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Fluorescent BV2 tool cell and construction method and application thereof

A construction method and BV2 technology, applied in the field of cell biology, can solve the problems of no fluorescence of BV2 cells, inconvenient in vivo observation, etc., and achieve the effects of convenient observation, good effect and high purity

Pending Publication Date: 2021-03-19
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, ordinary BV2 cells do not have fluorescence, and traditional observation methods require immunofluorescence staining before observation, which is inconvenient for in vivo observation. Therefore, there is an urgent need for a BV2 tool cell that can autofluoresce and can be stably passaged

Method used

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  • Fluorescent BV2 tool cell and construction method and application thereof
  • Fluorescent BV2 tool cell and construction method and application thereof
  • Fluorescent BV2 tool cell and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of a lentiviral gene carrier plasmid sequentially connected with a promoter and a green fluorescent marker gene

[0043]Design the DNA fragment used for the construction of the vector: PacI-CMV-EGFP-EcoRI, with PacI and EcoRI restriction sites at both ends of the fragment, and use two restriction endonucleases, PacI and EcoRI, to carry out digestion with the vector plasmid connection to obtain the objective lentiviral plasmid, the structure of the lentiviral gene carrier plasmid is as follows figure 2 As shown, it carries the CMV promoter followed by the EGFP fluorescent gene.

Embodiment 2

[0044] Construction and acquisition of embodiment 2 lentivirus

[0045] 1. Materials and reagents:

[0046] Full medium: DMEN+10%FBS+1% penicillin-streptomycin double antibody

[0047] Transfection medium: DMEM+10% FBS (without antibody)

[0048] Maintenance medium: DMEM + 2% FBS + 1% penicillin-streptomycin double antibody

[0049] Transfection reagent: Calcium Phosphate Mammalian Transfection Kit (Promega, E1200)

[0050] Other reagents: Chloroquine (Sigma, C6628, 25g)

[0051] Plasmid: lentiviral gene carrier plasmid (prepared in Example 1), pDelta 8.74 helper plasmid, pMD2.G plasmid (VSVg, coat protein), co-transfection of three kinds of plasmids to prepare lentivirus.

[0052] 2. Construction of lentivirus

[0053] Including the following steps:

[0054] 1. Culture 293T cells with full medium;

[0055] 2. 293T cells were inoculated into 20 15cm cell culture dishes, so that the cell fusion degree was about 50%;

[0056] 3. The next day until the cell confluency is ...

Embodiment 3

[0078] Example 3 Lentiviral transfection of BV2 cells

[0079] Use the lentivirus prepared in Example 2 to transfect BV2 cells, the specific operations are as follows:

[0080] 1. 18-24 hours before lentiviral transfection, inoculate adherent BV2 cells with 1×10 5 Spread / well into a 24-well plate and culture in high-sugar DMEM medium, so that the number of cells at the time of lentiviral transfection is 2×10 5 / hole left and right.

[0081] 2. On the second day, replace the original medium with 2 mL of fresh medium containing 6 μg / mL polybrene, and add an appropriate amount of lentivirus suspension with a transfection multiplier of 10-100. Incubate at 37°C. The transfection multiplicity is the number of viruses / the number of transfected cells. The transfection multiplicity is less than 10 and the transfection efficiency is low. If the transfection multiplicity is higher than 100, the cells cannot bear too much virus and cause death. The preferred transfection multiplicity ...

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Abstract

The invention discloses a fluorescent BV2 tool cell and a construction method and application thereof. According to the construction method, by taking a lentiviral tool as a vector, plasmids each witha fluorescent gene EGFP are integrated in a genome of a BV2 cell, and when blue light is used for excitation, it can be observed that the cell emit clear green fluorescence under a fluorescent microscope. The transgenic BV2 tool cell disclosed by the invention can spontaneously emit fluorescence, has high fluorescence intensity, can be observed conveniently without dyeing, and has important significance in the aspects of in vitro research on development, behaviors and functions of microglial cells.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a fluorescent BV2 tool cell and its construction method and application. Background technique [0002] Microglia is the smallest glial cell in the central nervous system (CNS), distributed throughout the central nervous system, accounting for about 5%-10% of the total glial cells. As immune effector cells resident in the central nervous system, microglia belong to the mononuclear phagocyte family and are widely considered to be the main immune effectors of the central nervous system. Nervous system injury and disease outcome process play a very important role, involved in human nervous system disorders such as HIV encephalopathy, Parkinson's disease, Alzheimer's disease, multiple sclerosis and so on. [0003] As an immune cell in the central nervous system, BV2 can not only protect neurons by phagocytizing pathogens and harmful particles in brain tissue, but also activate reactive mi...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/65C12N5/10C12Q1/02G01N21/64
CPCC12N15/86C12N15/65C12N5/0622G01N33/5026G01N33/5058G01N21/6486C12N2740/15043C12N2510/00C12N2800/107
Inventor 吕泽中詹阳
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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