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3MM Whatman paper
Whatman, Dassel, Germany
Autoclave
Integra Biosciences, Baar, Switzland
Benchtop gyroory incubator
New Brunswick Scientific, Edison
shaker for bacterium
N.J. USA
Bacteria incubator
Heraeus, Hanau, Germany
Bandelin Sonopuls HD 2070
South Yorkshire, England
Cell culture flasks/dishes
Greiner, Heidelberg, Germany
Cell culture incubator
Heraeus, Hanau, Germany
CELL locate glass cover slips with grids,
Eppendorf, Hamburg, Germany
– Grid size 55 μm
Cell scraper
Corning, Wiesbaden, Germany
Cell strainer (40μm)
Becton Dickinson, Heidelberg, Germany
FACS (Fluorescence-activated cell sorting)
FACScan, Beckton Dickinson, Heidelberg
Centrifuge
Cytocentrifuge
Thermo Shandon, Pittsburgh, USA
Rotina 48 RS Table top centrifuge
Hettich, Tuttlingen, Germany
-Sorvall RC- 5B refrigerated superspeed
centifuge and Biofuge 13R
Heraeus, Hanau, Germany
Table top centrifuge 5415C
Eppendorf, Hamburg, Germany
Megafuge 1.0 with Rotor BS4402/A
Heraeus, Hanau, Germany
Chemiluminescence detection
-Film BioMax
Eastman-Kodak, Rochester, USA
Corning 125 pH meter
Fisher Scientific, Shwerte, Germany
Cryotubes
Nalgene, Rochester, NY , USA
Cytofunnel
Shandon, Pittsburgh, USA
Electroporation:
Gene Pulser System with electroporation
Bio-Rad Laboratories, Munich, Germany
Cuvette 0.4 cm
Flow Chamber Kit
-Circular parallel plate
GlycoTech, Maryland, U.S.A
Gel electrophoresis system
-DNA-sub cell and Mini-Sub cell System
Gibco, Betheseida, USA
Power PAC 2000
BioRad, Munich, Germany
Irradiation of mice
-Betatron 500A
Siemens, Munich, Germany
Lumat LB 9507
Berthold, Bad Wildbad, Germany
Magnetic cell separation
-MiniMACS separator
Miltenyi, Bergisch Gladbach, Germany
-MidiMACS separator
Miltenyi, Bergisch Gladbach, Germany
-MACS separation columns: LD and LS
Miltenyi, Bergisch Gladbach, Germany
Microscope
-SZ40 Zoom Stereo Microscope
Olympus, Munich, Germany
Axioplan II-imaging Fluorescence microscope
Zeiss, Goettingen, Germany
Leitz Labovert inverted Microscope
Leitz, Wetzlar, Germany
Zeiss ID03 inverted microscope
Zeiss, Goettingen, Germany
Nitrocellulose membrane (0.45 μm)
Bio-Rad Laboratories, Munich, Germany
PCR
-Techne TC-312
Kisker , Steinfurt, Germany
Protein transfer
-Trans-Blot SD Semi-dry Transfer cell
BioRad, Munich, Germany
Spectrohotometer
-Gene Quant II
Pharmacia Biotech, Freiburg,
-Ultraspec® 2000
Germany
Steril bank
Heraeus, Hanau, Germany
Steril filter
-0.22μM
Millipore, Eschborn, Germany
-0.45μM
Millipore, Eschborn, Germany
Vortex
Eppendorf, Hamburg, Germany
Water bath
Julabo Labortechnik, Seelbach, Germany
Acetic acid glacia
Sigma, Steinheim, Germany
Acetone
Roth, Karlsruhe, Germany
Ammonium persulfate
Fluky, Deisenhofen, Germany
Ampicillin
Sigma, Steinheim, Germany
Anti-Sca-1 Microbeads
Miltenyi, Bergisch Gladbach, Germany
Biorad protein dye
