Efficient Control of Human G-CSF Gene Expression in the Primary Culture Cell using a FIV-Tet-On Vector System
2007
Kwon, M.S. (Catholic University of Daegu, Gyungsan, Republic of Korea) | Koo, B.C. (Catholic University of Daegu, Gyungsan, Republic of Korea) | Kim, T.A. (Catholic University of Daegu, Gyungsan, Republic of Korea), E-mail: [email protected]
In this study, using FIV-based lentivirus vector system, we tried to express hG-CSF in tetracycline-controllable manner. hG-CSF influences the proliferation, differentiation, and survival of cells in the neutrophil lineage. To enhance stability and translation of hG-CSF transcript, WPRE sequence was also introduced into FIV-Tet-On vector at downstream region of either the hG-CSF gene or the sequence encoding rtTA. Primary culture cells (CEF, chicken embryonic fibroblast; PFF, procine fetal fibroblast) infected with the recombinant FIV were cultured in the medium supplemented with or without doxycycline for 48 hours, and induction efficiency was measured by comparing the hG-CSF gene expression level using quantitative real-time PCR, Western blot and ELISA. Higher hG-CSF expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hG-CSF (in CEF) or rtTA (in PFF) gene. This FIV-Tet-On vector system may be helpful in solving serious physiological disturbance problems which has continuously hampered successful production of transgenic animals and gene therapy.
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