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Though mRNA vaccines against COVID-19 have revolutionized vaccinology and have been administered in billions of doses, we know incredibly little about how mRNA vaccines are metabolizedin vivo. Here we implemented enhanced nanopore Direct RNA sequencing (eDRS), to enable the analysis of single Moderna’s mRNA-1273 molecules, givingin vivoinformation about the sequence and poly(A) tails.We show that mRNA-1273, with all uridines replaced by N1-methylpseudouridine (mΨ), is terminated by a long poly(A) tail (~100 nucleotides) followed by an mΨCmΨAG sequence. In model cell lines, mRNA-1273 is swiftly degraded in a process initiated by the removal of mΨCmΨAG, followed by CCR4-NOT-mediated deadenylation. In contrast, intramuscularly inoculated mRNA-1273 undergoes more complex modifications. Notably, mRNA-1273 molecules are re-adenylated after mΨCmΨAG removal. Detailed analysis of immune cells involved in antigen production revealed that in macrophages, after mΨCmΨAG removal, vaccine mRNA is very efficiently re-adenylated, and poly(A) tails can reach up to 200A. In contrast, in dendritic cells, vaccine mRNA undergoes slow deadenylation-dependent decay. We further demonstrate that enhancement of mRNA stability in macrophages is mediated by TENT5 poly(A) polymerases, whose expression is induced by the vaccine itself. Lack of TENT5-mediated re-adenylation results in lower antigen production and severely compromises specific immunoglobulin production following vaccination.Together, our findings provide an unexpected principle for the high efficacy of mRNA vaccines and open new possibilities for their improvement. They also emphasize that, in addition to targeting a protein of interest, the design of mRNA therapeutics should be customized to its cellular destination.

Nucleic acids research / Special publication 48(16), 9387 - 9405 (2020). doi:10.1093/nar/gkaa647, Template-independent terminal ribonucleotide transferases (TENTs) catalyze the addition of nucleotide monophosphates to the 3′-end of RNA molecules regulating their fate. TENTs include poly(U) polymerases (PUPs) with a subgroup of 3′ CUCU-tagging enzymes, such as CutA in Aspergillus nidulans. CutA preferentially incorporates cytosines, processively polymerizes only adenosines and does not incorporate or extend guanosines. The basis of this peculiar specificity remains to be established. Here, we describe crystal structures of the catalytic core of CutA in complex with an incoming non-hydrolyzable CTP analog and an RNA with three adenosines, along with biochemical characterization of the enzyme. The binding of GTP or a primer with terminal guanosine is predicted to induce clashes between 2-NH$_2$ of the guanine and protein, which would explain why CutA is unable to use these ligands as substrates. Processive adenosine polymerization likely results from the preferential binding of a primer ending with at least two adenosines. Intriguingly, we found that the affinities of CutA for the CTP and UTP are very similar and the structures did not reveal any apparent elements for specific NTP binding. Thus, the properties of CutA likely result from an interplay between several factors, which may include a conformational dynamic process of NTP recognition., Published by Information Retrieval Ltd.21517, London

The 3'-end poly(A) tail is an important and potent feature of most mRNA molecules that affects mRNA fate and translation efficiency. Polyadenylation is a posttranscriptional process that occurs in the nucleus by canonical poly(A) polymerases (PAPs). In some specific instances, the poly(A) tail can also be extended in the cytoplasm by noncanonical poly(A) polymerases (ncPAPs). This epitranscriptomic regulation of mRNA recently became one of the most interesting aspects in the field. Advances in RNA sequencing technologies and software development have allowed the precise measurement of poly(A) tails, identification of new ncPAPs, expansion of the function of known enzymes, discovery and a better understanding of the physiological role of tail heterogeneity, and recognition of a correlation between tail length and RNA translatability. Here, we summarize the development of polyadenylation research methods, including classic low-throughput approaches, Illumina-based genome-wide analysis, and advanced state-of-art techniques that utilize long-read third-generation sequencing with Pacific Biosciences and Oxford Nanopore Technologies platforms. A boost in technical opportunities over recent decades has allowed a better understanding of the regulation of gene expression at the mRNA level. This article is categorized under: RNA MethodsRNA Analyses In Vitro and In Silico.