Biorad, Munich, Germany
Borax
Sigma, Steinheim, Germany
BrdU
Sigma, Steinheim, Germany
β-Mercaptoethanol
Fluka , Deisenhofen, Germany
Bromophenol blue
Sigma, Steinheim, Germany
Calcium chloride
Sigma, Steinheim, Germany
Chlorofor
Fluka , Deisenhofen, Germany
Cumaric acid
Sigma, Steinheim, Germany
ddH2O
Sigma, Steinheim, Germany
DEPC
Sigma, Steinheim, Germany
DMSO
Sigma, Steinheim, Germany
EDTA, disodium, dihydrate
Sigma, Steinheim, Germany
Ethanol
Merck, Darmstadt, Germany
Ethidiumbromid
Sigma, Steinheim, Germany
Formaldehyd
Merck, Darmstadt, Germany
Forene (Isofluran)
Abbott GmbH, Wiesbaden, Germany
Glucose
Sigma, Steinheim, Germany
Glutathione sepharose
TM
Amersham/Pharmacia Biotech, Freiburg,
Germany
Glycerol
Sigma, Steinheim, Germany
Glycin
Merck, Darmstadt, Germany
Isopropylthio-β-D-Galactoside (IPTG)
Sigma, Steinheim, Germany
HCL
Merck, Darmstadt, Germany
Isobutanol
Sigma, Steinheim, Germany
Isopropanol
Fluka , Deisenhofen, Germany
Kanamycin
Sigma, Steinheim, Germany
Lineage cell depletion kit
Miltenyi, Bergisch Gladbach, Germany
Luminol
Fluka , Deisenhofen, Germany
Sigma, Steinheim, Germany
MgSO4
Fluka , Deisenhofen, Germany
NaN3
Sigma, Steinheim, Germany
NaCl
Merck, Darmstadt, Germany
NaH2PO4
Sigma, Steinheim, Germany
NaOH
Roth GmbH, Karlsruhe, Germany
Natriumacetat
Fluka , Deisenhofen, Germany
Paraformaldehyd
Merck, Darmstadt, Germany
p-Coumaric acid
Sigma, Steinheim, Germany
PD-10 columns
Amersham/Pharmacia Biotec, Freiburg,
Germany
Phenol
Fluka , Deisenhofen, Germany
PKH26 Red fluorescence kit
Sigma, Steinheim, Germany
Polybrene
Sigma, Steinheim, Germany
Ponceau S
Sigma, Steinheim, Germany
Propedium iodide
Sigma, Steinheim, Germany
Protease-inhibitors
Roche Basel, Switzerland
Pre-stained SDS-PAGE standards
-Broad range
Bio-Rad Laboratories, Munich, Germany
Retronectin
®
Takara Bio Inc., Otsu, Japan
Sca-1 Isolation Kit
Miltenyi, Bergisch Gladbach, Germany
SDS
Sigma, Steinheim, Germany
STREPtactin sepharose
TM
IBA, Goettingen, Germany
Protein elution buffer
IBA, Goettingen, Germany
TEMED
Sigma, Steinheim, Germany
Tritriplex III, 1,1%
University teaching hospital Frankfurt
Trizma HCl
Sigma, Steinheim, Germany
Triton X-100
Sigma, Steinheim, Germany
Tween20
Sigma, Steinheim, Germany
Vivapore (For concentrating protein)
VivaScience, Stonehouse, UK
Xylencyanol
Sigma, Steinheim, Germany
7-aminoactinomycin D (7-AAD)
Sigma, Steinheim, Germany
BSA, V solution
Sigma, Steinheim, Germany
Chloroquine
Sigma, Steinheim, Germany
DMEM, -High glucose (4.5g/l)
Invitrogen, Karlsruhe, Germany
DMSO
Sigma, Steinheim, Germany
FBS
Gibco BRL, Paisley, Schottland
Ficoll separating solution
Biochrom (# L-6115), Berlin, Germany
Gelatin
Sigma, Steinheim, Germany
GNF-2
Sigma, Steinheim, Germany
HBSS
Gibco, Karlsruhe, Germany
HEPES solution
Gibco, Karlsruhe, Germany
Horse serum
Gibco, Karlsruhe, Germany
Imatinib
