Rapid identification of 3’ UTR sequences by means of Stem-Loop 3’ UTR RACE PCR (SLURP).

<p>Section A: Figure shows stem-loop primer (SLURP rt) binding and priming of the reverse transcription reaction. As highlighted in green and yellow, the 'SLURP rt' provides two primer binding sites for a nested PCR that are hidden by the secondary structure. Section B: The use of two batched gene specific primers (GSP rev i and GSP rev ii) together with 'fw i and fw ii' binding to the sites provided by the 'SLURP rt' allowed for a nested PCR. Section C: Identified miR-29a target sites within the 3’ UTR of porcine CASP7. SLURP based sequence revealed that target site 1 was highly conserved between humans and pigs. The predicted target site 2 suggested non-canonical binding of miR-29a as also shown in humans.</p

Sequential mutagenesis of multiple miR-29a target sites within the 3’ UTR of CASP7 by means of SMAP.

<p>Section A: Full length 3’ UTR of CASP7 was generated harbouring both identified target sites of miR-29a. Section B: For individual but also combined target site mutagenesis, the 3’ UTR was theoretically divided in three segments defined by locations of target sites. Sections C-E: Modular assembly of amplicons resulted in sequential mutagenesis of target sites. Section F: Gel image shows five amplicons after mutagenesis as well as assembled products for reporter gene fusion. NC represents the no template control of the assembly PCR. Relative luciferase activity (Luc _Gaussia : Luc _Cypridina) was determined using a miR-29a mimic compared with a nonsense miRNA control together with the mutated target site 1 (pTKGhCASP7m1), mutated target site 2 (pTKGhCASP7m2), mutated target sites 1 and 2 (pTKGhCASP7m1+2) as well as the wild type control (pTKGhCASP7) . The columns show means of normalised luciferase activity of three biological replicates each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (*: P < 0.05; ***: P < 0.001, paired t test).</p

SMAP reveals the transcription factor NKX3.1 as a miR-155 target.

<p>Section A: Luciferase reporter assays after mutagenesis proved NKX3.1 as a miR-155 target. Relative luciferase activity (Luc _Gaussia : Luc _Cypridina) was determined using a miR-155 mimic compared with a nonsense miRNA control together with the mutated seed (pTKGhNKX3.1m) as well as wild type control (pTKGhNKX3.1) . The columns show means of normalised luciferase activity of three biological replicates each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (****: P < 0.001, unpaired t test). Section B: mRNA degradation assays by means of RT-qPCR experiments are shown detecting relative NKX3.1 levels (reference gene: GAPDH). U937 were transfected with miR-155, nonsense miRNA and NKX3.1 siRNA. RNA was isolated at 24 and 48 h post transfection. Columns show mean relative NKX3.1 transcript levels (± SD) of three biological replicates each measured in triplicates compared with nonsense transfected controls. Section C: Western Blots detecting NKX3.1 and GAPDH (reference protein) are shown using the monocytic U937 cells transfected with miR-155, nonsense miRNA and NKX3.1 siRNA. Intrinsic NKX3.1 levels are decreased after miR-155 transfection compared with nonsense controls and siRNA (48 h post transfection). The bar graph shows the luminescence-based relative quantification of protein (NKX3.1:GAPDH) of three individual biological replicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (*: P < 0.05, unpaired t test). Transfection efficiency was evaluated by transfecting Cy3 labelled nonsense siRNA and fluorescence microscopy. While NKX3.1 translation is markedly inhibited by miR-155, cellular NKX3.1 mRNA levels remained stable after miR-155 at 24 h and decreased after 48 h suggesting slow cellular turnover rates of miR-155 directed NKX3.1 inhibition. Section D: Endogenous miR-155 expression leads to decreased luciferase activity of transfected reporters. The bar graph on the left hand side shows relative miR-155 expression in U937 cells at 5 and 24 h after LPS stimulation. The other bar graph shows that endogenous miR-155 expression results in clear repression of luciferase activity (nonsense transfected) while ectopically introduced miR-155 results in elevated repression and mutated controls remain unaffected. The columns show means of three biological replicates each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (**: P < 0.01,***: P < 0.001, unpaired t test).</p

Concept of Seed Mutagenesis Assembly PCR (SMAP).

<p>Figure shows seed mutagenesis of miR-155 targets SHIP1 and NKX3.1. Gel images show amplicons obtained after each step applying a gradient PCR, while NC represents the no template control at the lowest annealing temperature. Section A: 3’ UTR amplicons were generated harbouring the predicted target site. Section B: Oligonucleotides were used for seed mutagenesis and generation of overlapping termini that possessed approximately 12 nucleotides at the 3’ as well as 5’ ends flanking the mutated site. Section C: Assembly PCR of amplicons with mutated termini provided 3’ UTRs including the mutated seed. The amplicon was fused to a luciferase gene for reporter gene assays. Relative luciferase activity (Luc _Gaussia : Luc _Cypridina) was determined using a miR-155 mimic compared with a nonsense miRNA control together with the mutated seed (pTKGhSHIP1m) as well as wild type control (pTKGhSHIP1) . The columns show means of normalised luciferase activity of three biological replicates each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (****: P < 0.001, unpaired t test).</p

Data_Sheet_1.DOCX

<p>Background: Global as well as specific expression profiles of selected rat tissues were characterized to assess the safety of genetically modified (GM) maize MON810 containing the insecticidal protein Cry1Ab. Gene expression was evaluated by use of Next Generation Sequencing (NGS) as well as RT-qPCR within rat intestinal tissues based on mandatory 90-day rodent feeding studies. In parallel to two 90-day feeding studies, the transcriptional response of rat tissues was assessed as another endpoint to enhance the mechanistic interpretation of GM feeding studies and/or to facilitate the generation of a targeted hypothesis. Rats received diets containing 33% GM maize (MON810) or near-isogenic control maize. As a site of massive exposure to ingested feed the transcriptomic response of ileal and colonic tissue was profiled via RT-qPCR arrays targeting apoptosis, DNA-damage/repair, unfolded protein response (UPR). For global RNA profiling of rat ileal tissue, we applied NGS.</p><p>Results: No biological response to the GM-diet was observed in male and in female rat tissues. Transcriptome wide analysis of gene expression by RNA-seq confirmed these findings. Nevertheless, gene ontology (GO) analysis clearly associated a set of distinctly regulated transcripts with circadian rhythms. We confirmed differential expression of circadian clock genes using RT-qPCR and immunoassays for selected factors, thereby indicating physiological effects caused by the time point of sampling.</p><p>Conclusion: Prediction of potential unintended effects of GM-food/feed by transcriptome based profiling of intestinal tissue presents a novel approach to complement classical toxicological testing procedures. Including the detection of alterations in signaling pathways in toxicity testing procedures may enhance the confidence in outcomes of toxicological trials. In this study, no significant GM-related changes in intestinal expression profiles were found in rats fed GM-maize MON810. Relevant alterations of selected cellular pathways (apoptosis, DNA damage and repair, UPR) pointing toward intestinal toxicity of the diets were not observed. Transcriptomic profiles did not reveal perturbations of pathways associated with toxicity, underlining the study results revealed by classical OECD endpoints.</p