Deciphering 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.

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28Deciphering a function of a given protein requires investigating various biological aspects. Usually, the protein 29 of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation 30 or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology 31 opened many possibilities but in many cases it is restricted to non-essential genes. It may also be time-32 consuming if a homozygote is needed. Recombinase-dependent gene integration methods, like the Flp-In 33 system, are very good alternative. The system is widely used in different research areas, which calls for the 34 existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and creation 35 of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of 36 stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent 37 DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR 38 primers. The collection allows tetracycline-inducible expression of proteins with various tags suitable for 39 protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies 40 (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a 41 bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and 42 its product replaced by a mutated miRNA-insensitive version. We demonstrate the efficacy of our vectors by 43 creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). 44We have analysed transgene expression over time to provide a guideline for future experiments and compared 45 the utility of commonly used inducers of tetracycline-responsive promoters. We determined the protein 46 interaction network of the exoribonuclease XRN2 and examined the role of the protein in transcription 47 termination by RNAseq analysis of cells devoid of its ribonucleolytic activity. In total we created more than 48 500 DNA constructs which proves high efficiency of our strategy. 49 50 KEYWORDS 51 FLP recombinase, Flp-In system, stable cell lines, cellular model, sequence-independent cloning, SLIC, 52 tetracycline-regulated gene expression, XRN2 ribonuclease, transcription termination, protein-protein 53 interactions 54 55Deciphering a protein's function requires investigating its subcellular localisation, identifying its 56 binding partners, and performing multiple functional assays. There are many ways to achieve these goals, with 57 different amounts of required time and effort as well as variable biological relevance of the results obtained. 58The usual course of action is to express the protein of interest with a fusion tag, a short peptide or a domain, 59 that aids or allows biochemical, cellular or functional analysis. Study of one protein often leads to follo...