Novartis, Basel Switzerland
IMD
Gibco, Karlsruhe, Germany
L-Glutamine
Gibco, Karlsruhe, Germany
Methoculttm GF M3434
StemCell Technologies, Vancouver,
Canada
Mycosa
Gibco, Karlsruhe, Germany
PBS
Gibco, Karlsruhe, Germany
Penicillin-Streptomycin solution
Gibco, Karlsruhe, Germany
Retinoic acid
Sigma, Steinheim, Germany
Retronectin
Takara, Shiga, Japan
RPMI 1640
Gibco, Karlsruhe, Germany
Trypan blue stain (0.4%)
Gibco, Karlsruhe, Germany
Trypsin EDTA, 0.25% solution
Gibco, Karlsruhe, Germany
rmG-CSF
Peprotech, Offenbach, Germany
rmSCF
Peprotech, Offenbach, Germany
rmIL-6
Peprotech, Offenbach, Germany
rmIL-3
Peprotech, Offenbach, Germany
rmGM-CSF
Peprotech, Offenbach, Germany
Calf intestinal phosphatase (CIP)
NEB, Frankfurt, Germany
Gateway LR clonase enzyme mix
Invitrogen, Karslruhe, Germany
Klenow-Fragment DNA-polymerase I
NEB, Frankfurt, Germany
Restriction endonucleases
NEB, Frankfurt, Germany
RNAse
Sigma, Steinheim, Germany
Superscript II
Invitrogen, Karlsruhe, Germany
T4 DNA-ligase
NEB, Frankfurt, Germany
Taq-DNA-polymerase
Biosystems, Weiterstadt, Germany
Proteinase K
Stratagene, La Jolla, USA
Taq-DNA-polymerase
Biosystems, Weiterstadt, Germany
DNTPs
Fermentas, St. Leon-Rot, Germany
PCR Buffer
Fermentas, St. Leon-Rot, Germany
MgCl2
Fermentas, St. Leon-Rot, Germany
PfU turbo polymerase
Stratagene, La Jolla, USA
Primers
Sigma, Steinheim, Germany
Mouse anti-β-tubulin (Ab-4)
Neo Markers, Asbach, Germany
Mouse lineage panel
Pharmingen, San Diego, CA, USA
Rabbit anti-c-ABL (11/24)
Santa Cruz, Heidelberg, Germany
Rabbit anti-p-ABL-Tyr-245
Upstate-Biotechnology, Lake Placid, NY,
USA
Rabbit anti-p-ABL-Tyr-412
Cell Signalling, Boston, USA
Rabbit anti-p-BCR-Tyr-177
Cell Signalling, Boston, USA
Rabbit anti-BCR(C-20)
Santa Cruz, Heidelberg, Germany
Mouse anti- GFP
Upstate-Biotechnology, Lake Placid, NY,
USA
Mouse anti-phospho-STAT5
Cell Signalling, Boston, USA
Rabbit anti -STAT5
Cell Signalling, Boston, USA
Rabbit anti-phospho-CrkL (Tyr207)
Cell Signalling, Boston, USA
Mouse anti-CrkL
Cell Signalling, Boston, USA
Mouse anti-phosphotyrosine (clone 4G10)
Upstate-Biotechnology, Lake Placid, NY,
USA
Anti-mouse IgG-HRP
Santa Cruz, Heidelberg, Germany
Anti-rabbit IgG-HRP
Santa Cruz, Heidelberg, Germany
Mouse-anti-human-PE-LNGFR
BD, San Jose, CA, USA
Rat-anti- mouse-PE-Gr-1
BD, San Jose, CA, USA
Rat-anti- mouse-PE-Mac-1
BD, San Jose, CA, USA
Rat-anti- mouse-PE-B220
BD, San Jose, CA, USA
Mouse-anti-PE-IgG 2a, K
BD, San Jose, CA, USA
Mouse-anti-PE-IgG 2b, K
BD, San Jose, CA, USA
Rat –anti-Mouse FITC-Sca-1
BD, San Jose, CA, USA
Rat-anti- mouse-PE-Sca-1
BD, San Jose, CA, USA
MACS buffer
20% (w/v) BSA
12.5 ml
EDTA
1mmol
Penicillin/Streptomycin
5ml
Add PBS up to
500ml
2M CaCl2 solution
The component was dissolved in ddH2O and sterile filtered with 0.22 μm filter and stored at –
20°C in aliquots until use.
