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Blood, Vol. 113, Issue 7, 1432-1443, February 12, 2009
Loss of Mll5 results in pleiotropic hematopoietic defects, reduced neutrophil immune function, and extreme sensitivity to DNA demethylation
Blood Heuser et al.
113: 1432
Supplemental materials for: Heuser et al
Gene targeting in embryonic stem cells and genotyping
The gene-targeting strategy engineered a germ-line deletion of 106 bp in exon 3 of Mll5 designated Mll5tm1Apa (Fig. S1A). The 5′ target arm was amplified from isogenic 129S6(SvEv) genomic DNA using the primers 5′armFII and 5′armR and the 3′ arm homology arm was amplified using the primers 3′armF and 3′armR (Table S1). The homology arms were cloned into pKO which has an IRES β-Gal and a selectable cassette (MC-neo) within LoxP sites as shown in Fig. S1A. Correctly targeted 129S6 embryonic stem (ES) cell clones were selected by Southern blotting using PvuII-digested genomic DNA and probing with a radiolabelled 5′ probe (generated by PCR using primers 5′prFII and 5′prFII) or the 3′ probe (generated by PCR using the primers 3′prF and 3′prR). Five correctly targeted clones were generated by this strategy and one was transmitted to the germline. Mice were backcrossed twice to parental 129S6 wild type mice before analysis. Offspring from Mll5+/tm1Apa crosses and individual embryos were genotyped by PCR using the primers HetF, HetR and Asc403 as indicated in Fig. S1A. Expression analysis by RT-PCR and Western blotting
RNA was extracted and reverse transcribed as previously described.1 Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) was done as previously described using the 7900HT Fast Real-Time PCR system (Applied Biosystems, Foster City, CA).1,2 Relative expression was determined with the 2−ΔΔCT method,3 and the housekeeping gene transcript Abl1 was used to normalize the results. Semi-quantitative RT-PCR was performed using epGradient S Thermocycler (Eppendorf, Hamburg, Germany) for 25–28 cycles (95°C/30sec, 55°C/30 sec and 72°C/30 sec) and visualized on a 1.5% agarose gel. Primers were manufactured by Invitrogen (Burlington, Canada). Primer sequences (5′ to 3′) are provided in Table S1. For protein expression cells or tissues were homogenized and sonicated in RIPA lysis buffer (150mM NaCl, 50mM Tris-HCl pH 7.5, 1mM EDTA, 1% Triton X-100, 1% sodium deoxycolate. 0.1% SDS, 0.5 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF and protein inhibitor cocktail (Roche Complete)). Protein extracts were fractionated by 6% SDS-PAGE, transferred to Trans-Blot pure nitrocellulose membranes (pore size 0.45µm) (Biorad, Hercules, CA) and blotted with a rabbit IgG polyclonal antibody raised against MLL5 (Fig. S1D). Antibody generation
The peptide SRVPKVTDKRRKKSGEKEQN (corresponding to residues 218–237 of murine MLL5) was synthesised and conjugated to keyhole limpet hemocyanin (KLH) via an amino-terminal residue and injected into rabbits by Alpha Diagnostics. Bleeds from rabbits were screened by ELISA (Alpha Diagnostics, TX) and by western blotting with purified peptide or purified fragments of MLL5 containing the epitope, and the serum from rabbit 9760 was used in further studies. FACS analysis, cell cycle analysis, and apopotosis measurement
Freshly isolated cells from murine bone marrow or spleen were used for fluorescence activated cell sorting (FACS) of hematopoietic progenitor or differentiated cell populations. Cells were stained as described1 using the following antibodies: For lin−Sca1+c-kit+ (LSK)-FLT3 populations PE-Cy5 conjugated anti-mouse Gr1 (clone Ly6G-6C, BioLegend, San Diego, CA), PE-Cy5 conjugated anti-mouse Ter119 (clone Ter119, BioLegend), PE-Cy5 conjugated anti-mouse B220 (clone RA3-6B2, Pharmingen) PE-Cy5 conjugated anti-mouse CD3 (clone 145-2C11, Pharmingen) PE-Cy5 conjugated anti-mouse CD4 (clone GK1.5, Pharmingen) PE-Cy5 conjugated anti-mouse CD8 (clone 53-6.7, Pharmingen), PE-Cy5 conjugated anti-mouse Mac-1 (clone M1/70, eBioscience, San Diego, CA) for lineage cocktail, APC conjugated anti-mouse c-kit (clone 2B8, Pharmingen), PE conjugated anti-mouse FLT3 (clone A2F10, eBioscience), FITC conjugated anti-mouse Sca-1 (clone E13—161.7, Pharmingen); CMPs and MEPs were stained as described.4 Lineage marker expressing cells were isolated using the following antibodies: APC-conjugated anti-mouse Mac-1 (clone M1/70, eBioscience), PE-conjugated anti-mouse Ter119 (clone TER119, Pharmingen), FITC-conjugated anti-mouse CD71 (clone C2, Pharmingen), PE-conjugated anti-mouse CD41 (clone MWReg30, Pharmingen), PE-conjugated anti-mouse B220 (clone RA3-6B2, Pharmingen), PE-conjugated anti-mouse CD4 (clone L3T4, Pharmingen), and PE-conjugated anti-mouse CD8a (clone 53-6.7, Pharmingen). Cell cycle and apoptosis analysis in LSK cells were performed using PE-conjugated anti-mouse Mac-1 (clone M1/70, eBioscience), PE-conjugated anti-mouse Gr-1 (clone RB6-8C5, Pharmingen), PE-conjugated anti-mouse Ter119, PE-conjugated anti-mouse B220, PE-conjugated anti-mouse CD3 (clone 17A2, Pharmingen), PE-conjugated anti-mouse CD4, and PE-conjugated anti-mouse CD8a, biotin conjugated anti-mouse Sca-1 (clone D7, Pharmingen), PETexasRed-conjugated Streptavidin (Pharmingen), APC-conjugated anti-mouse c-kit, FITC-conjugated anti-BrdU for cell cycle analysis (Pharmingen), FITC-conjugated annexin V for apoptosis analysis (Pharmingen), 7AAD for cell cycle analysis (Pharmingen), and DAPI for apoptosis analysis. Cell populations as specified in the results section were sorted or analyzed with a FACSDIVA flow cytometer (Becton Dickinson, Mississauga, Canada). Purity of cell populations was verified to be above 90 percent. Mice and retroviral infection of primary bone marrow cells
