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Rapid induction of GFP expression by the nitrate reductase promoter in the diatom Phaeodactylum tricornutum
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SYSNO ASEP 0468464 Druh ASEP J - Článek v odborném periodiku Zařazení RIV J - Článek v odborném periodiku Poddruh J Článek ve WOS Název Rapid induction of GFP expression by the nitrate reductase promoter in the diatom Phaeodactylum tricornutum Tvůrce(i) Chu, L. (DE)
Ewe, Daniela (MBU-M) ORCID
Bártulos, C.R. (DE)
Kroth, P. G. (DE)
Gruber, A. (DE)Zdroj.dok. PeerJ. - : PeerJ - ISSN 2167-8359
Roč. 4, AUG 25 (2016), e2344Poč.str. 16 s. Jazyk dok. eng - angličtina Země vyd. GB - Velká Británie Klíč. slova Flow cytometry ; Nitrogen source ; Nitrate Vědní obor RIV EE - Mikrobiologie, virologie Institucionální podpora MBU-M - RVO:61388971 UT WOS 000383262300001 EID SCOPUS 84992047608 DOI 10.7717/peerj.2344 Anotace An essential prerequisite for a controlled transgene expression is the choice of a suitable promoter. In the model diatom Phaeodactylum tricornutum, the most commonly used promoters for trans-gene expression are the light dependent lhcf1 promoters (derived from two endogenous genes encoding fucoxanthin chlorophyll a/c binding proteins) and the nitrate dependent nr promoter (derived from the endogenous nitrate reductase gene). In this study, we investigated the time dependent expression of the green fluorescent protein (GFP) reporter under control of the nitrate reductase promoter in independently genetically transformed P. tricornutum cell lines following induction of expression by change of the nitrogen source in the medium via flow cytometry, microscopy and western blotting. In all investigated cell lines, GFP fluorescence started to increase 1 h after change of the medium, the fastest increase rates were observed between 2 and 3 h. Fluorescence continued to increase slightly for up to 7 h even after transfer of the cells to ammonium medium. The subsequent decrease of GFP fluorescence was much slower than the increase, probably due to the stability of GFP. The investigation of several cell lines transformed with nr based constructs revealed that, also in the absence of nitrate, the promoter may show residual activity. Furthermore, we observed a strong variation of gene expression between independent cell lines, emphasising the importance of a thorough characterisation of genetically modified cell lines and their individual expression patterns. Pracoviště Mikrobiologický ústav Kontakt Eliška Spurná, eliska.spurna@biomed.cas.cz, Tel.: 241 062 231 Rok sběru 2017
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