Unknown,Transcriptomics,Genomics,Proteomics

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Cre-ERT mediated antiviral induction

ABSTRACT: In this work, we aimed at studying the impact of microRNAs on antiviral responses, using inducible deletion of one or two copies of Dicer1 (in Dicer-floxed x EsrCRE+ MEFs). Unexpectedly, we discovered that Cre-ERT activation by hydroxytamoxifen (OHT) alone, independent of Dicer1 deletion, had a strong impact on antiviral responses. These microarrays were performed on SV40 immortalized Dicer1 fl/fl EsrCRE+ MEFs, Dicer1 fl/wt EsrCRE+ MEFs, 96 h after initial OHT treatment, and SV40 immortalized Dicer1 wt/wt EsrCRE+ MEFs, Dicer1 wt/wt EsrCRE-neg MEFs (Wild Type MEFs), 48 h after initial OHT treatment. These analyses revealed the strong induction of several antiviral genes (Rsad2, Cxcl10...), following Cre-ERT activation. SV40 immortalized mouse embryonic fibroblasts from various genotypes were stimulated 24h with 500nM hydroxytamoxifen, and further expanded for 24 or 72h. Each sample was analyzed on Agilent-028005 SurePrint G3 Mouse GE 8x60K Microarray Each sample is in simplicate - no biological replicates; sample 1-4 were processed/analyzed on a different microarray slide to 5-8 (different day/operator)

ORGANISM(S): Mus musculus

SUBMITTER: Michael Gantier

PROVIDER: E-GEOD-72024 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Cre-dependent DNA recombination activates a STING-dependent innate immune response.

Pépin Geneviève G Ferrand Jonathan J Höning Klara K Jayasekara W Samantha N WS Cain Jason E JE Behlke Mark A MA Gough Daniel J DJ Gough Daniel J DJ G Williams Bryan R BR Hornung Veit V Gantier Michael P MP

Nucleic acids research 20160510 11

Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, ...[more]

PMID: 27166376

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Project description:Analysis of the transcriptional profiles of mRNA and microRNA in Rasless fibroblasts. 4-Hydroxy-tamoxifen (4-OHT) treatment triggers removal of K-Ras expression in [H-Ras-/-;N-Ras-/-;K-Raslox/lox;RERTert/ert] mouse fibroblasts (named K-Raslox) generating M-bM-^@M-^\RaslessM-bM-^@M-^] MEFs which are unable to proliferate, but recover proliferative ability after ectopic expression of constitutively active downstream kinases such as BRAF and MEK1. Using Affymetrix microarrays, we compared the transcriptional profiles of Rasless fibroblasts to those of K-Raslox (MEFs lacking only H-Ras and N-Ras). We also compared the transcriptional profile of Rasless cells with those of BRAF and MEK1-transfected Rasless cells which recovered proliferative ability. Re-entry of the Rasless cells into cell cycle progression through ectopic expression of activated BRAF or MEK1 resulted in full reversion of most transcriptional alterations identified in cells lacking the three canonical Ras proteins. Pre-confluent cell cultures corresponding to the different Ras genotypes were harvested for extraction of total RNA and miRNA samples, and subsequent hybridization on Affymetrix microarrays. The different sets of experimental mRNA samples analyzed here included (i) 8 samples K-Raslox (proliferating cells expressing only K-Ras, considered as Control): 4 biological replicates, technical duplicates each; (ii) 7 samples Rasless: the same cells after treatment with 4-OHT for 12 days (to become non-proliferating fibroblasts), including 4 biological replicates and technical duplicates from 3 of them; (iii) 3 samples BRAF-rescued MEFs (Rasless cells harboring activated BRAF constructs, with regained proliferative ability): 3 biological replicates; (iv) 3 samples MEK1-rescued MEFs (Rasless cells harboring activated MEK1 constructs, with regained proliferative ability): 3 biological replicates; and their respective controls harbouring the vempty vectors (v) 2 samples Hygromycin-resistant (control for BRAF-transfected Rasless cells) including 2 biological replicates, and (vi) 4 samples Puromycin-resistant (control for MEK1-transfected Rasless cells) including 2 biological replicates, technical duplicates each. The gene expression study contains a total of 27 samples. Regarding the miRNA expression study, the experimental samples analyzed included (i) 4 samples samples K-Raslox (used as Control): 2 biological replicates, technical duplicates each; (ii) 8 samples Rasless: the same cells after treatment with 4-OHT for 6 days (2 biological replicates, technical duplicates each) and 12 days (2 biological replicates, technical duplicates each); (iii) 4 samples BRAF-rescued, including 2 biological replicates, technical duplicates each; (iv) 4 samples MEK1-rescued,including 2 biological replicates, technical duplicates each, and (v) 4 samples Puromycin-resistant empty vector, containing 2 biological replicates, technical duplicates each. The miRNA expression study contains a total of 24 samples.

Dataset Information

Cre-ERT mediated antiviral induction

Publications

Cre-dependent DNA recombination activates a STING-dependent innate immune response.

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