Lentivirus vector expression system of luciferase and application thereof

A technology of luciferase and expression system, which is applied in the field of lentiviral vector expression system of luciferase, which can solve the problems of cumbersome process, inability to monitor metastasis, and omission of micrometastases

Inactive Publication Date: 2012-05-30
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method has two obvious defects: one is that it can only detect the state of the animal at the end of the experimental observation, and cannot monitor the location of metastasis in real time during tumor progression. As mentioned above, metastasis is a continuous and complicated process. A model that can observe a point in time obviously cannot show the full picture of the transfer process to the inventor, and if the quantitative data of tumor transfer can be obtained in real time, a better platform can be provided for the inventor's research on transfer; The identification of metastases can generally only be carried out by immunohistochemistry, which requires a large-scale section of all possible metastatic organs. The process is relatively cumbersome. If the section is incomplete, potential micro-metastases may be missed. The use of more sensitive and Efficient detection means is the only way to improve
[0005] Most of the currently established in vivo imaging models use cell lines transfected with luciferase-expressing plasmids or infected with luciferase-expressing viruses, but there are few reports using clinical tumor tissues

Method used

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  • Lentivirus vector expression system of luciferase and application thereof
  • Lentivirus vector expression system of luciferase and application thereof
  • Lentivirus vector expression system of luciferase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Construction of a lentiviral vector expression system for luciferase

[0082] Compared with the general plasmid transfection method, the lentiviral expression system has the advantages of high transfection efficiency, stable integration, and continuous expression. Therefore, the inventors constructed a luciferase lentiviral vector expression system. The present inventors replaced the U6 promoter in the FG12 vector in our laboratory with the CMV promoter suitable for eukaryotic expression, and then inserted the improved luciferase reporter gene (Luc2) behind the CMV promoter, while maintaining The original GFP reporter gene module remains unchanged, that is, the FG12 lentiviral vector co-expressing GFP and luciferase is obtained. By co-transfecting 293T cells with the corresponding packaging plasmid, collecting the cell culture supernatant and purifying and concentrating, high-titer virus particles can be obtained for in vivo and in vitro labeling experiments....

Embodiment 2

[0083] Example 2: Establishment of efficient luciferase-labeled tumor tissue method

[0084] After the construction of the lentiviral expression system was completed, the inventors marked the liver cancer tissue. For a long time, in vivo labeling of tumor tissue has been a recognized problem, and the relevant literature available for reference is very limited, and some existing reports, such as electroporation, have not achieved satisfactory results. After many attempts and optimizations, the inventors established a more efficient labeling method (see "Materials and Methods"). Experimental data show that this method can effectively infect most of the cells in the tumor tissue block, and the positive rate can reach more than 90%. Moreover, successfully infected tumors can continue to grow in nude mice (including subcutaneous and liver in situ), and become a good model for studying tumor growth, progression, and migration in vivo ( figure 2 ).

[0085] figure 2 Growth of l...

Embodiment 3

[0086] Example 3: Establishment of a nude mouse model for orthotopic transplantation of human liver cancer

[0087] The inventors planted fresh samples of liver cancer on the livers of nude mice in time to establish a nude mouse model of orthotopic transplantation of human liver cancer. Compared with liver cancer models generated from in vitro cell lines, liver cancer models based on patient tumor tissue are closer to the in vivo physiological state of the tumor. In the liver cancer model constructed by the present inventors, the samples used come from liver cancer tissues with a high degree of malignancy, and one of the important features is that the patients have liver portal vein metastasis of liver cancer cells. Therefore, this liver cancer model includes the liver cancer tissue from the primary tumor and the corresponding portal vein tumor thrombus, which can metastasize into blood vessels, and shows a strong ability to metastasize, providing an ideal model for the study ...

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Abstract

The invention relates to a lentivirus vector expression system of luciferase and an application thereof. Particularly speaking, in the invention a luciferase lentivirus expression system with FG12-Luc2 as core plasmid is successfully constructed, and a PVTT-1 cell line from tumor thrombus tissue of liver cancer patients is established by the system. Through the lentivirus expression system, liver cancer tissue is successfully marked; and by experimental methods such as subcutaneous transplantation and orthotopic liver cancer transplantation, a mouse orthotopic transplantation liver cancer metastasis model is established which is suitable for serial passage, is applicable to in-vivo imaging, and can realize real-time monitoring of the growth and metastasis conditions of tumors in the body.

Description

technical field [0001] The invention relates to a luciferase lentiviral vector expression system and its application. Background technique [0002] With the continuous development of tumor molecular biology, the research on the mechanism of tumor initiation and progression has made great progress. The discovery of more and more oncogenes and tumor suppressor genes has enabled people to have a relatively clear understanding of the biological events in the early stages of the development of many tumors. However, the late events of tumor development—invasion of peripheral organs and metastasis to distant tissues—remain an unexplored area, and they are the main causes of death in cancer patients. Data show that the death of patients caused by tumor metastasis accounts for 90% of the deaths caused by tumors. [0003] Tumor metastasis is a complex biological behavior, which includes many related sequential steps such as invasion of blood vessels, transport with blood, and distan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N15/65C12N15/52C12N5/10
Inventor 谢东冯宇雄李晶晶程树群
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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