Ghaderi Shahrooz, Alidadiani Neda, Soleimani Rad Jafar, Heidari Hamid Reza, Dilaver Nafi, Mansoori Behzad, Rhabarghazi Reza, Parvizi Rezayat, Khaze Shahgoli Vahid, Baradaran Behzad
Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Department of Molecular Medicine, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
Adv Pharm Bull. 2018 Mar;8(1):29-38. doi: 10.15171/apb.2018.004. Epub 2018 Mar 18.
Cardiovascular gene therapy is a sophisticated approach, thanks to the safety of vectors, stable transgene expression, delivery method, and different layers of the heart. To date, numerous expression vectors have been introduced in biotechnology and biopharmacy industries in relation to genetic manipulation. Despite the rapid growth of these modalities, they must be intelligently designed, addressing the cardiac-specific transgene expression and less side effects. Herein, we conducted a pilot project aiming to design a cardiac-specific hypoxia-inducible expression cassette. We explored a new approach to design an expression cassette containing cardiac specific enhancer, hypoxia response elements (HRE), cardiac specific promoter, internal ribosome entry site (IRES), and beta globin poly A sequence to elicit specific and inducible expression of the gene of interest. Enhanced green fluorescent protein (eGFP) was sub-cloned by BglII and NotI into the cassette. The specificity and inducible expression of the cassette was determined in both mouse myoblast C2C12 and mammary glandular tumor 4T1 as 'twin' cells. eGFP expression was evaluated by immunofluorescence microscope and flow cytometry at 520 nm emission peak. Our data revealed that the designed expression cassette provided tissue specific and hypoxia inducible (O<1%) transgene expression. It is suggested that cardiac-specific enhancer combined with cardiac-specific promoter are efficient for myoblast specific gene expression. As well, this is for the first time that HRE are derived from three well known hypoxia-regulated promoters. Therefore, there is no longer need to overlap PCR process for one repeated sequence just in one promoter.
心血管基因治疗是一种复杂的方法，这得益于载体的安全性、稳定的转基因表达、递送方法以及心脏的不同层次结构。迄今为止，在生物技术和生物制药行业中，已引入了许多与基因操作相关的表达载体。尽管这些方法发展迅速，但仍需进行智能设计，以实现心脏特异性转基因表达并减少副作用。在此，我们开展了一个试点项目，旨在设计一个心脏特异性缺氧诱导表达盒。我们探索了一种新方法来设计一个表达盒，该表达盒包含心脏特异性增强子、缺氧反应元件（HRE）、心脏特异性启动子、内部核糖体进入位点（IRES）和β珠蛋白聚腺苷酸序列，以引发目的基因的特异性和诱导性表达。通过BglII和NotI将增强型绿色荧光蛋白（eGFP）亚克隆到该表达盒中。在小鼠成肌细胞C2C12和乳腺肿瘤4T1这两种“孪生”细胞中测定了该表达盒的特异性和诱导性表达。通过免疫荧光显微镜和流式细胞术在520nm发射峰处评估eGFP表达。我们的数据表明，所设计的表达盒可提供组织特异性和缺氧诱导性（氧含量<1%）的转基因表达。研究表明，心脏特异性增强子与心脏特异性启动子相结合对于成肌细胞特异性基因表达是有效的。此外，这是首次从三个著名的缺氧调节启动子中获得HRE。因此，不再需要仅针对一个启动子中的一个重复序列进行重叠PCR过程。
