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Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system

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dc.abstract.enDeciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternatives. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and generation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. Our collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. Our toolkit and protocols have allowed us to create more than 500 constructs with ease. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the effectiveness of commonly used inducers for tetracycline-responsive promoters. As proof of concept we examined the role of the exoribonuclease XRN2 in transcription termination by RNAseq.
dc.affiliationUniwersytet Warszawski
dc.contributor.authorChlebowski, Aleksander
dc.contributor.authorKlosowska-Kosicka, Kamila
dc.contributor.authorKowalska, Katarzyna
dc.contributor.authorTomecki, Rafał
dc.contributor.authorDziembowski, Andrzej
dc.contributor.authorWarkocki, Zbigniew
dc.contributor.authorKrawczyk, Paweł
dc.contributor.authorAffek, Kamila
dc.contributor.authorOwczarek, Ewelina
dc.contributor.authorAdamska, Dorota
dc.contributor.authorSzczęsny, Roman
dc.contributor.authorBorowski, Łukasz
dc.contributor.authorJedroszkowiak, Agata
dc.contributor.authorKuliński, Tomasz
dc.contributor.authorKotrys, Anna
dc.date.accessioned2024-01-26T11:46:23Z
dc.date.available2024-01-26T11:46:23Z
dc.date.issued2018
dc.description.accesstimeAT_PUBLICATION
dc.description.financeNie dotyczy
dc.description.number3
dc.description.versionFINAL_PUBLISHED
dc.description.volume13
dc.identifier.doi10.1371/JOURNAL.PONE.0194887
dc.identifier.issn1932-6203
dc.identifier.urihttps://repozytorium.uw.edu.pl//handle/item/124604
dc.identifier.weblinkhttps://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194887
dc.languageeng
dc.pbn.affiliationbiological sciences
dc.relation.ispartofPLoS ONE
dc.relation.pages1-29
dc.rightsCC-BY
dc.sciencecloudnosend
dc.titleVersatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system
dc.typeJournalArticle
dspace.entity.typePublication