Establishment And Verification Of Human Lung Cancer Cell For Application In Vivo Imaging System

Objective:To establish a human lung cancer cell line stably expressing firefly luciferase and verify the relationship between luminescent and tumor in Blab/C mice. So a mouse model can be prepared for continuous observation of tumor progress.Materials and Methods:Firefly luciferase gene was obtained by digesting PGL3-Promoter plasmid with restriction enzyme Hindâ…¢and Xbaâ… , then the gene fragment was cloned into eukaryotic expression plasmid pRc/CMV₂ . Recombinant plasmid pRc/CMV₂-luc⁺ was transfected into A549 cells by lipofectmine 2000 and single clones were obtained by screening with G418. The positive clones stably expressing firefly luciferase were identified from single clones selected with G418 by the assay of luciferase activity. Bioluminescence of positive clones was further detected in vitro by an in vivo imaging system and the relationship between the luminescent and cell number were analyzed. Luciferase gene expression of two different positive clone was detected by quantitive RT-PCR. The intergration of luciferase gene was identified by PCR. Bioluminescence of xenograft in Blab/c mice was further analyzed using an in vivo imaging system. After the observation of five weeks, the xenograft was excised for HE staining and histopathology analysis.Results:1. two fragments of about 1.7 kbp and 5.5 kbp can be found when pRc/CMV₂-luc⁺ was digested with Hindâ…¢and Xbaâ… , so the recombinant plasmid pRc/CMV₂-luc⁺ was successfully constructed.2. Positive clones stably expressing Firefly luciferase were obtained through screening with G418 and the assay of luciferase activity, and luminescent ability of positive clones was showed to correlated to the number of cell when detecting by an in vivo imaging system(R²=0.99).3.The luciferase activity in different positive clone is related to the expression of luciferase gene. And the stable expression of luciferase in positive clone was based on the intergration of luciferase gene in genomic DNA.4., Bioluminescent signal intensity of xenograft in Blab/c mouse model was showed to correlated to the progress of tumor.5. Histopathology of xenograft show the characteristic of human lung adenocarcinoma.Conclusion:A549 cell lines with stable expression of luciferase gene were successfully established, and the cell line was proved to show the stable luminescent ability in vivo and in vitro, it can be used to continuously monitor the growth and progression of the tumor in alive mouse.