Constructing 9LLUC cell strain of expressing luciferase stably, and application

A luciferase and stable expression technology, applied in the field of biomedicine, can solve the problems such as the inability to obtain stable expression, the inability to complete the preparation and real-time observation of glioma animal models, and the inability to establish stable expression cell lines, and achieves a promising application prospect. Effect

Inactive Publication Date: 2007-07-25
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Claims
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Problems solved by technology

[0004] The luminescent intensity of luciferase in vivo is linearly related to the number of labeled cells. In the past, transferring luciferase or other tracers into 9L cells through adenovirus vectors, retrovirus vectors and other vectors could not obtain stable expression, nor could it The establishment of stable expression cell lines cannot be completed for the preparation and real-time observation of glioma animal models

Method used

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  • Constructing 9LLUC cell strain of expressing luciferase stably, and application
  • Constructing 9LLUC cell strain of expressing luciferase stably, and application

Examples

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Effect test

Embodiment 1

[0019] Example 1. Construction of lentiviral expression plasmid pBPLV-LUC.

[0020] The lentiviral expression vector pBPLV was constructed according to the method introduced by Mario Amendola et al. (Nature Biotechnology, 2005, 23: 108-116). After digesting the MA1 expression plasmid with PstI restriction enzyme and SalI restriction endonuclease, the multi-cloning site sequence (5'-GGCTAGCATGCTCTAGAGCGCTG-3', 3'-ACGTCCGATCGTACGAGATCTCGCGACAGCT-5') was added to construct the lentiviral expression plasmid pBPLV.

[0021] Firefly luciferase PGL 3 -Basic plasmid was purchased from Promega Company, passed NheI restriction endonuclease (Takara Company, D1162A), HpaI restriction endonuclease (Takara Company, D1064A) each 2 μl, PGL 3 -Basic plasmid 30 μl, 10×T Buffer 5 μl, incubate at 37°C for 2 hours to recover the LUC gene fragment, the fragment size is 1881bp; lentiviral expression vector pBPLV 30 μl, NheI restriction endonuclease 2 μl, Aor51 HI restriction endonuclease ( Takara...

Embodiment 2、293

[0022] The packaging of embodiment 2, 293FT cells

[0023] 293FT cells were cultured in high glucose DMEM containing 10% fetal bovine serum (FBS). In a sterile 5ml tube, add 1.5ml of serum-free DMEM medium, and then add the packaging plasmid mixture 4.2μg pLP1, 2μgpLP2, 2.8μg pLP / VSVG (Virapower TM Lentiviral Packaging Mix, Invitrogen Company, 4975-00) and 5 μg lentiviral expression plasmid, mixed gently. In another sterile 5ml tube, dilute 42μl Lipofectamine 2000 (Invitrogen, 11668-027) in 1.5ml serum-free DMEM medium, mix gently, and place at room temperature for 5min. Mix the above 2 tubes of liquid and mix gently. Incubate at room temperature for 20 min to form DNA-Lipofectamine 2000 complex. During DNA-lipid complex formation, 293FT cells were digested with 0.25% trypsin, counted, and resuspended in high-glucose DMEM containing 10% serum to a density of 1.2×10 6 pieces / ml. Add the DNA-Lipofectamine 2000 complex to a 10cm cell culture dish containing 5ml of growth me...

Embodiment 3

[0024] Example 3, 9L LUC Establishment of cell lines.

[0025] 9L cells in good growth state were routinely cultured for 24 hours and then digested with 0.25% trypsin. They were added to the culture dish together with 1ml of the prepared lentivirus solution and Polybrene (final concentration: 8 μg / ml), and were routinely cultured overnight. Fresh growth medium was replaced every The medium was changed once every 3 days. After 14 days of culture, the cells were digested to prepare a single cell suspension, and the 9L cells with green fluorescent protein were sorted by flow cytometry. LUC After the cells were cultured to 80-90% confluence, they were subcultured at a ratio of 1:2 (results shown in Figure 3).

[0026] Example 4, 9L LUC detection of luciferase. Will 9L LUC The cells were serially diluted in a 96-well plate, respectively 50000, 40000, 20000, 10000, 5000, 2000, 1000, 500, 200, 100, 50, media / well, and the firefly luciferase substrate D-luciferin (150 μg / ml, Sig...

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Abstract

This invention relates to a method for constructing 9LLUC cell strain capable of stably expressing firefly luciferase PGL3. This invention provides a cell strain 9LLUC, which is obtained by transfecting glioma cell 9L with lentivirus vector containing firefly luciferase PGL3 gene, and screening. Cell strain 9LLUC can be transplanted into different animals to obtain relative glioma animal models. Though stable expression of PGL3, the generation, development, infiltration, metastasis of glioma cell, and the curative effects of relative therapies can be real time observed. The glioma animal models can be used for studying the development of glioma, and screening relative drugs and therapies.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a stable expression firefly luciferase (PGL 3 ) of 9L LUC Establishment of cell lines. Background technique [0002] Luciferase gene (LUC)-tagged genes, cells, and animals allow researchers to directly monitor cellular activity and gene behavior in living organisms through in vivo imaging systems in living animals. Especially in various types of tumor research, it can directly and quickly measure the growth and metastasis of tumor cells in various tumor models, and can observe and evaluate the changes of tumor cells in real time during tumor treatment. It can detect and measure the in situ tumor, metastatic tumor and spontaneous tumor of the whole animal quantitatively without trauma, and even small migration can be detected. [0003] As one of the tools for gene transfection, lentiviral vectors have been widely favored in recent years because they can infect non-dividing cells in v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/86C12Q1/66C12Q1/04
Inventor 裴雪涛杨丽平赵敬湘袁红丰白慈贤闫舫
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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