2X HBS solution
Na2HPO4
0.315g (1.5 mM final)
HEPES
19.5g (0.05M final)
NaC
24g (0.28M final)
Water added up to 1200ml, three clean bottles were filled with exactly 400 ml of the solution,
the pH to 6,95/ 7,0 / 7,05 was adjusted respectively, each bottle was filled to 500 ml, pH was
controlled again. The buffer was filter-sterilized and stored at -20°C in aliquots.
1x TAE Electrophoresis buffer
Tris-Acetate
0,04M
EDTA
1 mM
Buffer W for lysis of bacteria
Tris-HCl pH 8.0
100 mM
NaCl
150 mM
EDTA
1 mM
Tween
0.1%
Protease inhibitor
1x
1X SDS gel-loading buffer
Tris-HCl, pH 6.8
50mM
SDS
2 % (w/v)
Glycerol
10% (v/v)
β-mercaptoethanol
100mM
Bromophenol blue
0.1 %( v/v)
1X SDS gel-loading buffer lacking dithiothretitol was stored at room temperature. β-
mercaptoethanol from a 14 M stock was added just before the buffer is used.
For protein electrophoresis
(Invitrogen life technologies, Germany)
NuPAGE Novex Bis-Tris
Tris-Acetate Pre-Cast Gels
NuPAGE Novex MES and Tris-Acetate SDS Running Buffers
NuPAGE Transfer Buffer
Precision plus protein (PPP)
NuPAGE LSD Sample Buffer
NuPAGE Reducing Agent
NuPAGE Antioxidant
10X TBS
Trizma HCl, pH 7.5
volume was adjusted to 1 L with wate.
TBST
1X TBS+ 0.05% Tween 20
Blocking Buffer for immunoblot
5% Carnation non-fat dry milk in TBST
Protein transfer buffer
TrisHCl
48 mM
Glycine
Coomassie staining solution
Coomassie brilliant blue R250
0.1 % (w/v)
Methanol
50 % (v/v)
Glacial acetic acid
10 % (v/v)
Coomassie destaining Solution
Methanol
50 % (v/v)
Glacial acetic acid
10 % (v/v)
Ponceau S staining solution
Ponceau S
2 % (w/v)
Trichloroacetic acid
30 % (v/v)
Sulfosalicylic acid.
30 % (v/v)
ECLsolution I:
1 M TrisHCl, pH 8,5
10ml
90mM p-Coumaric acid (in DMSO) 0.44ml
250mM Luminol (in DMSO) 1ml
Water was dded up to 100ml; aliquots were made and stored in the dark at -20°C.
ECLsolution II:
30 % H2O2
64μl
1 M TrisHCl, pH 8,5
10 ml
Water was added up to 100ml, aliquat solution and store in the dark at -20°C.
Stripping buffer
Tris-HCl, pH8.0
62.5 mM
2-mercaproethanol
100 mM
SDS
2 % (w/v)
FACS Wash Buffer
PBS + 1%FCS + 1% NaN3
FACS Fixative Solution
PBS + 2% formaldehyde solution
Phosphate-Buffered Saline (PBS)
NaCl
137 mM
KCl
2.7 mM
Na2HPO4
10 mM
KH2PO4
2 mM
The above components were dissoolved in water and adjusted pH to 7.4 with HCl.
Tris-HCl (1M)
121.1g of Tris base was dissolved in 800ml of water. The pH was adjusted to the desired
10X Tris EDTA (TE)
pH 7.4
100 mM Tris-Cl (pH 7.4)
10 mM EDTA (pH 8.0)
pH 7.6
100 mM Tris-Cl (pH 7.6)
10 mM EDTA (pH 8.0)
pH 8.0
100 mM Tris-Cl (pH 8.0)
10 mM EDTA (pH 8.0)
SDS (20% w/v)
200 g of electrophoresis-grade SDS was dissolved in 900 ml of water. The mixture was
heated to 68°C and stirred with a magnetic stirrer to assist dissolution. The pH was adjusted to
7.2 by adding a few drops of concentrated HCl. The volume was adjusted to 1 liter with
water.
