title: The strategy of fusion genes construction determines efficient expression of introduced transcription factors.
abstract: 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.
authors.affiliation: Department of Transplantation, Jagiellonian University Medical College, Kraków, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Adamus
authors.affiliation: Department of Transplantation, Jagiellonian University Medical College, Kraków, Poland.
authors.forename: Paweł
authors.initials: P
authors.lastname: Konieczny
authors.affiliation: Department of Transplantation, Jagiellonian University Medical College, Kraków, Poland.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Sekuła
authors.affiliation: Department of Transplantation, Jagiellonian University Medical College, Kraków, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Sułkowski
authors.affiliation: Department of Transplantation, Jagiellonian University Medical College, Kraków, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Majka
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; C000604407:SNAI1 protein, human; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; D049452:Green Fluorescent Proteins