All mice were bred and maintained as approved by the University of British Columbia Animal Care Committee or under the authority of a U.K. Home Office Project License (PPL80/1503). The transgenic mice (Mll5tm1Apa) were maintained as an inbred stock on a 129S6/SvEv genetic background on high-fat sterile diet. Breeders were supplemented with dough diet and sunflower seeds. Primary mouse BM cells were transduced as previously described.5 Briefly, bone marrow cells were harvested from stock mice and stimulated for 48 h in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 10 ng/mL of human interleukin-6 (hIL-6), 6 ng/mL of murine interleukin-3 (mIL3), and 20 ng/mL of murine stem cell factor (mSCF; all from StemCell Technologies Inc., Vancouver, Canada). The cells were infected by cocultivation with irradiated (4,000 cGy) GP+ E86 viral producer cells in the presence of 5 µg/mL protamine sulfate (Sigma, Oakville, Canada). Bone marrow transplantation and monitoring of mice
Bone marrow cells were injected into the tail vein of lethally irradiated recipient mice that were exposed to a single dose of 750 cGy total-body irradiation accompanied by a life sparing dose of 1 × 105 freshly isolated bone marrow cells from syngenic mice, if not otherwise stated. Viability of mice was monitored daily. Engraftment of donor cells was monitored by tail vein bleeds and FACS analysis of GFP or YFP positive cells every four weeks. Bone marrow in viable animals was aspirated from the femur in anesthetized animals as stated in the text. Blood counts with differential WBC analysis were performed using an ABC Vet Automated Blood Counter (VetNovations Canada, Barrie, ON). Blood cell counts in non-transplanted animals were obtained through cardiac puncture of euthanized animals and by using the automated cell counter. Lineage distribution was determined by FACS analysis (FACSCalibur, Becton Dickinson, Mississauga, Canada) as previously described.5 Monoclonal antibodies used were phycoerythrin (PE)-labeled Gr-1-PE, Mac-1-APC, B220PE, CD4-PE/CD8-APC, Ter119-PE, c-kit-APC, and Sca-1PE (Pharmingen, San Diego, CA). Morphologic and histologic analysis of PB and BM was performed as previously described.5 Briefly, mouse tissues were dissected and fixed for four hours to overnight in 10% neutral buffered formalin solution, embedded in paraffin and sectioned at 4 to 7 µm. Tissue sections were stained with hematoxylin and eosin. Images were visualized using a Nikon Eclipse 80i microscope (Nikon, Mississauga, Canada) and a 20×/0.45 or 40×/0.65 numerical aperture objective. A Nikon Coolpix 4500 camera (Nikon) and Canon ZoomBrowser EX 2.0 software (Canon, Ontario, Canada) were used to capture images. Clonogenic progenitor assay
Colony-forming cells (CFCs) were assayed in methylcellulose (Methocult M3234; StemCell Technologies Inc., Vancouver, Canada) supplemented with 10ng/mL G-CSF. 1 × 105 viable cells/well were plated in duplicate. Colonies were evaluated microscopically 10 days after plating using standard criteria. Oxidative burst assay
In order to determine the ability of murine blood neutrophils to generate oxygen radicals commercially available flow cytometry based assays were used according to the manufacturer’s instructions (Phagoburst®, ORPEGEN Pharma, Heidelberg, Germany). The Phagoburst® assay uses dihydrorhodamine (DHR) 123 as a fluorogenic substrate for oxidative products within the cell and determines the percentage of active cells and their enzymatic activity. Cells were stimulated with either unlabeled opsonized bacteria (E. coli) (6.7 × 108 bacteria/ml), phorbol 12-myristate 13-acetate (PMA) (0.7 µM), or the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP) (6.4 µM) for 10 min in a 37°C water bath and processed afterwards according to manufacturers instructions. Cells were analyzed with a FACSCalibur flow cytometer (BD Biosciences) using CellQuestPro (BD Biosciences). Forty thousand cells were measured for each experimental point in a forward/side scatter (FSC/SSC) dot plot, gates were set on granulocytes to analyze with regard to proportion of positive cells and mean fluorescence intensity. REFERENCES 1. Heuser M, Beutel G, Krauter J et al. High meningioma 1 (MN1) expression as a predictor for poor outcome in acute myeloid leukemia with normal cytogenetics. Blood. 2006;108:3898–3905.
2. Palmqvist L, Glover CH, Hsu L et al. Correlation of murine embryonic stem cell gene expression profiles with functional measures of pluripotency. Stem Cells. 2005;23:663–680.
3. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–408.
4. Heuser M, Argiropoulos B, Kuchenbauer F et al. MN1 overexpression induces acute myeloid leukemia in mice and predicts ATRA resistance in AML patients. Blood. 2007.
5. Pineault N, Buske C, Feuring-Buske M et al. Induction of acute myeloid leukemia in mice by the human leukemia-specific fusion gene NUP98-HOXD13 in concert with Meis1. Blood. 2003;101:4529–4538.
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