EDTA (0.5 M, pH 8.0)
186.1 g of disodium EDTA was added to 800 ml of H2O. The mixture was stired vigorously
on a magnetic stirrer. The pH was adjusted to 8.0 with NaOH. The disodium salt of EDTA did
not go into solution until the pH of the solution was adjusted to ~ 8.0 by the addition of
NaOH.
 
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Materials
49
Solutions for plasmid minipreparation
Resuspension solution (Sol I)
Glucose
50 mM
Tris-HCl pH 8.0
25 mM
EDTA pH 8.0
10 mM
Autoclaved and stored the solution at 4°C
Alkaline lysis solution (Sol II)
NaOH
0.2 M
SDS
1 % (w/v)
Sol II was prepared fresh and used at room temperature
Neutralization solution (Sol III)
5 M Potassium acetate
60 ml
Glacial acetic acid
11.5 ml
ddH2O 28.5 ml
The solution was stored at 4°C and transferred to ice before use
0.1% Diethylpyrocarbonate (DEPC)
1 g DEPC was dissolved in 1 L water and mixed it vigiously. It let stand with loose lid
overnight at room temperature or at 37°C for 1h and autoclaved for 15 minutes at 15 psi on
liquid cycle. DEPC cannot be used to treat Tris buffer.
2.3.7 Plasmids and vectors
pCDNA 3.1 vector
Invitrogen GmbH, Karlsruhe, Germany
pEntr1A
Invitrogen GmbH, Karlsruhe, Germany
PINCO
Retroviral hybrid vector with LTR derived from Moloney
murine Leukemia virus. The main characteristics of this vector
is the presence of the EBV origin of replication and the EBNA-
1 gene and the presence of the cDNAs that encodes for the
EGFP, controlled by a cytomegalovirus promoter.This vector
allows high-efficiency gene transfer (Grignani et al., 1998).
PAULO
This vector is a modified pinco vector in which GFP is replaced
by LNGFR
PIDE
This vector is based on pinco vector in which GFP is deleted and
Hind III site can be used for cloning gene instead of GFP
Pinco∆CMV
Retroviral pinco based vector without any reporter gene
 
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Materials
50
pPRIBA2
IBA, Gottingen , Germany
pGEX4T3
Amersham Biosciences, Freiburg, Germany
2.3.8 Bacterial E.Coli Strain and genotype
E. coli – HB101, BL21, JM83, DB 3.1,
Invitrogen, Karlsruhe, Germany
2.3.9 Medium for bacterium
LB medium
Bacto-Tryptone
1 % (w/v)
Bacto-Yeast-Extrac
0.5 % (w/v)
NaCl
1.5 % (w/v)
Adjuted pH to 7.4 with NaOH and autoclaved
LB agar plates
Bacto-Agar
1.5 % (w/v)
Bacterium freezing solution
Glycerin
65 % (v/v)
MgSO4
0.1 M
TrisHCl, pH 8.0
0.025M
SOC
Invitrogen, Karlsruhe, Germany
2.3.10 Cell lines
2.3.10.1 Ph+ cells
CML blast cell lines
In CML cells, the translocation product encodes for p210
BCR/ABL
.
BV173: Human B cell precursor leukaemia cell line, established from the peripheral blood of
a patient with chronic myeloid leukaemia (CML) in lymphoid blast crisis. Contains the t(9;22)
b2-a2 fusion gene. Obtained from DSMZ, Germany
K562: Established from the pleural effusion of a patient with chronic myeloid leukaemia
(CML) in myeloid blast crisis. Cells carry the Ph chromosome with the Bcr-Abl b3-a2 fusion
gene. Obtained from DSMZ, Germany
Ph+ ALL
In Ph+ cells, the translocation product encodes for p185
BCR/ABL
.
 
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51
Sup-B15: Human B cell precursor leukemia cell line, established from the bone marrow of a
patient with acute lymphoblastic leukemia, carrying the ALL-variant (m-bcr) of the
BCR/ABL fusion gene (e1-a2). Obtained from DSMZ, Germany
Tom-1: human B cell precursor leukemia. Established from the bone marrow of a patient with
refractory Ph chromosome acute lymphoblastic leukemia (ALL); described to carry the ALL-
variant (m-BCR) of the BCR/ABL fusion gene (e1-a2) obtained from DSMZ, Germany.
