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| Construction of A Cell Line EGFP-Luc-Hepa1-6 and Its Application in the Mouse Model of the Hepatoma |
| YE Yu-chen1, ZHAO Jun-long2, WANG Lin1, DUAN Juan-li1, GAO Chun-chen2, QIN Hong-yan2, DOU Ke-feng1 |
1. Department of Hepatobiliary Surgery, Xijing Hospital, Xi'an 710032, China;
2. State Key Laboratory of Cancer Biology, Department of Medical Genetics and Developmental Biology, Fourth Military Medical University, Xi'an 710032, China |
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Abstract Objectives: We aimed to construct an eukaryotic expressive vector pCMV-Luciferase-IRES2-EGFP which contains luciferase and enhanced green fluorescent protein(EGFP) and steadily screen Hepa1-6 cell line transfected with this recombinant vector,with the purpose of stably labeling and in vivo tracking the hepatoma cells in orthotopic hepatoma model established using this cell line. Methods: pGL3-Basic vector was digested by Xba I,end-repaired by Klenow Fragment and then redigested by Xho I to acquire the luciferase gene,which was ligated with the larger fragment of pIRES2-EGFP vector through Xho I/Sma I double digestion to construct the recombinant vector. Authenticated by endonuclease digestion and sequencing,the recombinant product presented the correct sequence. The expression of EGFP and activity of luciferase of Hepa1-6 cells transfected with this vector were,respectively,dectected by fluorescent microscope and dual-luciferase reporter assay and LuminaⅡ imaging system.Confirming the expression of the vector in the cell line, we inoculated this cell line orthotopically in the liver of C57BL/6 mouse to establish a hepatoma model.The growth of the hepatoma was consecutively monitored in vivo using LuminaⅡ imaging system and both paraffin sections(HE staining) and frozen sections (immunofluorescent staining) were prepared from the ex vivo tumor tissue to observe the pathological features and EGFP expression of the hepatoma cells. Results: Hepa1-6 cell line expressing the recombinant vector pCMV-Luciferase-IRES2-EGFP(EGFP-Luc-Hepa1-6) was successfully constructed.Using this cell line,an orthotopic hepatoma model in mice was established with the capability of in vivo tracking and ex vivo labeling the hepatoma cells. Conclusions: The EGFP-Luc-Hepa1-6 cell line was constructed successfully and the orthotopic hepatoma model established using this cell line can satisfy the needs of detecting the progression of the hepatoma both in vivo and ex vivo.
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Received: 13 February 2015
Published: 25 May 2015
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