title: IFN_r2
organism: Mus musculus
description: Protocols: C57BL/6 female mice (8 weeks old) were irradiated and transplanted with mouse hematopoietic progenitor/stem cells transduced with a control mTie2-GFP-mirT LV. At 20 weeks of age mice were sacrificed. Spleen tissue was dissociated and cells subjected to FACS sorting. F4/80+ macrophages were recovered. IFN treatment was performed by reconstituting lethally irradiated mice with hematopoietic stem and progenitor cells (HSPCs) that were previously transduced with a lentiviral vector (LV) containing the cDNA sequence of the IFN-alpha gene under the control of the Tie2 promoter/enhancer sequences. Among differentiated hematopoietic cells, Tie2 is selectively expressed in a population of tumor-infiltrating monocytes (TEMs), thus resulting in tumor-specific IFN-alpha delivery. The LV design also incorporates into the IFN-alpha transgene multiple miR-126 and miR-130a target sequences to suppress post-transcriptionally transgene expression in HSPCs limiting potential transgene in this compartment. C57BL/6 female mice (8 weeks old) were irradiated and transplanted with mouse HSPC transduced with a control mTie2-GFP-mirT LV. At 16 weeks of age mice were injected intravenously with 10-100 thousand B-ALL cells. At 20 weeks of age mice were sacrificed. Spleen tissue was dissociated and cells subjected to FACS sorting. F4/80+ macrophages were recovered. C57BL/6 female mice (8 weeks old) were irradiated and transplanted with mouse HSPC transduced with a control mTie2-IFN-mirT LV. At 16 weeks of age mice were injected intravenously with 10-100 thousand B-ALL cells. At 20 weeks of age mice were sacrificed. Spleen tissue was dissociated and cells subjected to FACS sorting. F4/80+ macrophages were recovered. ReliaPrep RNA MiniPrep System (Promega) RNA-Seq libraries were prepared with the SMART-Seq2 protocol, with minor modification. Briefly, RNA (1-5 ng) was reverse transcribed using custom oligodT and template-switching LNA oligos (sequences), followed by PCR amplification and clean-up (Ampure XP beads, Beckman Coulter). The resulting cDNA (0,5- 1 ng) was tagmented at 55°C for 30 minutes and final RNA-Seq libraries generated using reagents from the Nextera XT DNA Library Prep Kit (Illumina).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-07-26T17:02:32Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-05-29T14:42:41Z
attributes.tag: External Id
attributes.value: SAMEA4696501
attributes.tag: INSDC center name
attributes.value: San Raffaele Telethon Institute for Gene Therapy.
attributes.tag: INSDC first public
attributes.value: 2018-07-26T17:02:32Z
attributes.tag: INSDC last update
attributes.value: 2018-05-29T14:42:41Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6482:IFN_r2
attributes.tag: age
attributes.value: 20
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: macrophage
attributes.tag: common name
attributes.value: house mouse
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: lentiviral expression of mTie2-IFN-mirT LV and Tie2-driven overexpression of IFNalpha
attributes.tag: organism part
attributes.value: spleen
attributes.tag: phenotype
attributes.value: F4/80 positive
attributes.tag: sample name
attributes.value: E-MTAB-6482:IFN_r2
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: female
attributes.tag: strain
attributes.value: C57BL/6