Development of a novel DsRed-NLS vector with a monopartite classical nuclear localization signal
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The nuclear localization signal (NLS) marks proteins for transport to the nucleus and is used in various applications in many fields. NLSs are used to achieve efficient and stable transport of biomolecules. Previously, commercial vectors used in NLS studies contained three iterations of the NLS sequence, but these sequences can affect experimental results and alter protein function. Here, we investigated a new vector using a single classical NLS sequence with a mutation in pDsRed2-C1-wt to reduce experimental artifacts. In the newly constructed pDsRed2-C1-1NLS vector, the NLS sequence is placed near the multiple cloning sites of pDsRed2-C1-wt, and the multiple cloning site region was designed to facilitate insertion of the desired gene by site-directed mutagenesis. Fluorescent protein expression in the nucleus can be visually confirmed. The results show that the fluorescent protein was bound to the transport protein. The constructed vector had a cell survival rate of 89–95% and a transfection efficiency of 39–56% when introduced into animal cells, which are similar to those of other NLS vectors. Additionally, the constructed NLS vector can be used to demonstrate complementary binding between target proteins, and that the target protein is transported by the NLS transport system. Especially, we show that the vector can be useful for experiments involving the S100A10 gene. In addition, the constructed vector is useful for studies of genes and proteins that show potential for gene therapy or drug delivery applications.
KeywordsNuclear localization signal Vector Single construction of sequence Viability Transfection efficiency S100A10
This research was supported by the Basic Science Research Program, through the National Research Foundation of Korea (NRF), funded by the Ministry of Science ICT & Future Planning (NRF-2014R1A1A1002349). This article was revised by a professional English proofreader (Editage).
HSY, YJO, EJL and SSK performed the conceptualization; HSY, YJO and EJL performed the data curation and formal analysis; SSK and SHH performed funding and acquisition and resources; HSY, YJO, EJL and SSK performed the experiments including of investigation, methodology, software; HSY wrote the manuscript including of data visualization, original draft, review and editing; SHH performed project administration, supervision and validation. All authors read and approved the final manuscript.
This research was supported by the Basic Science Research Program, through the National Research Foundation of Korea (NRF), funded by the Ministry of Science ICT & Future Planning (NRF-2014R1A1A1002349).
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Conflict of interest
The authors have no conflict of interest (commercial or otherwise) to declare regarding this study.
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