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

PLoS One. 2018 Mar 28;13(3):e0194887. doi: 10.1371/journal.pone.0194887. eCollection 2018.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular
  • DNA Nucleotidyltransferases / genetics
  • DNA Nucleotidyltransferases / metabolism*
  • Exoribonucleases / genetics
  • Exoribonucleases / metabolism
  • Gene Expression Regulation*
  • Genetic Vectors*
  • HeLa Cells
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Mutation
  • Nucleophosmin
  • Promoter Regions, Genetic
  • Proteins / genetics
  • Proteins / metabolism*
  • Recombination, Genetic*
  • Transcription Termination, Genetic*


  • NPM1 protein, human
  • Proteins
  • Nucleophosmin
  • DNA Nucleotidyltransferases
  • FLP recombinase
  • Exoribonucleases
  • XRN2 protein, human

Grants and funding

This work was mainly supported by the Ministry of Science and Higher Education of Poland (IP2012 046372 to RJS, and cosupported by the National Science Centre, Poland (UMO-2014/13/D/NZ2/01114 to RJS, UMO-2012/04/S/NZ1/00036 to ZW, and the Foundation for Polish Science (TEAM/2016-1/3 to AD, Experiments were carried out with the use of CePT infrastructure financed by the European Union – the European Regional Development Fund (Innovative economy 2007–13, Agreement POIG.02.02.00-14-024/08-00). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.