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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.

ORGANISM(S): Mus musculus

PROVIDER: GSE72024 | GEO | 2016/05/18

SECONDARY ACCESSION(S): PRJNA292756

REPOSITORIES: GEO

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Project description:MiRNAs have the potential to regulate cellular differentiation programs. However, miRNA-deficiency in primary hematopoietic stem cells (HSCs) results in HSC depletion in mice, leaving the question of whether miRNAs play a role in early-lineage decisions unanswered. To address this issue, we deleted Dicer1, which encodes an essential RNaseIII enzyme for miRNA biogenesis, in murine CCAAT/enhancer-binding protein alpha (C/EBPA)-positive myeloid-committed progenitors in vivo. In contrast to the results in HSCs, we found that miRNA depletion affected neither the number of myeloid progenitors nor the percentage of C/EBPA-positive progenitor cells. Analysis of gene-expression profiles from wild type and Dicer1-deficient granulocyte-macrophage progenitors (GMPs) revealed that 20 miRNA families were active in GMPs. Of the derepressed miRNA targets in Dicer1-null GMPs, 27% are normally exclusively expressed in HSCs or are specific for multi-potent progenitors and erythropoiesis, indicating an altered gene-expression landscape. Dicer1-deficient GMPs were defective in myeloid development in vitro and exhibited an increased replating capacity, indicating a regained self-renewal potential of these cells. In mice, Dicer1 deletion blocked monocytic differentiation, depleted macrophages and caused myeloid dysplasia with morphological features of Pelger-Huët anomaly. These results provide evidence for a miRNA-controlled switch for a cellular program of self-renewal and expansion towards myeloid differentiation in GMPs. To generate Cebpa-Cre;R26-LSL-Eyfp;Dicer1wt/fl/Dicer1fl/fl mice, we crossed mice that contain floxed Dicer1 alleles (Dicer1fl) with Cebpa-Cre;R26-LSL-Eyfp reporter mice 2. Fetal livers were obtained on embryonic day (E) 13.5. Routine genotyping of Dicer1; Cebpa-Cre;R26-LSL-Eyfp mice was performed by PCR assays of DNA from tail or toe biopsies. For transplantation, 6 to 8-week-old recipient mice (C57Bl/6, Jackson Laboratories) were irradiated (8.5 Gy) and tail-vein injected with fetal liver single-cell suspensions. Typically, cells from each fetal liver were transplanted into two recipient mice. Hematopoietic tissues were analyzed 6-10 weeks post transplantation. EYFP positive GMPs from the bone marrow of Dicer wt control (n=3), Dicer -/wt (n=3 and Dicer fl/fl (n=3) were sorted and analyzed for gene expression profiles.

Project description:DNA polymerase zeta (Polz) plays a key role in translesion DNA synthesis. Inactivation of the gene encoding its catalytic subunit (Rev3L) leads to embryonic lethality and abrogates cell proliferation in primary fibroblasts highlighting an essential role of Polz in mammalian cells. However, its biological function remains poorly understood. Here, we examined if the absence of Rev3L may impact on transcriptional program by performing microarray-based transcriptome profiling from Rev3L+/+ and Rev3L-/- MEFs. For this study, we used mouse embryonic fibroblasts (MEF) that are derived from mice harboring one allele with essential exons flanked by loxP sites, and either one inactivated allele of Rev3L (Rev3L−/lox) or, one wild type allele (Rev3L+/lox). Upon introduction of an adenovirus expressing Cre, floxed copy of Rev3L was deleted thus generating Rev3L−/Δ cells, and cell lines were SV40 Tag-immortalized (Lange et al., NAR 2012). We then used Rev3L−/Δ and Rev3L+/lox MEFs (named Rev3L-/- and Rev3L+/+ MEFs, respectively in this study). From two independent cell culture (A and B), total RNAs were extracted from Rev3L+/+ and Rev3L-/- MEFs and processed to determine the transcriptional program in these cell lines. For this study, we used mouse embryonic fibroblasts (MEF) that are derived from mice harboring one allele with essential exons flanked by loxP sites, and either one inactivated allele of Rev3L (Rev3L−/lox) or, one wild type allele (Rev3L+/lox). Upon introduction of an adenovirus expressing Cre, floxed copy of Rev3L was deleted thus generating Rev3L−/Δ cells, and cell lines were SV40 Tag-immortalized (Lange et al., NAR 2012). We then used Rev3L−/Δ and Rev3L+/lox MEFs (named Rev3L-/- and Rev3L+/+ MEFs, respectively in this study). From two independent cell culture (A and B), total RNAs were extracted from Rev3L+/+ and Rev3L-/- MEFs and processed to determine the transcriptional program in these cell lines with Agilent Mouse Genomic array (8x60k) with two replicates in dual color.

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Cre-ERT mediated antiviral induction

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