Efficiency of CDS cloning into pKK-RNAi vectors.

<p>Efficiency of CDS cloning into pKK-RNAi vectors.</p

The pKK vector series.

<p>(A) Nucleotide sequences of the TEV-L and TEV-R. Translation to protein and TEV protease cleavage site are shown. Shaded letters indicate nucleotides common to both sequences. (B) Cloning sites of selected pKK vectors. Potentially useful unique restriction sites are marked. For all pKK vectors BshTI and NheI restriction enzymes are used for vector linearization before DNA cloning with the help of our universal SLIC protocol. All pKK vectors have promoters with the TetR repressor binding site. (C) Example of a pKK-BI16 vector. Map of pKK-BI16-TEV-mCherry vector and its cloning region (bottom diagram). Useful unique restriction sites are marked. The tetracycline operator sequences are present in all vectors of pKK-BI16 series, thus, transcription in both directions is regulated by the tetracycline repressor.</p

Comparison of gene expression inducers.

<p>(A-D) Cells were treated with different concentrations of tetracycline or doxycycline and gene expression was monitored by western blot (A: anti-EGFP, B: anti-THOC5 antibodies, Ponceau S staining of the membrane was performed as a loading control) or flow cytometry (C, D: EGFP fluorescence). (D) Quantitative representation of data shown in panel C. Data are represented as mean ± SD (n = 3). (E) Analysis of the kinetics of expression of the indicated transgenes. Cells were treated with tetracycline, collected after indicated time and analyzed by flow cytometry. Mean fluorescent intensity of EGFP positive cells is shown (mean ± SD, n = 3).</p

pKK-RNAi vectors as a tool for generation of a cellular model for functional studies.

<p>(A) Diagram of cloning regions. Potentially useful unique restriction sites are marked. (B) Principles of the approach. Three levels of gene expression are shown. The plasmid integrated into a genome contains: 1) a gene for miRNAs that target the mRNA of a gene of interest; 2) an allele of the gene of interest where the CDS contains silent mutations so that it is insensitive to the miRNAs. As a result the endogenous version of the protein of interest is depleted whereas its ectopic form expressed. NLS marks a nuclear localization signal.</p

Efficiency of stable cell line generation.

<p>(A) Influence of plasmid quantity and selection stringency on the number of colonies obtained following stable transfection of 293 Flp-In T-REx cells. 1.0 μg of pOG44 was mixed with the indicated amounts of pcDNA5/FRT/TO and used for transfection. Cells were selected by treatment with the indicated concentration of hygromycin B and constant concentration of blasticidin S (10 μg/ml). Colonies were stained with crystal violet. (B) Comparison of stable transfection efficiency with pcDNA5/FRT/TO or its pKK derivatives. Cells were transfected with 300 ng of indicated plasmids and 1.0 μg of pOG44 and subjected to selection with hygromycin B (50 μg/ml) and blasticidin S (10 μg/ml).</p

Intracellular localization of EGFP tagged proteins.

<p>Live cell imaging of HeLa-derived stable cell lines expressing EGFP fusions of the indicated proteins. Nuclei were stained with Hoechst 33342.</p

Influence of different FBS on transgene expression.

<p>293 Flp-In T-REx cells stably transfected with a plasmid encoding firefly and renilla luciferase under control of a TetR-regulated bidirectional promoter were cultured in medium supplemented with different fetal bovine sera (FBS), and transgene expression was assessed by measurement of luciferase activity. Two FBS certified for absence of tetracycline or its derivatives were compared to regular FBS. Cells were treated with the indicated concentrations of tetracycline to measure induction response on different sera. Luciferase activity was normalized to the number of cells, which was assessed using AlamarBlue. Data are represented as mean ± SD.</p

Involvement of XRN2 in transcription termination.

<p>(A) Flow cytometry measurement of transgenes expression after 24 hours of induction (EGFP tags XRN2, mCherry is a reporter of miRNA expression). (B) Confocal live cell imaging of EGFP tagged XRN2 and Hoechst 33342 stained nuclei. (C) Western blot analysis of XRN2 protein with anti-XRN2 antibodies. Parental 293 cells and their derivatives analyzed in panel A and B were treated with tetracycline for 72 hours and subjected to western blot. Ponceau S staining of the membrane was performed as a loading control. (D) Meta-gene analysis of transcriptional read-through in wild-type and mutant XRN2 cells. Strand-specific read densities were averaged across 250-bp genomic windows placed directly downstream of 3' ends of highly expressed (TPM > 10), spliced transcripts. The signal is normalized to the average expression detected in the last 250 nt of the analyzed transcripts (250-bp windows upstream to the expected termination site). The shaded part of the graph marks transcripts downstream of transcription termination site (products of transcriptional read-through). It is important to note that lines representing RNA steady-state levels overlay in the part of the graph which correspond to RNAs originating from the transcription upstream of the transcription termination site. This is in contrast to the part of the graph which represent RNA resulting from the unsuccessful transcription termination (shaded part of the graph).</p

SLIC-based DNA cloning strategy.

<p>See main text for detailed description. RE–restriction enzymes used for vector linearization. These are BshTI and NheI in our protocol for universal SLIC. EGFP is an example of tag that can be used. A detailed protocol for the SLIC procedure can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194887#pone.0194887.s007" target="_blank">S2 Supporting Information</a>.</p

Efficiency of the miRNA cassette subcloning into pKK-RNAi vectors.

<p>Efficiency of the miRNA cassette subcloning into pKK-RNAi vectors.</p