Abstract
Purpose: 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.Methods: 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.Results: Our data revealed that the designed expression cassette provided tissue specific and hypoxia inducible (O2<1%) transgene expression.Conclusion: 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.
Highlights
Since identification and description of hypoxia-inducible factor 1 (HIF-1) by Wang et al.,[1] more than 200,000 articles have been published in scientific literatures
Results Enhanced green fluorescent protein (eGFP) Cloning confirmation with HindIII digestion To confirm eGFP cloning, the expression cassette was digested by HindIII and positive colony was determined by three sharp plasmid DNA band, including 3000, 2000 and 1500 bps on gel electrophoresis
Tissue-specific cis-regulatory elements and correlated transcription factors, including myogenic regulatory factors (MRFs) like myogenic differentiation 1 (MYOD1), myogenic factor 5 (MYF5), myogenin and myogenin regulator factor 4 (MRF4), myogenic factor 6 (MYF6) interact with other transcription factors, T box protein 2 (TBX2) and NK2 homeobox 5 (Nkx2.5).,[21,22,23,24] which play a pivotal role in the correct differentiation and progressive formation of cardiac muscle (Table 3)
Summary
Since identification and description of hypoxia-inducible factor 1 (HIF-1) by Wang et al.,[1] more than 200,000 articles have been published in scientific literatures. HIF1 is an inducible transcription factor, which binds to hypoxia response elements (HREs) or enhancer elements during hypoxia. HIF-1 regulates several genes, such as vascular endothelial growth factor (VEGF) and erythropoietin (EPO).[2] hypoxia inducible factor-1 alpha (HlF-1) is comprised of a heterodimer basic helix-loophelix (bHLH) transcriptional complex and is divided into distinct subsets, including HIF-1α and aryl hydrocarbon receptor nuclear translocator (ARNT), encoding a protein that is referred to beta (β) subunit. HIF-1 belongs to a conserved subfamily of PER-ARNT-SIM (PAS), which functions as oxygen sensors. The PAS domain is a subfamily of bHLH, which is a transcription factor. HIF-1α has an essential role both in physiologic and pathologic conditions, including, but not limited to, myocardial ischemia, coronary artery disease, organ rejection and some cancers.[5,6]
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