RT Journal Article
SR Electronic
T1 Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 160101
DO 10.1101/160101
A1 Roman J. Szczesny
A1 Katarzyna Kowalska
A1 Kamila Klosowska-Kosicka
A1 Aleksander Chlebowski
A1 Ewelina P. Owczarek
A1 Zbigniew Warkocki
A1 Tomasz M. Kulinski
A1 Dorota Adamska
A1 Kamila Affek
A1 Agata Jedroszkowiak
A1 Anna V. Kotrys
A1 Dominik Cysewski
A1 Rafał Tomecki
A1 Pawel S. Krawczyk
A1 Lukasz S. Borowski
A1 Andrzej Dziembowski
YR 2017
UL http://biorxiv.org/content/early/2017/07/06/160101.abstract
AB 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.