Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes / 한국실험동물학회지

Genetically engineered mouse models are commonly preferred for studying the human disease due to genetic and pathophysiological similarities between mice and humans. In particular, Cre-loxP system is widely used as an integral experimental tool for generating the conditional. This system has enabled researchers to investigate genes of interest in a tissue/cell (spatial control) and/or time (temporal control) specific manner. A various tissue-specific Cre-driver mouse lines have been generated to date, and new Cre lines are still being developed. This review provides a brief overview of Cre-loxP system and a few commonly used promoters for expression of tissue-specific Cre recombinase. Also, we finally introduce some available links to the Web sites that provides detailed information about Cre mouse lines including their characterization.

Recombinant AAV Vector with MITF-M Promoter for Melanoma Gene Therapy

We have developed the recombinant adeno-associated virus (AAV) carrying the EGFP gene under the control of the microphtalmia-associated transcription factor-M (MITF-M) promoter region for melanoma-specific expression. MITF-M distal enhancer (MDE) region enhances the specific expression of the reporter gene specifically in cultured melanoma cells. Expression of EGFP protein was very high in AAV-CMV-EGFP infected cells but relatively low in cells infected with AAV-Mitf(Enh/Pro)-EGFP. After an in vitro infection by a recombinant AAV carrying the EGFP gene under the control of human MITF-M promoter, the reporter gene was expressed in MITF-M producing melanoma cell lines (SK-28 and G361), but not in MITF-M non-producing cell lines (HaCat). These results suggest that the utilization of the MITF-M promoter in a recombinant AAV vector could provide benefits in gene therapy applications.

Radioiodine Therapy of Liver Cancer Cell Following Tissue Specific Sodium Iodide Symporter Gene Transfer and Assessment of Therapeutic Efficacy with Optical Imaging / 핵의학분자영상

PURPOSE: Cancer specific killing can be achieved by therapeutic gene activated by cancer specific promotor. Expression of sodium iodide symporter (NIS) gene causes transportation and concentration ofiodide into the cell, therefore radioiodine treatment after NIS gene transfer to cancer cell could be a form of radionuclide gene therapy. luciferase (Luc) gene transfected cancer cell can be monitored by in vivo optical imaging after D-luciferin injection. Aims of the study are to make vector with both therapeutic NIS gene driven by AFP promoter and reporter Luc gene driven by CMV promoter, to perform hepatocellular carcinoma specific radiodiodine gene therapy by the vector, and assessment of the therapy effect by optical imaging using luciferase expression. MATERIALS AND METHODS: A Vector with AFP promoter driven NIS gene and CMV promoter driven Luc gene (AFP-NIS-CMV-Luc) was constructed. Liver cancer cell (HepG2, Huh-7) and non liver cancer cell (HCT-15) were transfected with the vector using liposome. Expression of the NIS gene at mRNA level was elucidated by RT-PCR. Radioiodide uptake, perchlorate blockade, and washout tests were performed and bioluminescence also measured by luminometer in these cells. In vitro clonogenic assay with I-131 was performed. In vivo nuclear imaging was obtained with gamma camera after I-131 intraperitoneal injection. RESULTS: A Vector with AFP-NIS-CMV-Luc was constructed and successfully transfected into HepG2, Huh-7 and HCT-15 cells. HepG2 and Huh-7 cells with AFP-NIS-CMV-Luc gene showed higher iodide uptake than non transfected cells and the higher iodide uptake was totally blocked by addition of perchlorate. HCT-15 cell did not showed any change of iodide uptake by the gene transfection. Transfected cells had higher light output than control cells. In vitro clonogenic assay, transfected HepG2 and Huh-7 cells showed lower colony count than non transfected HepG2 and Huh-7 cells, but transfected HCT-15 cell did not showed any difference than non transfected HCT-15 cell. Number of Huh-7 cells with AFP-NIS-CMV-Luc gene transfection was positively correlated with radioidine accumulation and luciferase activity. In vivo nuclear imaging with I-131 was successful in AFP-NIS-CMV-Luc gene transfected Huh-7 cell xenograft on nude mouse. CONCLUSION: A Vector with AFP promoter driven NIS and CMV promoter driven Luc gene was constructed. Transfection of the vector showed liver cancer cell specific enhancement of I-131 cytotoxicity by AFP promoter, and the effect of the radioiodine therapy can be successfully assessed by non-invasive luminescence measurement.

Transcription Activity of Ectogenic Human Carcinoembryonic Antigen Promoter in Lung Adenocarcinoma Cells A549 / 华中科技大学学报（医学）（英德文版）

The transcription activity of ectogenic human carcinoembryonic antigen (CEA) promoter in lung adenocarcinoma cells A549 was investigated for the further gene-targeting therapy. The reporter gene green fluorescent protein (GFP) driven by CEA promoter and human cytomegalovirus (CMV) promoter were relatively constructed and named plasmid pCEA-EGFP and pCMV-GFP respectively. The intensity of fluorescence was detected by fluorescence microscope and flow cytometry analysis after the pCEA-GFP and pSNAV-GFP plasmids were transfected into A549 cells through liposome respectively. The results showed (4.08±0.63) % of the A549 cells transfected with pCEA-AFP plasmid expressed, significantly lower than that of the A549 cells transfected with pCMV-GFP [(43.27±3.54) %]. It was suggested that ectogenic human CEA promoter in lung adenocarcinoma cells A549 was weakly expressed. The distinct specificity of CEA promoter in CEA high expression cells was regarded as a tool in selective gene therapy, but the transcription activity of ectogenic human CEA promoter was needed to increase in the future.