A new coordination compound of copper(II) with a tolfenamate ligand of the paddle-wheel-like structure [CuII2(Tolf)4(MeOH)2]∙2MeOH was obtained and structurally characterized. Chemical bonds of Cu(II)∙∙∙Cu(II) and Cu(II)–O were theoretically analyzed and compared with the results for selected similar structures from the CSD database. QTAIM analysis showed that the Cu(II)∙∙∙Cu(II) interaction has a strength comparable to a hydrogen bond, as indicated by the electron density at a critical point. The remaining QTAIM parameters indicate stability of the Cu(II)∙∙∙Cu(II) interaction. Other methods, such as NCI and NBO, also indicate a significant strength of this interaction. Thus, the Cu(II)∙∙∙Cu(II) interaction can be treated as one of the noncovalent interactions that affects the structure of the coordination compound, the packing of molecules in the crystal, and the general properties of the compound.

Biogenesis of most eukaryotic mRNAs involves the addition of an untemplated polyadenosine (pA) tail by the cleavage and polyadenylation machinery. The pA tail, and its exact length, impacts mRNA stability, nuclear export, and translation. To define how polyadenylation is controlled in S. cerevisiae, we have used an in vivo assay capable of assessing nuclear pA tail synthesis, analyzed tail length distributions by direct RNA sequencing, and reconstituted polyadenylation reactions with purified components. This revealed three control mechanisms for pA tail length. First, we found that the pA binding protein (PABP) Nab2p is the primary regulator of pA tail length. Second, when Nab2p is limiting, the nuclear pool of Pab1p, the second major PABP in yeast, controls the process. Third, when both PABPs are absent, the cleavage and polyadenylation factor (CPF) limits pA tail synthesis. Thus, Pab1p and CPF provide fail-safe mechanisms to a primary Nab2p-dependent pathway, thereby preventing uncontrolled polyadenylation and allowing mRNA export and translation.

In order to improve pharmaceutical properties of drugs, complexes are synthesized as combinations with other chemical substances. The complexes of fenamic acid and its derivatives, such as mefenamic-, tolfenamic- and flufenamic acid, with acridine were obtained and the X-ray structures were discussed. Formation of the crystals is determined by the presence of the intermolecular O–H…N hydrogen bond that occur between fenamic acids and acridine. Intermolecular interactions stabilizing the crystals such as π…π stacking, C–H…X (X = O, Cl) intermolecular hydrogen bonds as well as C–H…π and other dispersive interactions were analyzed by theoretical methods: the quantum theory of atoms in molecules (QTAIM) and noncovalent interaction (NCI) approaches.

Summary: Osteoblasts orchestrate bone formation through the secretion of type I collagen and other constituents of the matrix on which hydroxyapatite crystals mineralize. Here, we show that TENT5A, whose mutations were found in congenital bone disease osteogenesis imperfecta patients, is a cytoplasmic poly(A) polymerase playing a crucial role in regulating bone mineralization. Direct RNA sequencing revealed that TENT5A is induced during osteoblast differentiation and polyadenylates mRNAs encoding Col1α1, Col1α2, and other secreted proteins involved in osteogenesis, increasing their expression. We postulate that TENT5A, possibly together with its paralog TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization. Importantly, the Tent5a knockout (KO) mouse line displays bone fragility and skeletal hypomineralization phenotype resulting from quantitative and qualitative collagen defects. Thus, we report a biologically relevant posttranscriptional regulator of collagen production and, more generally, bone formation.

Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3’ terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development., TUTase mediated uridylation of mRNA promotes degradation. Here, Scheer, de Almeida et al. show that Arabidopsis TUTase URT1 interacts directly with the translation inhibitor and decay factor DECAPPING5 and suppresses siRNA biogenesis by preventing accumulation of deadenylated mRNAs

Osteoblasts orchestrate bone formation by secreting dense, highly cross-linked type I collagen and other proteins involved in osteogenesis. Mutations in Col1α1, Col1α2, or collagen biogenesis factors lead to the human genetic disease, osteogenesis imperfecta (OI). Herein, we show that the TENT5A gene, whose mutation is responsible for poorly characterized type XVIII OI, encodes an active cytoplasmic poly(A) polymerase regulating osteogenesis. TENT5A is induced during osteoblast differentiation and TENT5A KO osteoblasts are defective in mineralization. The TENT5A KO mouse recapitulates OI disease symptoms such as bone fragility and hypomineralization. Direct RNA sequencing revealed that TENT5A polyadenylates and increases expression of Col1α1 and Col1α2 RNAs, as well as those of other genes mutated in OI, resulting in lower production and improper folding of collagen chains. Thus, we have identified the specific pathomechanism of XVIII OI and report for the first time a biologically relevant post-transcriptional regulator of collagen production. We further postulate that TENT5A, possibly together with its paralogue TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization.

