Shiue Shih-Chang, Huang Miao-Zeng, Tsai Ting-Fen, Chang Alice Chien, Choo Kong Bung, Huang Chiu-Jung, Su Tsung-Sheng
Institute of Microbiology & Immunology, National Yang-Ming University, Taipei, Taiwan.
Department of Medical Research, Taipei Veterans General Hospital, Taipei, 112, Taiwan.
J Biomed Sci. 2015 Jan 23;22(1):10. doi: 10.1186/s12929-015-0114-6.
Argininosuccinate synthetase (ASS) participates in urea and nitric oxide production and is a rate-limiting enzyme in arginine biosynthesis. Regulation of ASS expression appears complex and dynamic. In addition to transcriptional regulation, a novel post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. Moreover, many cancers, including hepatocellular carcinoma (HCC), have been found not to express ASS mRNA; therefore, they are auxotrophic for arginine. To study when and where ASS is expressed and whether post-transcriptional regulation is undermined in particular temporal and spatial expression and in pathological events such as HCC, we set up a transgenic mouse system with modified BAC (bacterial artificial chromosome) carrying the human ASS gene tagged with an EGFP reporter.
We established and characterized the transgenic mouse models based on the use of two BAC-based EGFP reporter cassettes: a transcription reporter and a transcription/post-transcription coupled reporter. Using such a transgenic mouse system, EGFP fluorescence pattern in E14.5 embryo was examined. Profiles of fluorescence and that of Ass RNA in in situ hybridization were found to be in good agreement in general, yet our system has the advantages of sensitivity and direct fluorescence visualization. By comparing expression patterns between mice carrying the transcription reporter and those carrying the transcription/post-transcription couple reporter, a post-transcriptional up-regulation of ASS was found around the ventricular zone/subventricular zone of E14.5 embryonic brain. In the EGFP fluorescence pattern and mRNA level in adult tissues, tissue-specific regulation was found to be mainly controlled at transcriptional initiation. Furthermore, strong EGFP expression was found in brain regions of olfactory bulb, septum, habenular nucleus and choroid plexus of the young transgenic mice. On the other hand, in crossing to hepatitis B virus X protein (HBx)-transgenic mice, the Tg (ASS-EGFP, HBx) double transgenic mice developed HCC in which ASS expression was down-regulated, as in clinical samples.
The BAC transgenic mouse model described is a valuable tool for studying ASS gene expression. Moreover, this mouse model is a close reproduction of clinical behavior of ASS in HCC and is useful in testing arginine-depleting agents and for studies of the role of ASS in tumorigenesis.
精氨琥珀酸合成酶（ASS）参与尿素和一氧化氮的生成，是精氨酸生物合成中的限速酶。ASS表达的调控似乎复杂且具有动态性。除转录调控外，还报道了一种影响核前体RNA稳定性的新型转录后调控。此外，已发现包括肝细胞癌（HCC）在内的许多癌症不表达ASS mRNA；因此，它们对精氨酸营养缺陷。为了研究ASS在何时何地表达，以及转录后调控在特定的时空表达以及诸如HCC等病理事件中是否受到破坏，我们建立了一个转基因小鼠系统，该系统带有携带用EGFP报告基因标记的人类ASS基因的修饰细菌人工染色体（BAC）。
我们基于使用两个基于BAC的EGFP报告盒建立并表征了转基因小鼠模型：一个转录报告盒和一个转录/转录后偶联报告盒。使用这样的转基因小鼠系统，检测了E14.5胚胎中的EGFP荧光模式。发现荧光图谱与原位杂交中的Ass RNA图谱总体上吻合良好，但我们的系统具有灵敏度和直接荧光可视化的优点。通过比较携带转录报告盒的小鼠和携带转录/转录后偶联报告盒的小鼠之间的表达模式，发现在E14.5胚胎脑的脑室区/室下区周围存在ASS的转录后上调。在成年组织的EGFP荧光模式和mRNA水平中，发现组织特异性调控主要在转录起始阶段受到控制。此外，在年轻转基因小鼠的嗅球、隔膜、缰核和脉络丛的脑区中发现了强烈的EGFP表达。另一方面，与乙型肝炎病毒X蛋白（HBx）转基因小鼠杂交时，Tg（ASS-EGFP，HBx）双转基因小鼠发生了HCC，其中ASS表达如临床样本中一样下调。
所描述的BAC转基因小鼠模型是研究ASS基因表达的宝贵工具。此外，该小鼠模型与HCC中ASS的临床行为非常相似，可用于测试精氨酸消耗剂以及研究ASS在肿瘤发生中的作用。
