The strategy of fusion genes construction determines efficient expression of introduced transcription factors
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The main goal in gene therapy and biomedical research is an efficient transcription factors (TFs) delivery system. SNAIL, a zinc finger transcription factor, is strongly involved in tumor, what makes its signaling pathways an interesting research subject. The necessity of tracking activation of intracellular pathways has prompted fluorescent proteins usage as localization markers. Advanced molecular cloning techniques allow to generate fusion proteins from fluorescent markers and transcription factors. Depending on fusion strategy, the protein expression levels and nuclear transport ability are significantly different. The P2A self-cleavage motif through its cleavage ability allows two single proteins to be simultaneously expressed. The aim of this study was to compare two strategies for introducing a pair of genes using expression vector system. We have examined GFP and SNAI1 gene fusions by comprising common nucleotide polylinker (multiple cloning site) or P2A motif in between them, resulting in one fusion or two independent protein expressions respectively. In each case transgene expression levels and translation efficiency as well as nuclear localization of expressed protein have been analyzed. Our data showed that usage of P2A motif provides more effective nuclear transport of SNAIL transcription factor than conventional genes linker. At the same time the fluorescent marker spreads evenly in subcellular space.
- Arbab AS, Yocum GT, Wilson LB, et al., (2004) Comparison of transfection agents in forming complexes with ferumoxides, cell labeling efficiency, and cellular viability. Mol Imaging 3: 24-32.
- Batlle E, Sancho E, Francí C, et al., (2000) The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol 2: 84-89.
- Chudakov DM, Matz M V, Lukyanov S, Lukyanov KA (2010) Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev 90: 1103-1163.
- Gao SY, Jack MM, O'Neill C (2012) Towards optimising the production of and expression from polycistronic vectors in embryonic stem cells. PLoS One 7: e48668.
- Guo Q-S, Zhu M-Y, Wang L, et al., (2012) Combined transfection of the three transcriptional factors, PDX-1, NeuroD1, and MafA, causes differentiation of bone marrow mesenchymal stem cells into insulin-producing cells. Exp Diabetes Res 2012: 672013.
- Kim JH, Lee S-R, Li L-H, et al., (2011) High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6: e18556.
- Lee JM, Dedhar S, Kalluri R, Thompson EW (2006) The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol 172: 973-981.
- Mokrejs M (2006) IRESite: the database of experimentally verified IRES structures (www.iresite.org). Nucleic Acids Res 34: D125-130.
- Radcliffe PA, Mitrophanous KA (2004) Multiple gene products from a single vector: 'self-cleaving' 2A peptides. Gene Ther 11: 1673-1674.
- Seibel NM, Eljouni J, Nalaskowski MM, Hampe W (2007) Nuclear localization of enhanced green fluorescent protein homomultimers. Anal Biochem 368: 95-99.
- Szymczak AL, Workman CJ, Wang Y et al. (2004) Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nat Biotechnol 22: 589-594.
- Yamasaki H, Sekimoto T, Ohkubo T et al. (2005) Zinc finger domain of Snail functions as a nuclear localization signal for importin beta-mediated nuclear import pathway. Genes Cells 10: 455-464.
- Yeh JE, Toniolo PA, Frank DA (2013) Targeting transcription factors: promising new strategies for cancer therapy. Curr Opin Oncol 25: 652-658.
- Yook JI, Li X-Y, Ota I, et al., (2005) Wnt-dependent regulation of the E-cadherin repressor snail. J Biol Chem 280: 11740-11748.
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