This article is a report from a workshop on clinical research methods organised by the European College of Neuropsychopharmacology (ECNP), which took place between 7 and 9 November 2018 in Barcelona. The programme of the workshops and an outline of the content of the speeches were presented here. The author made an effort to synthetically present issues concerning methods used in clinical research design, analysis of published results and creation of scientific publications, which were discussed during the workshop.

1. Opracowanie rekordu w ramach umowy 509/P-DUN/2018 ze środkow MNiSW przeznaczonych na dzialalnośc upowszechniającą nauke (2019).

A new methoxo bridged dinuclear Mn(III) coordination compound, [Mn2(µ-OMe)2(L)2(MeOH)2]Cl2∙2MeOH (1), with tridentate N2O-donor half-Salen ligand was synthesized by the reaction of MnCl2·4H2O and (Z)-3-((2-aminocyclohexyl)amino)-1-phenylbut-2-en-1-one (HL) in methanol and characterized by spectroscopic methods and elemental analysis. The structure of compound 1 was also determined by single crystal X-ray diffraction studies which indicated it is a dinuclear Mn(III) coordination compound. Two Mn(III) are connected together by two methoxo bridges and have distorted octahedral geometry. The coordination compound is ionic and two chloride anions appear as the counter anions which are involved in strong hydrogen bond interactions. The catalytic activity of compound 1 was investigated in oxidation of styrene in the presence of hydrogen peroxide as a green oxidant. The effects of influencing parameters like molar ratio of the reagents, temperature and the presence of different solvents were investigated. The results of catalytic studies indicated the selectivity and activity of this catalytic system considerably depend on the reaction condition. Temperature and molar ratio of oxidant to substrate have high influence on both conversion and selectivity of the oxidation products. The best selectiviity toward styrene epoxide was obtained at 40 °C and the best TOF was 66.4 h−1. The results indicate half-salen Mn(III) coordination compound can act as selective and efficient catalyst for oxidation of styrene under mild condition

New multifunctional 1,2,4-triazole based allyl-derivative as the ligand L (3-allylsulfanyl-4-allyl-5-phenyl-4H-1,2,4-triazole) revealed an ability to form stable binuclear copper(I) π-complexes. A series of related Cu(I) halides π-complexes, [Cu2(L)2X2] (X = Cl, 1a; Br, 1b; I, 1c), [Cu2(L)2I2]2 (1c’), [Cu2(L)2(CuCl2)2] (2), [Cu2(L)2Cu2Br4] (3) was obtained by alternating-current electrochemical synthesis, starting from acetonitrile solutions of the ligand L and CuX2 (in the case of 1a, 1b, 2, 3) or I2 (1c, 1c’). This is the first notice of using iodine being reduced at copper electrodes under alternating current in the presence of a ligand for copper(I) iodide complexes synthesis. The crystal structures were determined by means of single crystal X-ray diffraction. The organic ligand acts as multidentate chelating σ,π-donor using triazole (tr) and η2-allyl moieties, resulting in a formation of the dimeric {Cu2N4} core with a trigonal-pyramidal (2 N, C C + X) environment of the Cu cation. Packing polymorphism was revealed for the iodine complex. In the case of 2 and 3 the Cu centers participate in a formation of both – metal-organic cations and copper(I) halide anions. In structure 3, the copper(I)-halide subunit is organized in the Cu2(μ2-Br)2Br2 cluster that links organometallic moieties leading to a 1D ladder fragment. High inertia of complexes in acetonitrile solutions enabled a comparative evaluation of a strength of the Cu(I)–(C C) interaction by means of 1H NMR spectra. NMR spectroscopic studies are consistent with the structural analysis.

TENT5C is a non-canonical cytoplasmic poly(A) polymerase highly expressed by activated B cells to suppress their proliferation. Here we measure the global distribution of poly(A) tail lengths in responsive B cells using a Nanopore direct RNA-sequencing approach, showing that TENT5C polyadenylates immunoglobulin mRNAs regulating their half-life and consequently steady-state levels. TENT5C is upregulated in differentiating plasma cells by innate signaling. Compared with wild-type, Tent5c−/− mice produce fewer antibodies and have diminished T-cell-independent immune response despite having more CD138high plasma cells as a consequence of accelerated differentiation. B cells from Tent5c−/− mice also have impaired capacity of the secretory pathway, with reduced ER volume and unfolded protein response. Importantly, these functions of TENT5C are dependent on its enzymatic activity as catalytic mutation knock-in mice display the same defect as Tent5c−/−. These findings define the role of the TENT5C enzyme in the humoral immune response., Regulating polyadenylation is important for mRNA stability, which can in turn affect B cell maturation and humoral immune responses. Here the authors use Nanopore poly(A) sequencing to explore the importance of the cytoplasmic poly(A) polymerase TENT5C, particularly in the production of immunoglobulins.

