentivirus technology, applied in the field of biomedicine, can solve the problems of inconvenient living cell division, motion observation, research, inability to achieve permanent nuclear labeling, easy quenching of fluorescent signals, etc., and achieve good application prospects and development space, Wide application and wide range of effects

Inactive Publication Date: 2018-12-28
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Organic fluorescent dyes have the advantage of being more convenient to use, but at the same time there are disadvantages such as easy quenching of the fluorescent signal, non-renewable, and inconvenient observation and research on the division and movement of living cells; at the same time, organic dyes can quickly enter living cells Combined with DNA, it is also regarded as a toxic substance and carcinogen for organisms. During use, it is necessary to pay attention to the handling and disposal procedures to avoid harm to experimenters and pollution to the environment
[0004] With the development of biomedical research, the requirements for cell and nucleus labeling are getting higher and higher, and the existing organic fluorescent dyes cannot meet the requirements of permanent cell nucleus labeling

Method used

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  • Lentiviral expression vector for permanently labeling nuclei and labeling method
  • Lentiviral expression vector for permanently labeling nuclei and labeling method
  • Lentiviral expression vector for permanently labeling nuclei and labeling method

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1 lentiviral expression vector construction

[0044] Design the primers of the nucleotide sequence of EGFP, and add the nuclear localization signal (NLS) sequence shown in SEQ ID NO:4 at the 5' end of the upstream primer, and send it to Shanghai Yingjun Company for synthesis, and the sequence of the upstream primer is as SEQ ID Shown in NO:6, the downstream primer sequence is shown in SEQ ID NO:7. Use the PEGFP-C2 vector as a template to carry out PCR amplification. After the product is subjected to 1% agarose electrophoresis, the target band is recovered and connected to the PCDH-CMV-MCS-EF1-RFP vector treated with XbaI and EcoRI. After transformation Positive clones were extracted and sequenced. The sequencing results were consistent with those shown in SEQ ID NO:5, and the plasmids were extracted. That is, the lentiviral expression vector for labeling cell nuclei of the present invention is obtained.

Embodiment 2

[0045] Embodiment 2 packaging virus

[0046] Passage 293FT cells to 6-well plates one day in advance, so that the cell density is 80% the next day. The next day for transfection: Take a new 1.5ml EP tube, add 100μL Opti-medium, then add 1μg of target plasmid, 0.5μg of packaging plasmid; take a new set of 1.5ml EP tube, add 5μl Liposome 2000 , gently flick the centrifuge tube to mix; after standing still for 5 minutes, add the plasmid mixture to the liposome mixture, flick the centrifuge tube gently to mix, and stand still for 30 minutes. The medium of the 293FT cells was replaced with 2 ml of fresh complete medium without antibiotics, and the liposome-plasmid complex was added. After 6 hours, the medium was changed again. After 48 hours of transfection, observe the fluorescence of 293FT cells, such asfigure 1 As indicated, 293FT cell culture supernatants were collected and centrifuged at 4°C for 5 min to remove cells. Filter the virus with a 0.45 μm filter, aliquot the viru...

Embodiment 3

[0048] Example 3 Obtaining Stable Nuclear Fluorescently Labeled Glioma Cells

[0049] The glioma cells were passaged into 6-well plates one day in advance, so that the cell density was 30% the next day. Replace the medium of the cells in the 6-well plate. Add 500 μl of virus liquid into each 6-well plate, and add Polybrene at the same time, the final concentration is 8 μg / ml, and infect the cells overnight. Discard the medium containing the virus liquid and replace it with the normal complete medium. After obtaining a sufficient amount of cells one week later, the cells positive for EGFP and RFP were sorted by flow cytometry for expanded culture. At the same time, the cell slides were performed to observe the situation of the green fluorescently labeled nuclei, such as figure 2 shown. Obtain fluorescently labeled glioma cells.

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Abstract

The invention belongs to the field of biomedicine, and particularly relates to a lentiviral expression vector for permanently labeling nuclei and a labeling method. A cell is infected with a lentivirus, a nucleus is labeled with a fluorescent protein with a nuclear localization signal sequence, and the entire cell is labeled with another fluorescent protein that emits a different fluorescent signal on a viral vector. According to the method, the nucleus and the cell are marked simultaneously, and the survival, mitosis process and movement of the cell in vitro and in vivo can be safely, permanently and accurately observed. The method has no influence on the growth of cells, and has a good application prospect and development space.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a lentiviral expression vector for permanently marking cell nuclei and a marking method. Background technique [0002] In traditional fluorescent labeling techniques for cell nuclei, organic fluorescent dyes such as 4',6-diamidino-2-phenylindole (DAPI) and internalidine iodide (PI) are mostly used for nuclear labeling. Both DAPI and PI can strongly bind to the chromosomal DNA in the nucleus, so they can be used to localize the nucleus and observe changes in structure and shape. [0003] Organic fluorescent dyes have the advantage of being more convenient to use, but at the same time there are disadvantages such as easy quenching of the fluorescent signal, non-renewable, and inconvenient observation and research on the division and movement of living cells; at the same time, organic dyes can quickly enter living cells Combined with DNA, it is also regarded as a toxic substa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/65C12N5/10
CPCC12N5/0603C12N5/0686C12N15/65C12N15/86C12N2510/00C12N2740/15043
Inventor 谈娟
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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