Construction of Designed Expression Vectors for Analysis of Reciprocal Interactions Between RAX And E2F1 Transcription Factors in HEK-293T Cells

Construction of Designed Expression Vectors for Analysis of Reciprocal Interactions Between RAX And E2F1 Transcription Factors in HEK-293T Cells
Shima Ziaei,¹ pendar Shojaei,² kianoush Dormiani,^3,* Mohammad hosein Nasr Esfahani,⁴
1. 1. Department of Biology, Faculty of Science and Technology, ACECR Institute of Higher Education (Isfahan), Isfahan, Iran 2. Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
2. Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
3. Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
4. Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
Introduction: Vision is one of the vital senses of human beings that is involved in most of the daily activities of life. Retinal Progenitor Cells (RPCs) are multipotent progenitors derived from retinal stem cells, with the potential to be differentiated into various types of retinal cells. Previous studies have demonstrated that retinal development is controlled by coordinated interactions between three key groups including transcription factors, cell cycle components, and regulators of signaling pathways. Hence, it is important to identify regulatory interactions between these components. E2F family, are conserved transcription factors (TFs) with dual activities like a double edge sword in different biological process and play roles in both proliferation and apoptosis. This family is divided into three groups including activators (E2F1, E2F2, and E2F3), repressor (E2F4, E2F5, and E2F6), and atypical groups (E2F7 and E2F8). E2F1, A member of the activator group, is a crucial player involved in cell cycle progression from G1 to S phase by binding to the promoters of target genes and induction of their transcription. Homeobox genes are a group of transcription factors that are critical for the development of many organs, including brain and eye of vertebrates. Retina and anterior neural fold homeobox (RAX) is one of the important Homeobox genes with significant roles in the forebrain and eye development of vertebrates. RAX expression leads to commitment of retinal progenitor cells by regulating their initial specification and proliferation. It has been shown that knockout of RAX gene in mice led to no eye formation and also abnormal formation of the forebrain.
Methods: Bioinformatic studies have predicted several binding sites of E2F1 approximately within 3 kb upstream of human RAX gene. Furthermore, based on in silico analysis, a 1658 bp fragment upstream of the human E2F1 gene was considered as E2F1 promoter. The coding sequences of RAX and E2F1 and also regulatory region of E2F1 gene were amplified by PCR. These segments were then cloned into target expression vectors harboring EGFP and mCherry reporters, which later was used for monitoring of transcription factor plasmids transfection. Next, the effect of E2F1 transcription factor on RAX promoter activity and the putative reciprocal interaction of RAX protein with E2F1 regulatory region were evaluated by transient transactivation assay in HEK-293T cells. For this purpose, target expression vectors were transfected into HEK-293T cells using lipofectamine LTX reagent.
Results: Results: The integrity of the expression vectors were also examined by restriction digestion and PCR. Prior cloning into the expression vectors, the accuracy of the amplified sequences were also confirmed by sequencing analysis. The results indicated that all target fragments were inserted into mammalian expression vectors. The successful expression of EGFP in HEK-293T cells was confirmed by microscopic analysis. 48 h post co-transfection of vectors which were target promoters and expressing TFs, cells were lysed, total RNA was extracted and incorporated in cDNA synthesis. In order to investigate the interaction of RAX and E2F1, the level of EGFP mRNA derived by target promoters were analyzed using q-PCR.
Conclusion: Conclusion: Retinal degeneration is an irreversible condition in which progressive death of retinal cells leads to deterioration of the retina. As E2F1 and RAX are critical modulators of RPC proliferation, analysis of their interactions might provide better insight into the mechanisms underlying retinal progenitor maintenance which is important to have better therapeutic approaches in regenerative medicine for treatment of retinal degenerative disease. This study aimed to evaluate the mutual interactions between RAX and E2F1. Construction and transfection of vectors expressing RAX and E2F1 CDS and promoters into HEK-293T cells were successfully accomplished, and in vitro evaluation of interactions between these two transcription factors were studied by EGFP expression analysis using q-PCR.
Keywords: Retina Progenitor Cells, Transcription Factor, E2F1, RAX, Proliferation