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Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein
Published on: September 23, 2010
[Preparation and characterization of the recombinant hFGF21-containing lentiviral]
Shuaiyao Lu1, Min Yan, Xiaonan Wang
1Institute of Medical Biology, Peking Union Medical College, Chinese Academy of Medical Science, Kunming 650118, China.
Objective:
To package the lentiviral particles carrying human fibroblast growth factor 21 (hFGF21) and identify the morphological characteristics and transduction capability for the target gene of human embryo kidney 293T (HEK293T) cells.
Methods:
After sequencing identification, the lentiviral recombinant plasmid Lv105-hFGF21 was packaged and concentrated in HEK293T cells, and then identified by RT-PCR and electron microscopy. The titer of recombinant lentivirus was determined by real-time quantitative PCR. The expression of hFGF21 at both mRNA and protein levels was detected by RT-PCR and immunofluorescence, respectively in lentivirus-infected Vero cells.
Results:
RT-PCR demonstrated that hFGF21 mRNA could be transcribed in Lv105-hFGF21 infected HEK293T cells. The typical lentiviral particle was seen under the electron microscope. The diameter of virus particles was 40-70 nm. The title of the virus was substantially elevated from 3.68 × 10(8); VG/mL to 1.25 × 10(9); VG/mL after concentration. The expression of hFGF21 was detected in Lv-hFGF21 infected Vero cells by means of both RT-PCR and immunofluorescence.
Conclusion:
The hFGF21 lentiviral particles have been packaged successfully and hFGF21 can be expressed in the infected Vero cells.