SummaryTENT5C is a non-canonical cytoplasmic poly(A) polymerase (ncPAP) upregulated in activated B cells and suppressing their proliferation. Herein we measured the global distribution of poly(A) tail lengths in responsive B cells using a modified Nanopore direct RNA-sequencing approach and revealed that TENT5C polyadenylates immunoglobulin mRNAs regulating their steady-state levels. Consequently, TENT5C deficient B cells secrete less antibodies and KO mice have diminished gamma globulin concentrations despite the increased number of CD138high plasma cells as a consequence of accelerated differentiation. TENT5C is explicitly upregulated in differentiating plasma cells by innate signaling. Importantly, TENT5C deficiency in B lymphocytes impairs the capacity of the secretory pathway through the reduction of ER volume and downregulation of unfolded protein response.Our findings define the role of the TENT5C enzyme in B cell physiology and discover the first ncPAP engaged in the regulation of immunoglobulin mRNA poly(A) tails, thus serving as a regulator of humoral immunity.

The reaction between tridentate NNN donor ligands, (E)-2-(2-(pyridin-2-ylmethylene)hydrazinyl)benzo[d]thiazole (HL1) and (E)-2-(2-(phenyl(pyridin-2-yl)methylene)hydrazinyl)benzo[d]thiazole (HL2), with VO(acac)2 in ethanol gave [VO2(L1)] (1) and [VO2(L2)] (2) complexes. Complex [VO2(L2)]·0.2(H2O) (3) was produced from the reaction of HL2 and V2O5 in ethanol. Molecular structures of complexes were determined by single-crystal X-ray diffraction. All of the investigated compounds were further characterized by elemental analysis, FT-IR, UV–vis and NMR spectroscopy. The dioxidovanadium(V) species were used as catalysts for olefin oxidation in the presence of hydrogen peroxide (H2O2) as an oxidant. Under similar experimental conditions with equal vanadium loading, the presence of the [VO2(L1)] (1) resulted in higher conversion than in the case of using [VO2(L2)] (2) and [VO2(L2)]·0.2(H2O) (3) as catalytic agents.

The first cyanide rhenium(I)–silver(I) clusters were synthesized in the course of simple one-pot high-yielding reactions. This new class of obtained self-assembled, cyclic octanuclear complexes is composed of pseudo-square-shaped {Re4Ag4(μ-CN)8} units, which, along with PPh3 ligands, adopt an approximately block-like overall geometry. We discovered that the studied cavity-shaped clusters feature a channeling crystal network capable of hosting smaller molecules and exhibit the ability to undergo reversible guest solvent sorption. Depending on the reaction conditions, the tetranuclear complex [Re2Ag2(μ-CN)4(CO)4(PPh3)6] and the species of the formal motif {ReAg1.5(CN)2.5(CO)2(PPh3)2} can be formed.

Introduction: This case report illustrates the use of a connective tissue (CT) graft harvested from a distal wedge procedure for root coverage. The retromolar area could be an alternative donor site, especially when a distal wedge procedure is planned. This approach offers an alternative CT donor site for the treatment of a localized gingival recession (GR).Case Presentation: A healthy 22-year-old female patient presented with a localized Miller Class II GR defect on a mandibular incisor (tooth #25) caused by trauma from a lip piercing. Probing depths of 6 mm were also noted bilaterally over the distal aspect of the mandibular second molars. A root coverage procedure was performed, together with a distal wedge procedure for pseudopocket reduction. A CT graft harvested from a distal wedge was used for the root coverage. Complete root coverage with stable follow-up was documented up to 2 years, 5 months. Tissue from a contralateral distal wedge was submitted for histologic evaluation. Histopathologic examin...

The reaction of equimolar amounts of 2-hydroxy-5-iodobenzaldehyde and 5-(methylthio)-1H-1,2,4-triazol-3-amine in methanol yielded a new 1,2,4-triazole based Schiff base ligand, H2L, which was characterised by spectroscopic methods. The combination of this Schiff base ligand with Mn(OAc)2·4H2O and KSCN in methanol yielded dark brown crystals of [KMn5(L)4(O)2(OAc)4(CH3OH)2(H2O)]·5.25(CH3OH)·2.33(H2O) (1), which were characterised by elemental analysis, spectroscopic methods and single-crystal X-ray diffraction. The compound contains Mn(III) pentanuclear clusters in which two oxido, four acetate and four dinegative Schiff base ligands link the Mn(III) ions. The structure of the cluster consists of two perpendicular Mn3O units and one Mn(III) ion acting as a bridge between them. The study of the magnetic properties revealed the occurrence of antiferromagnetic intramolecular interactions among the Mn(III) atoms. Since this type of compounds are candidates to exhibit SMM-behavior, the presence of SMM behavior is also investigated.

