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Lentiviral vector and application thereof in construction of immortalized cells

A technology of immortalization and lentivirus, applied in the field of genetic engineering, can solve problems such as complex mechanism, decay, and difficulty in control, and achieve the effects of expanding the application range, reducing the possibility of canceration, and improving the efficiency of immortalization

Inactive Publication Date: 2019-02-01
湖南丰晖生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some primary cells can temporarily gain the ability to proliferate continuously under the action of viral genes, and pass through the M1 phase smoothly; but when the cells are passed to the 20th to 30th generation, they cannot pass through the M2 phase and begin to decline
Moreover, once the genes in the virus that are not related to immortalization are integrated into the cell genome, by-products may be produced, disrupting the normal metabolic pathways of cells
[0009] 2. Immortalization methods are commonly used now, and almost all of them are suitable for their own cells. They cannot be applied to all cells, and their use has limitations.
[0010] 3. Whether it is virus infection or the introduction of proto-oncogenes, tumor suppressor genes, etc., there is a risk of forming tumor cells, which has certain safety hazards
[0011] 4. The mechanism involved in the existing immortalization process is complex and difficult to control. So far, there has been no case of constructing a controllable immortalization carrier

Method used

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  • Lentiviral vector and application thereof in construction of immortalized cells
  • Lentiviral vector and application thereof in construction of immortalized cells
  • Lentiviral vector and application thereof in construction of immortalized cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] 1. Using the lentiviral vector pLV-CMV-SV40LT as the base expression vector, construct the pLV-CMV-HPV16E6 vector.

[0090] 2. Synthesize the HPV16E6 sequence (NC_001526.4, SEQ ID NO: 4), and add Pme I (GTTTAAAC) and Sma I (CCCGGG) restriction site sequences at both ends

[0091] 3. The pLV-CMV-SV40LT vector ( figure 1 ) in the SV40LT sequence is replaced by the HPV16E6 sequence to obtain the pLV-CMV-HPV16E6 vector ( figure 2 ).

[0092] 4. Synthesize the TRE3G sequence (SEQ ID NO: 2), and add BamH I (GGATCC) and EcoR I (GAATTC) restriction site sequences at both ends.

[0093] 5. Construct the TRE3G sequence on the pLV-CMV-HPV16E6 vector by double enzyme digestion to obtain the pLV-TRE3G-CMV-HPV16E6 vector.

[0094] 6. Synthesize hTERT sequence (NM_198253.2, SEQ ID NO: 1), and add EcoR I (GAATTC) and Xba I (TCTAGA) restriction site sequences at both ends.

[0095]7. Construct the hTERT sequence into pLV-TRE3G-CMV-HPV16E6 by means of double enzyme digestion to obta...

Embodiment 2

[0097] 1. Using the lentiviral vector pLV-CMV-SV40LT as the base expression vector, construct the pLV-TRE3G-hTERT-CMV-SV40LT vector.

[0098] 2. Synthesize the TRE3G sequence (SEQ ID NO: 2), and add BamH I (GGATCC) and EcoR I (GAATTC) restriction site sequences at both ends.

[0099] 3. The TRE3G sequence was constructed on the pLV-CMV-SV40LT vector by double enzyme digestion to obtain the pLV-TRE3G-CMV-SV40LT vector.

[0100] 4. Synthesize hTERT sequence (NM_198253.2, SEQ ID NO: 1), and add EcoR I (GAATTC) and Xba I (TCTAGA) restriction site sequences at both ends.

[0101] 5. The hTERT sequence was constructed into pLV-TRE3G-CMV-SV40LT by means of double enzyme digestion to obtain the pLV-TRE3G-hTERT-CMV-SV40LT vector ( image 3 ).

Embodiment 3

[0103] The pMD2.G vector and psPAX2 vector will be used to package the pLV-TRE3G-hTERT-CMV-HPV16E6 vector into a lentivirus. The packaging method is:

[0104] 1. Divide 293T cells into 7×10 6 Each dish was inoculated, and a total of 3 dishes were inoculated. Add DMEM medium containing 10% fetal bovine serum, place at 37°C, 5% CO 2 cultured in an incubator.

[0105] 2. After 24 hours, 10 μg pMD2.G, 12 μg psPAX2, and 10 μg pLV-TRE3G-hTERT-CMV-HPV16E6 plasmids were co-transfected into 293T cells.

[0106] 3. Collect the cell supernatant 48h and 72h after transfection, filter to remove impurities, concentrate to 1ml with an ultrafiltration tube, divide into 5 tubes, and store at -80°C.

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Abstract

The invention relates to the technical field of genetic engineering, in particular to a lentiviral vector and application thereof in construction of immortalized cells. According to the invention, bymeans of lentivirus infection, different immortalized key genes are selectively and targetedly integrated into a target cell genome, thus not only improving the immortalization efficiency, but also generating no harmful by-product in the cells. Because of simultaneous introduction of two immortalized genes, the plasmid vector has expanded application scope, can be applicable to more cell types, also the two groups of genes have different mechanisms in inducing immortalization, can complement each other to improve the immortalization efficiency. By adding an inducible promoter, TERT expressioncan be controlled by an inducer, the cell canceration probability is reduced, and the immortalization process of cells can be better controlled.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a lentiviral vector and its application in constructing immortalized cells. Background technique [0002] Cell immortalization refers to the process in which cells cultured in vitro are affected by the external environment or their own genes are changed, and have the ability to proliferate indefinitely, thereby escaping the normal cell aging and death mechanism. The chance of spontaneous immortalization of cells is extremely low, rodents in 10 -5 ~10 -6 Between, human cells have only 10 -12 . The construction of immortalized cells has far-reaching significance for the research of drug toxicity, bioartificial organ construction and tissue engineering. [0003] To establish an immortalized cell line so that normal cells cultured in vitro can proliferate indefinitely, the most common method at present is through exogenous virus infection, introduction of telomerase r...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10C12N7/01
CPCC12N9/1276C12N15/86C12N2510/04C12N2740/15043C12Y207/07049
Inventor 许澎黄巧胡清云
Owner 湖南丰晖生物科技有限公司
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