2.3.10.2 Other Cell lines
Nalm-6: Human lymphoblastic B cell line; Ph-
Ba/F3: IL-3 dependent murine pro B cell line established from peripheral blood; apparently
derived from BALB/c mouse.
32D: IL-3 dependent murine myeloid cell line established from peripheral blood.
Rat-1: Rat fibroblast cells line
293T: Transformed human embryonal kidney cell line
Phoenix: Based on the 293T cell line (transformed human embryonal kidney cell line).
Expresses the retroviral structural genes gag, pol und env. It is also known as a packaging cell
line. The ecotropic packaging cell line delivers genes only to dividing murine or rat cells.
All cells were obtained from the “Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH” (DSMZ) (German Collection of Microorganisms and Cell Cultures),
with the exception of the ecotrophic Phoenix cells which were obtained from Nolan lab,
Standford, USA.
2.3.11 Medium for Cell culture
L-glutamine
1 % (v/v)
FBS
10 % (v/v)
Penicillin/Streptomycin
1 % (v/v)
Added to DMEM and RPMI medium for adherent and suspension cells respectively
Medium for 32D/Baf3 cells
L-glutamine
1 % (v/v)
FBS
10 % (v/v)
Penicillin/Streptomycin
1 % (v/v)
mIL-3
10 ng /ml
Added the above to RPMI medium
Medium for Nalm-6, BV-173 and K562
L-glutamine
1 % (v/v)
 
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52
FBS
10 % (v/v)
Penicillin/Streptomycin
1 % (v/v
Added the above to RPMI medium
Medium for Tom-1
L-glutamine
1 % (v/v)
FBS
20 % (v/v)
Penicillin/Streptomycin
1 % (v/v
Added the above to RPMI medium
Medium for Sup-B15
L-glutamine
1 % (v/v)
FBS
20 % (v/v)
Penicillin/Streptomycin
1 % (v/v
Added the above to Mycosa medium
Medium for Sca1+/lin- mouse BM cells
L-glutamine
1 % (v/v)
FBS(Hyclon)
10 % (v/v)
Penicillin/Streptomycin
1 % (v/v)
mIL-3
20 ng /ml
mIL-6
20 ng /ml
mSCF
100 ng /ml
Added the above to ISCOVE medium
Cell freezing medium
Solution I
RPMI/DMEM
70 % (v/v)
FBS
30 % (v/v)
Solution II
RPMI/DMEM
80 % (v/v)
DMSO
20 % (v/v)
2.3.12 Materials for animal experiments
2.3.12.1 Mice
The C57BL/6N mice (age between 6-8 weeks, all female) were purchased from Charles River
Laboratories GmbH in Munich, Germany and served as the recipient mice for all the animal
experiments.
 
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53
2.4 Miscellaneous
Nucleospin
R
Mini plasmid DNA isolation Kit
Macherey-Nagel, Dueren Germany
Nucleospin
R
Maxi plasmid isolation kit
Macherey-Nagel, Dueren Germany
Gel cleaning kit
Macherey-Nagel, Dueren Germany
PCR cleaning kit
Macherey-Nagel, Dueren Germany
Qiagen gel extraction Kit
Qiagen, Duesseldorf, Germany
Qiagen Plasmid kit Mini, Midi and Maxi
Qiagen, Duesseldorf, Germany
Qiagen PCR purification Kit
Qiagen, Duesseldorf, Germany
Quick change site mutagenesis kit
Stratagene, La Jolla, USA
Endo Toxin removal kit
Profos AG, Regensburg, Germany
Cell proliferation (XTT) kit
Amersham/Pharmacia Biotech, Freiburg,
Germany
Protein concentrator (Vivapore)
VivaScience, Hannover, Germany
 
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54
3 Methods
3.1 Preparation of plasmid DNA
3.1.1 Transformation of E.coli
A frozen vial of competent E.coli bacteria was thawed on ice. The transforming DNA (up to
25ng per 50ul competent bacterium or 10ul ligation product) was pipetted directly to
competent E.coli bacteria and mixed by swirling the tubes gently several time.