Saprochaete suaveolens is an ascomycetous yeast that produces a range of fruity flavors and fragrances. Here, we report the high-contiguity genome sequence of the ex-holotype strain, NRRL Y-17571 (CBS 152.25)., Saprochaete suaveolens is an ascomycetous yeast that produces a range of fruity flavors and fragrances. Here, we report the high-contiguity genome sequence of the ex-holotype strain, NRRL Y-17571 (CBS 152.25). The nuclear genome sequence contains 24.4 Mbp and codes for 8,119 predicted proteins.

Tetranuclear cyanide rhenium(I)–copper(I) coordination compounds were synthesized in the course of simple one-pot reactions in mild conditions. A new class of mixed-metal compounds based on rhenium(I) clusters that are comprised of the pseudo-rhombic-shaped {Re2Cu2(μ-CN)4} unit were found. Two kinds of geometries adopted by the self-assembled cyclic clusters are observed, both of which can be described as approximately block-like overall geometries with different coordination modes. The unique polynuclear structure {ReCu(CH3CN)(μ-CN)2(CO)2(PPh3)2}n can be formed under the reaction conditions. Presented compounds were characterised by single crystal X-ray diffraction and spectroscopic studies.

The one-dimensional polymeric structure of sodium diaquafenamate–water (1/1) was studied by X-ray diffraction. The sodium cation is coordinated to one oxygen atom of the carboxylate group and to four water oxygen atoms. To characterize the Na—O bonds, the quantum theory of atoms in molecules (QTAIM) and noncovalent interaction (NCI) approaches have been used. Both methods confirmed that the Na—O bonds are very weak, comparable with the weak N—H...O intramolecular hydrogen bond. The polymeric structure is stabilized by the interaction of the sodium cation with the surrounding water molecules.

In the European Waters there is still a huge amount of Unexploded Ordnance (UXO) in place. Different causation scenarios from war hostilities and military actions have led to an unknown amount of different UXO in the Baltic Sea, as well as in the North Sea. Sediment movement, currents or missing information make it difficult to determine the exact position or the amount of remaining UXO with geophysical methods. Since the upcoming and growing economic use of the Baltic Sea (windfarms, pipelines, cables), an extensive Risk Mitigation and the development of working procedures are becoming more and more inevitable. There are well established procedures to perform a historical investigation for hazard identification on land. This stepwise procedure is defined in a German working guide on explosive ordnance clearance. It is transferred to be used in German waters and can also be used for other countries around the Baltic Sea. The historical investigation prepares a geophysical investigation previously to offshore construction work and will reduce the costs and minimize the risk for personnel, equipment and the environment.

A comparative study of the Knoevenagel condensation of an aromatic aldehyde with an active methylene compound (e.g., malononitrile or ethyl cyanoacetate), using solvent free grindstone conditions and microwave irradiation is reported. Our solvent free approaches led these reactions to proceed faster, more cleanly and in higher yields. Moreover, they also allowed the use of nitrobenzaldehydes, which failed to yield the desired products under traditional and water based approaches. Thus, an efficient procedure for the Knoevenagel condensation of aryl and heteroaromatic aldehydes with malononitrile. using the complex [CdNa2(mu-L-2)](n)center dot 6.34H(2)O or commercial CdO as catalyst under solvent-free and very mild conditions has been developed. (C) 2017 Elsevier B.V. All rights reserved. info:eu-repo/semantics/acceptedVersion

The origin of oxygen atoms in oxido-bridged cubo-octahedral hexanuclear rhenium clusters [Re6(μ-O)12L6]+ (L – aromatic amine) has been investigated thoroughly. We confirmed that water is a crucial agent in the creation of the Re6(μ-O)12 core. The reaction of ReI3 with H218O and 3-methylpyridine was carried out resulting in the formation of [Re6(μ-18O)12(3-Mepy)6]I complex.

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.

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. It may also be time-consuming if a homozygote is needed. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternative. 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 creation 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. The 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. 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 utility of commonly used inducers of tetracycline-responsive promoters. We determined the protein interaction network of the exoribonuclease XRN2 and examined the role of the protein in transcription termination by RNAseq analysis of cells devoid of its ribonucleolytic activity. In total we created more than 500 DNA constructs which proves high efficiency of our strategy.