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Characterization of an Osmr Conditional Knockout Mouse Model

ABSTRACT: Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines. OSM has been found to have distinct anti-inflammatory and pro-inflammatory properties in various cellular and disease contexts. OSM signals through a receptor complex that includes OSMRbeta and activates downstream signaling through the JAK/STAT, MAPK/ERK and PI3K/AKT pathways. To investigate the anti- and/or pro-inflammatory roles of OSM signaling in the context of adult hematopoiesis, we utilized the readily available Cre-driven conditional knockout Osmrfl/fl mouse model B6;129-Osmrtm1.1Nat/J. This model contains loxP sites flanking exon 2, which is the first coding exon of the Osmr gene. We crossed Osmrfl/fl mice to an interferon-inducible Mx1-Cre allele that is classically utilized to study adult hematopoiesis given its high efficiency of expression and recombination in adult hematopoietic cells. We developed a PCR assay to evaluate recombination of the Osmrfl allele. In Osmrfl/fl Mx1-Cre mice, we observed complete recombination of the Osmrfl allele and loss of exon 2 in hematopoietic (bone marrow) as well as non-hematopoietic (liver, lung, kidney) tissues. To evaluate Osmr mRNA expression downstream of exon 2, real-time PCR was utilized with TaqMan probes that anneal to an amplicon spanning Osmr exons 17 and 18. Using this assay, reduced Osmr transcript was observed in kidney samples from Osmrfl/fl Mx1-Cre mice compared to Mx1-Cre controls. However, Osmr transcript levels were similar in bone marrow, lung, and liver samples from Osmrfl/fl Mx1-Cre compared to Mx1-Cre control mice, suggesting that transcript is being produced despite loss of exon 2. Western blots revealed that liver cells from Osmrfl/fl Mx1-Cre mice had complete loss of OSMR protein. In contrast, bone marrow, kidney, and lung cells from Osmrfl/fl Mx1-Cre mice had reduced levels of OSMR protein. Together, our data suggest that the B6;129-Osmrtm1.1Nat/J model should be utilized with caution as loss of exon 2 of Osmr variably impacts mRNA and protein expression in a tissue-dependent context.

ORGANISM(S): Mus musculus

PROVIDER: GSE244544 | GEO | 2024/02/18

REPOSITORIES: GEO

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Project description:Mutations in spliceosomal genes are commonly found in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). These mutations occur at highly restricted amino acid residues and perturb normal splice site and exon recognition. Spliceosomal mutations are always heterozygous and rarely co-occur with one another, suggesting that cells may only tolerate a partial deviation from normal splicing activity. To test this hypothesis, we generated mice with inducible hemizygous expression of the commonly occurring SRSF2P95H mutation in the hematopoietic system. These mice rapidly developed lethal bone marrow failure upon activation of the Srsf2P95H mutation with concomitant deletion of the wildtype Srsf2 allele, demonstrating that Srsf2-mutant cells depend on the wildtype Srsf2 allele for survival. We next tested whether spliceosomal-mutant leukemias display greater sensitivity to pharmacologic splicing inhibition induced by the small molecule E7107. Treatment of isogenic murine leukemias as well as patient-derived xenograft (PDX) AMLs showed significant reductions in leukemic burden specifically in samples carrying spliceosomal mutations. Collectively, these data provide genetic and pharmacologic evidence that leukemias with spliceosomal mutations are preferentially susceptible to additional splicing perturbations in vivo compared with wildtype counterparts. Modulation of spliceosome function may provide a novel therapeutic avenue in genetically defined subsets of MDS/AML patients. We created an isogenic murine leukemia model by retroviral overexpression of the MLL-AF9 fusion oncogene in Vav-Cre Srsf2+/+ or Vav-Cre Srsf2P95H/+ BM cells followed by transplantation into lethally irradiated recipient mice. In order to determine the mechanistic origins of the Srsf2 mutant-selective effects of E7107, we analyzed transcriptional changes after five consecutive days of E7107 or vehicle treatment in vivo. GFP+ Cd11b+ cells were purified from the BM of recipient mice exactly three hours after the last dose of E7107 and were subjected to paired-end 2x50bp RNA-seq. For the knock-in/knock-out experiments, we generated inducible, hemizygous Srsf2P95H mice to study the effects of wildtype Srsf2 deletion with concomitant activation of the Srsf2P95H allele. Mx1-cre Srsf2fl/+ mice were crossed to Srsf2P95H/+ mice to generate Srsf2 wildtype (Mx1-cre Srsf2+/+), Srsf2 heterozygous knockout (Mx1-Cre Srsf2+/-), Srsf2 heterozygous P95H mutant (Mx1-Cre Srsf2P95H/+), and Srsf2 hemizygous P95H mutant (Mx1-Cre Srsf2P95H/-) mice, which were subjected to single-end 101bp RNA-seq.

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Characterization of an Osmr Conditional Knockout Mouse Model

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