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Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System
Published on: May 24, 2012
[Construction of lentiviral expression vector containing Foxp3 gene and its expression in DC2.4 cell line]
Yu-bo Gong1, Yi-fei Huang, Yan Li
1Department of Ophthalmology, Chinese PLA General Hospital, Beijing 100853, China.
Aim:
To construct a Foxp3 lentiviral vector and transfer it into DC2.4 cells, which provides Foxp3+DC cells for further study on its immune modulation.
Methods:
We cloned mouse Foxp3 gene into lentiviral vector(pGC-FU) and acquired the plasmid pGC-FU-Foxp3. PCR and sequencing analysis were made for verifying the positive clones. The virus packaging plasmids(pGC-FU-Foxp3, pHelper1.0 and pHelper2.0) were contransfected into 293T cells, and the Lentivirus-Foxp3 was harvested from 293T cells. The Lentivirus-Foxp3 was used to infect DC2.4 cells in vitro and the expression of Foxp3 in infected DC2.4 cells was detected with flow cytometry(FCM).
Results:
PCR and sequencing revealed that the pGC-FU-Foxp3 plasmid was correctly constructed. The Lentivirus-Foxp3 with a titer of 2×10(8); TU/mL was successfully packaged. Foxp3 expression in DC2.4 cells infected with the Lentivirus-Foxp3 was increased significantly compared with negative control lentivirus.
Conclusion:
The pGC-FU-Foxp3 plasmid has been successfully constructed and the Lentivirus-Foxp3 has been successfully packaged. Foxp3 can be enhanced in DC cells infected with the Lentivirus-Foxp3.
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