Establishment Of OVA-Luc-GFP-H22 Cell Line Via Lentiviral Transduction And Construction Of An Orthotropic Tumor Model For Hepatocellular Carcinoma

Objective:(1)To establish a stable cell line expressing OVA-GFP-Luc,we construct recombinant lentiviral vector OVA-Luc-GFP-p LVA7,packaged lentivirus then infected H22 cells.(2)Using this cell line to construct the mouse liver cancer in situ,the expression of luciferase and green fluorescent protein was detected by using a small animal imaging instrument,explore the application of this model.Methods:(1)The recombinant lentiviral vector OVA-Luc-GFP-p LVA7 was constructed,Obtained the OVA-Luc fusion gene by using PCR,Eco RI-HF and Hind III-HF were used to separate the PCR product,purified the PCR product.simultaneously extracted lentiviral vector p LVA7 tagged with GFP,p LVA7 was digested by using Eco RI-HF and Hind III-HF.OVA-Luc fusion gene was loaded into p LVA7 lentiviral vector by DNA assembly mix ligase,and sequenced.Extracted the lentiviral vector p MD2.G and envelope protein ps PAX2,three plasmids were transfected into 293 T cells with PEI(polyethylenimine)to prepare the virus and infected H22 cells.Picked the monoclonal cell stable expression of OVA-Luc-GFPH22 by using microinjection apparatus,amplified.(2)The mouse model of orthotopic liver cancer was established by orthotopic injecting the cell line into the liver.The expression of GFP and luciferase were detected by using a small animal imaging system.Results:(1)The recombinant lentiviral vector OVA-Luc-GFP-p LVA7 was by enzyme digested and sequenced,consistented with the expected gene sequence.The H22 cell line was infected by lentivirus that was packaged from 293 T,the expression of GFP was detected by fluorescence microscopy,the expression of Luc was detected by using bioluminescence imaging.The monoclonal cell expressing OVA-GFP-Luc was successfully achieved by using a micro injector,amplified to cell line.(2)A mouse model of orthotopic liver cancer was successfully established with OVA-Luc-GFP-H22 cell line,the expression of luciferase was detected by small animal imaging instrument,but the expression of GFP was not detected.Conclusions:(1)The recombinant lentiviral vector OVA-Luc-GFP-p LVA7 was constructed,packaged lentivirus expressing OVA-GFP-Luc,infected the H22,we established a stable OVA-Luc-GFP-H22 cell line,revealed its expression in vitro.(2)The OVA-Luc-GFP-H22 cell line was used to construct the mouse model of liver cancer in situ,hepatocarcinoma was performed by in vivo tracting.