Abstract
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.
Highlights
Deciphering a protein’s function requires investigating its subcellular localisation, identifying its binding partners, and performing multiple functional assays
The multiple cloning site of the vector was modified by Streamlined approach for generation of stable mammalian cell lines for functional studies introducing two 21-nucleotide long sequences, the Sequence and ligation independent cloning (SLIC) arms (Fig 1)
We have created a series of vectors that facilitate various functional and biochemical studies of human proteins
Summary
Deciphering a protein’s function requires investigating its subcellular localisation, identifying its binding partners, and performing multiple functional assays. Ectopic expression is widely used for investigations of human proteins It can be achieved by transient or stable transfection of cultured cells with a plasmid or virus. One can perform targeted genomic manipulation to engineer the gene of interest in its natural locus This used to be difficult and time-consuming for most vertebrate cell lines before the advent of CRISPR-based approaches [1, 2, 3]. Genome editing has the crucial advantage in that the studied gene is expressed at its natural levels and naturally responds to all stimuli. This approach can prove to be problematic if control of gene expression is required or specific alleles are to be tested. Transfection, transient or stable, offers a lot of flexibility in transgene sequence, allowing for the use of stronger, weaker, or even regulated promoters
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