New trends in tumor biology: transfection of a human hepatoblastoma cell line with green fluorescent protein
| Autor: | Jens Mahrt, Jörg Fuchs, Claudia Treuner, Guido Seitz, Gerhard A. Müller, Johannes T. Wessels, Steven W. Warmann, Peter Ruck |
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| Rok vydání: | 2005 |
| Předmět: |
Hepatoblastoma
viruses Genetic Vectors Green Fluorescent Proteins Cell Biology Transfection Green fluorescent protein Flow cytometry Tumor Cells Cultured medicine Humans RNA Messenger Laser capture microdissection medicine.diagnostic_test Liver Neoplasms fungi General Medicine Flow Cytometry Molecular biology In vitro Reverse transcriptase Cell biology medicine.anatomical_structure Cell culture Liposomes embryonic structures Pediatrics Perinatology and Child Health Surgery |
| Zdroj: | Journal of Pediatric Surgery. 40:653-657 |
| ISSN: | 0022-3468 |
| DOI: | 10.1016/j.jpedsurg.2004.12.010 |
| Popis: | Background/Purpose Enhanced green fluorescent protein (eGFP) is widely used as a marker in different biologic systems. After vector transfection, eGFP is expressed by eukaryotic cells and can be visualized using fluorescent microscopy. The aim of this study was to establish an eGFP-transfected human hepatoblastoma (HB) cell line as tool for further basic research studies. Methods The HB cell line HUH6 was transfected with the pEGFP-N1 vector by liposomal transfection. Enhanced green fluorescent protein–positive cells were sorted out by fluorescence-activated cell sort and selected using G418 resistance. Expression of eGFP-messenger RNA was assessed by single-cell reverse transcriptase polymerase chain reaction after laser microdissection. Original and transfected cells were compared biologically and cytomorphologically. Results Vector transfection produced up to 15% eGFP-positive cells. After fluorescence-activated cell sort and G418 selection, a pure cell line was established with 100% eGFP-positive cells. Reverse transcriptase polymerase chain reaction revealed constant expression of eGFP-messenger RNA in fluorescending cells. Analysis of cell characteristics revealed no differences between transfected and original cells. Conclusions For the first time, the authors established an eGFP-transfected HB cell line. This cell line can serve as a promising tool for further studies investigating HB in vitro and in vivo. Our model might also be a basis for similar work on other pediatric solid tumors. |
| Databáze: | OpenAIRE |
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