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cDNA cloning, lentiviral expression construction and stable HepG2 cell line establishment of Wnt Antagonist Frzb(PDF)

Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

2014 05
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cDNA cloning, lentiviral expression construction and stable HepG2 cell line establishment of Wnt Antagonist Frzb
MA Yuling PEI Zhe WANG Jing WEI Xiao ZENG Zhongqiu CHEN Yuwen TANG Yaxiong
Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
Frzb HCC HepG2 cloning recombinant lentivirus stable cell line
Q78 : R966

Aberrant activation of the Wnt signaling pathway is closely linked to tumorigenesis in a variety of major human malignant tumors. However, the anticancer potential of Frzb/sFrp3, one of the secreted frizzled related protein (sFRP) families, in hepatocellular carcinoma is poorly understood. This research aimed to study the cDNA cloning, lentiviral expression construction and stable HepG2 cell line establishment of Wnt antagonist Frzb. The Frzb cDNA fragments were amplified by reverse transcription polymerase chain reaction (RT-PCR) from the total RNA of normal human liver HL-7702 (L02) cells, which were verified by the direct sequencing of PCR products. Secondly, the Frzb cDNA fragments were subcloned into the pLVX-IRES-ZsGreen1-Frzb or pLVX-Puro-Frzb, containing a fluorescent marker (GFP) or a puromycin resistance gene respectively; and the recombinant lentiviral expression plasmids were further confirmed by double digestion and sequencing. Thirdly, HEK293T cells were co-transfected with the lentiviral expression plasmid, plus packaging plasmid psPAX2 and envelope plasmid pMD2.G by using Lenti-X? Lentiviral Expression Systems to obtain recombinant lentiviral particles at 48 hours after transfection; then the overall titers were evaluated by fluorescence microscopy of GFP expression, and the expression of Flag-tagged Frzb successfully detected by Western blot in the infected HEK293T cells. Finally, HepG2 cells were infected with the recombinant lentiviral particles expressing Frzb and the stable Frzb-transducted HepG2 cell line was successfully established by antibiotic selection with puromycin (2 μg/mL). These findings help to lay a foundation for studies about anticancer activities and molecular mechanisms of Frzb in hepatocellular carcinoma.


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Last Update: 2014-10-23