Wicke et al.
Gene Therapy of Mpl deficiency
Supplementary material and methods
Murine bone marrow transplantation model
Lin- cells were isolated from complete BM by magnetic cell sorting using MACS lineage cell
depletion (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The lineage negative cell
population was then prestimulated for two days in StemSpan medium (Stem Cell
Technologies, France), containing 50 ng/mL mSCF, 100 ng/mL hFlt-3 ligand, 100 ng/mL hIL11, 20 ng/mL mIL-3, penicillin/streptomycin and 2 mM glutamine. Lin- cells were transduced
on two following days (day 3 and 4) using plates coated with Retronectin (TaKaRa, Otsu,
Japan) and vector. For preparation of plates, the surface of the wells was coated with 10
ng/cm2 Retronectin and virus loaded by centrifugation of viral supernatants at 1000 g for 30
min at 4° C. Virus preloading was repeated up to two times. Transgene expression was
measured on day five just prior to transplantation. 5 - 10 x 105 cells were transplanted into
lethally (10 Gy) irradiated mice.
Mouse analysis
PB was collected periodically from the retro-orbital cavity using EDTA-coated glass capillary
tubes and analyzed by automated complete differential blood cell counts (Scil ABC Vet Blood
Counter, ABX Diagnostics, France). Mice were sacrificed when symptomatic and
macroscopically examined for pathological abnormalities. Enlarged organs were weighed. A
panel of tissues was collected, fixed in 4% formalin, and paraffin-embedded for histological
examinations. Cells from BM, spleen and PB were subjected to flow cytometry (Becton
Dickinson, Heidelberg, Germany) and analyzed with CellQuest, FlowJo and Diva software.
After RBC lysis, cells were stained with lineage-specific antibodies against Gr1, CD11b,
TER119, CD71, CD19, CD3, Sca1, ckit or HA-Tag (BD Bioscience, Heidelberg, Germany;
eBioscience, San Diego, CA, USA; Roche Diagnostics, Mannheim, Germany). Antibodies
were usually directly linked to FITC, PE, APC, PerCP-Cy5.5 or PE-Cy7 fluorochromes. Dead
cells were excluded by propidium iodide (PI) or 4',6-Diamidino-2- phenylindoldihydrochlorid
(DAPI) staining. Leukocyte morphology was evaluated in Giemsa stained PB smears and
cytospins of BM and spleen cells.
The LSK cell fraction was analysed in total BM cells that were flushed form both hind limbs.
The lineage negative fraction was determined by staining for the lineage markers CD11b,
Gr1, CD19, CD3 and Ter119 and the Sca1/ckit double positive cell fraction within the lineage
marker negative cells was detected.
Western Blot Analysis
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Wicke et al.
Gene Therapy of Mpl deficiency
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32D cells were transduced with retroviral vectors and expanded to achieve suitable cell
numbers. Cells were then starved overnight without IL-3 and 0.5% BSA and stimulated with
different cytokines (w/o, IL-3, Thpo) for 10 min on the following day. Cells were collected
directly after stimulation and lysed in lysis buffer containing phosphatase inhibitors (50 mM
HEPES, 150 mM NaCl, 50 mM NaF, 10 mM Na4P2O7, 10% Glycerin, 1% NP-40). Protein
concentration was measured by Bradford Assay (BioRad, Munich, Germany) and 10-20 µg of
protein samples were separated by electrophoresis. Nitrocellulose membrane was blocked in
5% milk and incubated with one of the following antibodies: phos-ERK and phos-AKT. For
loading controls, blots were stripped and reprobed using anti-ERK and anti-AKT antibodies
(Cell Signaling Technologies, Boston, USA) as appropriate.
Vector copy number and mRNA expression levels
wPRE specific primers forward 5’-GAGGAGTTGTGGCCCGTTGT-3’ and reverse 5’TGACAGGTGGTGGCAATGCC-3’ and flk1 intron enhancer (AF061804, bases 352-459),
forward 5'-GGTTTCAATGTCCCGTATCCTT-3' and reverse 5'-CTTTGCCCCAGTCCCAGT
TA-3' were used.
For the quantification of mRNA expression of the genes Sfpi-1 (Cat. No. QT00098077) and
Fli-1 (Cat. No. QT00153559) mRNA was prepared from splenocytes and translated into
cDNA which was used for quantitative analysis using primers of Qiagen (Hilden, Germany).
LM-PCR
LM-PCR was performed as described 1. DNA (100-500 ng) was digested with 2.5 U Tsp509I
(New England BioLabs, UK). Primer extension was performed using 0.25 pmol biotinylated
primer LTR-1 5’-TGCGGTGACCATCTGTTCTTGGCCCCG-3’. The first PCR (95°C for 5 min;
95°C for 1 min, 55°C for 30 sec, 68°C for 2 min for 30 cycles; 68°C for 10 min) was
performed using Extensor Hi-Fidelity PCR Master Mix (ABgene, Hamburg, Germany) and
primers:
LTR-2
primer
5’-GACCTTGATCTGAACTTCTC-3’
and
linker
primer-1
5’-
GACCCGGGAGATCTGAATTCG-3’. The nested PCR was performed under identical
conditions: LTR-3 primer 5’-TCCATGCCTTGCAAAATGGCG-3’ and linker primer-2 5’AGTGGCACAGCAGTTAGGACG-3’. PCR products were isolated by gel electrophoresis,
purified using QIA quick Gel Extraction Kit (Qiagen, Hilden, Germany) and sequenced after
subcloning into TA cloning vector (Invitrogen, Karlsruhe, Germany). Recovered sequences
were screened using the NCBI mouse genome database (NCBI37, accessed February
2009). Gene classification followed database records and PubMed literature.
In vitro colony-forming assays (CFU)
Wicke et al.
Gene Therapy of Mpl deficiency
Myeloid colony forming assays were performed in methylcellulose-based medium containing
3 U/mL erythropoietin, 10 ng/mL IL-3, 10 ng/mL IL-6, and 50 ng/mL SCF per manufacturer’s
protocols (M3434, StemCell Technologies, France). BM cells from CMPD mice were plated
at 2.5 x 105 cells per dish, in duplicates. Plates were incubated at 37 °C for seven days and
colonies counted.
Histopathological evaluation
All necropsies were fixed in buffered methanol-formol (BM) or formalin solution (specimens
from the other organs) for at least 24 hours and embedded in paraffin (WAX) after
decalcification of the BM specimens by ethylene-diamine tetra-acetic acid. From all
necropsies, 3 µm thick sections were cut and stained with Hematoxylin-Eosin (HE), Giemsa,
PAS, Prussion blue and Gomori silver impregnation. Micrographs were taken with Olympus
Microscope BX51TF (Olympus, Japan).
Referrences
1.
Schmidt, M., Hoffmann, G., Wissler, M., Lemke, N., Mussig, A., Glimm, H., et
al. (2001). Detection and direct genomic sequencing of multiple rare unknown
flanking DNA in highly complex samples. Hum Gene Ther 12: 743-749.
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