| Summary: | 碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 99 === Argininosuccinate synthetase (ASS) participates in urea and nitric oxide (NO) production and is a rate-limiting enzyme in arginine biosynthesis. The ASS gene is subjected to developmental and hormonal regulation. In addition, a novel regulation affecting nuclear precursor RNA stability has been reported. On the other hand, many cancer types including hepatocellular carcinoma (HCC) have been found not to express the ASS mRNA; therefore, they are auxotrophic for arginine. As a result, a target therapy to control cancer of this type by arginine depletion has been developed. To study when and where the ASS expressed and whether post-transcriptional regulation is underlined in particular temporal and spatial expression and in pathological events such as HCC, we have set up a transgenic mouse system with modified BAC (bacterial artificial chromosome) carrying human ASS gene tagged with EGFP (enhanced green florescence protein). Two versions of BAC transgenic mouse were developed, one designated as Tg(ASS-Ex3-EGFP) where EGFP is under transcriptional control similar to that of the endogenous ASS gene, another as Tg(ASS-Ex16-EGFP) where EGFP is subjected to both transcriptional and post-transcriptional regulation as that of the endogenous ASS gene.
Using BAC transgenic mice system, in this study, we investigated EGFP expression pattern in liver, the organ for urea production and in intestine and kidney that responsible for arginine biosynthesis. The EGFP fluorescence of particular organ taken from embryo E14.5 to neonates to young adult was examined under fluorescence microscope either directly or after cryosection. Moreover, the levels of EGFP mRNA and endogenous mouse Ass mRNA during development were quantified by S1 nuclease mapping. We found that EGFP fluorescence and EGFP mRNA level in both liver and kidney increases progressively toward birth yet the expression profiles of these two organs are distinct. In contrast to liver and kidney, EGFP expression in intestine is higher during neonates and disappears at about 3 weeks after birth. These complex expression patterns are consistent with that of endogenous mouse Ass gene. Moreover, this study indicates such developmental and tissue specific regulations are controlled at the transcriptional level. Likewise, by examining EGFP fluorescence in liver tumor derived from Tg(ASS-Ex3-EGFP) and Tg(ASS-Ex16-EGFP) lines, we concluded that the diminish of ASS expression in HCC may be mainly controlled at the transcriptional level. On the other hand, we constructed ASS atlases in the E14.5 embryo and in the young adult mouse brain based on EGFP fluorescence. Profiles of fluorescence and Ass RNA in situ hybridization (http://www.informatics.jax.org/) of E14.5 embryo were found to be in good agreement in general, yet our system has the merit of sensitivity and visualization capacity. This study demonstrates that our BAC transgenic mouse is a valuable tool to study ASS regulation not only in physiological but also in pathological condition.
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