Determination of gene delivery and expression in different tumors by different gene constructs and reporter probes

• Main Authors: Lin Chi Ta, 林其達
• Other Authors: Chen Fu Du
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/09365840078670664182

碩士 === 國立陽明大學 === 放射醫學科學研究所 === 92 === A vital step in transgenic animal study and gene therapy is the ability to assay the extent of transgene expression. Conventional methods used by molecular biologists to monitor gene expression take advantage of reporter genes, such as β-galactosidase (β-gal) and chloramphenicol-actyltransferase, which require tissue samples for determining their expression levels. Other methods make use of optical reporter genes, such as luciferase, green fluorescent protein or β-galactosidase. The optical reporter genes have been used in living animal models as well but are not suitable for human application. Because of technologic innovations, such as position emission tomography (PET), it is now possible by using radiotracers (reporter probes), to image reporter gene expression in vivo both repeatedly and non invasively. In our study, we are aim at comparing and optimizing gene delivery efficiency and levels of gene expression for different tumors by different constructs, delivery systems, promoters and PET reporter probes. Human tumor cells from different organs have been inoculated to the immuno- suppressed NOD/SCID mice to grow into a solid tumor suitable for PET imaging or other imaging related procedures.We purposed to use non-viral plasmid vector together with the same synthetic HSV1tk for the entire study, so that the selective use of delivery system will become an important issue if one wish to achieve reasonable transfection in different tissue in vitro and in vivo.We decided to use jetPEI as our delivery reagentfor all experiments. Non-specific promoters such as CMV promoter have been used extensively in the past, however, it is not tissue specific, therefore, we were used the human species specific EF1α promoter to drive gene expression in the later experiments. Radiolabeled FHBG and FIAU have been used extensively in gene imaging research.In this study we used human hepatoma, Leiomyosarcoma and medulloblastoma NOD/SCID mice models to compare and find out the most suitable PET reporter probes for gene expression imaging,and also utilized γ-counting and luciferase assay systems to find out the difference among three different tumors. So far, we are able to demostrate the optimal gene delivery efficiency and level of gene expression in different tumors. The results provided good reference in the current studies and future applications. The current results demonstrated that the developed three animal tumor models provided good support for gene imaging research. Using plasmid vector combined with transfection reagent enable us to image gene expression in vivo successfully. Using bi-cistronic vector system, the target gene needs to be positioned upstream of IRES element for its expression to be robust. Reasonable correlation of the ratio of about 0.8 was obtained from data of region of interest (ROI) and γ-counting for animals with Tk positive and negative tumors, this indicates that imaging technologies such as region of interest is an effective way in evaluating the level of gene expression in vivo. Human tissue specific promoter (i.e EF1α) can provide better gene imaging in immuno- suppressed animals inoculated with human tumor cells. In three animal tumor models, TE671 medulloblastoma and SK-LMS-1 leiomyosarcoma have similar level of gene expression; they exhibited better gene expression than HepG2 hepatoma.The results from tumors with either stable clone or direct transfection show that FIAU is better than FHBG in terms of the quality of gene expression imaging.