GoKawiilAnimal studies
All animal studies were performed in accordance with UK Home Office regulations with approval by the University of Oxford Animal Welfare and Ethical Review Body (project license number 30/3359, PP2240412 and PP3246892) and the Francis Crick Institute Animal Welfare and Ethical Review Body (project license number PP8468807), as well as the Irish Health Products Regulatory Authority regulations License AE19136/P108 and P196, approved by Trinity College Dublin’s Animal Research Ethics Committee. NOD.Cg-KitW-41JTyr+PrkdcscidIl2rgtm1Wjl/ThomJ (NSGW41) mice45, Il1rl1-KO mice46, Gata2G320D knock-in mice30 and Gata1–eGFP BAC transgenic mice8 have been previously described. For IL-33 administration 9-week-old male C57BL/6J or Gata1–eGFP mice or short hairpin RNA (shRNA)-targeting Scramble or Lmo4, or control or St2−/− transplanted mice were intraperitoneally injected with 1 μg of recombinant mouse IL-33 (Peprotech) or PBS. For parasite infection, 9–12-week-old male C57BL/6J were used. Eight-nine-week-old Gata2D/D mice and wild-type littermates were infected with 200 H. polygyrus third-stage larvae, obtained from faecal cultures of H. polygyrus-infected mice, by oral gavage. Mice were maintained under specific pathogen-free conditions at 19–23 °C and 45–65% humidity on a 12-h light–12-h dark cycle. Sex and age-matched animals were randomly selected and allocated to experimental group, expect when assignment based on mouse genotype. Experiment and data collection were not performed blind to the condition of the experiment.
Plasmid construction
Flag–LMO2, MYC–LMO4, shRNA-targeting Scramble and shRNA-targeting Lmo4 constructs were obtained from Addgene (#64893, #22965, #59299 and #59292, respectively). The HA–FOG1 construct has been previously described17. Gata2 as well as LMO2/4 chimeras LMO-C1 and LMO-C2 cDNA were synthetized and subcloned into pLeGO-G (Addgene #27347) or pLeGO-C2 (Addgene #27339) under a mCherry-P2A reporter. For lentiviral transduction, Lmo4 cDNA was amplified and subcloned into pLeGO-C2 (Addgene #27339) or LT3-GEPIR (Addgene #111177) under a mCherry-P2A reporter.
Lentiviral production
HEK-293T (American Type Culture Collection CRL-11268) cells were plated in DMEM (Gibco) supplemented with 10% FBS (Gibco), 2 mM l-glutamine (Gibco) and 100 U ml−1 penicillin–streptomycin (Gibco), a day before co-transfection. Co-transfection was performed using PEIpro (Polyplus) according to the manufacturer’s recommendation, with psPAX2 (Addgene #12260), pMD2-G (Addgene #12259) and LeGO-C2-P2A-Lmo4/empty vectors. The viral supernatant was collected 48 h post-transfection, filtered using 0.45-µm cellulose acetate filter (Milipore) and concentrated using an Optima XPN-80 (Beckman Coulter) at 23.000 rpm for 1.5 h at 4 °C. HEK-293T cells were purchased from the supplier (Sigma) as a cell line authenticated by STR profiling, and was tested negative for mycoplasma.
Bone marrow transplantation
A total of 5 × 105 bone marrow cells were isolated from 12-week-old female Gata1–eGFP and Gata2+/+ or Gata2D/D mice and engrafted in lethally irradiated (10 Gy) 8–10-week-old female CD45.2 recipient mice by intravenous injection. To generate Il1rl1−/− chimeras, 5 × 105 total bone marrow cells from CD45.2 Il1rl1−/− or wild-type control mice were co-transplanted with an equal number of wild-type CD45.1 bone marrow cells into lethally irradiated (10 Gy) 8–10-week-old female CD45.1 recipient mice by intravenous injection.
Transplantation of genetically modified HSPCs
Bone marrow was isolated from 10–14-week-old female C57BL/6J or Gata1–eGFP mice8. Haematopoietic stem and progenitor cells (HSPCs) were enriched using CD117 MicroBeads and LS columns, according to the manufacturer’s recommendation (Miltenyi Biotec). Transduction of empty/Lmo4-expressing or shRNA-targeting Scramble/Lmo4 lentivirus was at a multiplicity of infection of 50 in StemSpan SFEM (STEMCELL Technologies) supplemented with 2 mM l-glutamine (Gibco), 100 U ml−1 penicillin–streptomycin (Gibco), 5 μg ml−1 polybrene (Merck), 100 ng ml−1 mouse SCF (Peprotech) and 100 ng ml−1 human TPO (Peprotech). Cells were spinfected at 33 °C and 700g for 1 h and incubated at 37 °C for 7 h or overnight. A total of 5 × 105 cells were injected intravenously into lethally irradiated (12 Gy; split dose) 8–10-week-old female CD45.2-recipient or CD45.1-recipient mice.
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