Supplementary information
Phenotype of leukemic mice
Recipient type leukemias
In observation time of 11 months we noted two cases of precursor T-cell leukemias, one in the
SRS.SF.IL2RG.pre transplanted mice in exp.I and one in the MFG.γC group of the exp.II. In both cases
no retroviral vector genomes could be detected in the leukemic cells by qPCR and Southern blot (1). We
conclude that both of these leukemias were not vector related but irradiation induced, as also observed
by others (2). No symptoms of vector-associated leukemia were found in primary recipients, as diagnosed
by histopathology, distribution of the leukocyte lineages in bone marrow and blood analysis by FACS and
differential cell counts.
Lymphoblastic leukemia (M32-1 and M32-2 and M35-1 and M35-2)
Four mice developed leukocytosis, enlarged lymph nodes (cervical, inguinal, popliteal, mesenterial),
enlarged spleen, infiltrations in liver, kidneys and lung (exp.II/M32-1 and exp.III/M32-2, exp.III/M35-1 and
exp.III/M35-2). The leukemic cells were negative for most lineage markers tested (CD11b, Gr1, CD19,
CD3, CD4, CD8, C25, Ter119, CD41), but positive for B220, Sca-1, ckit, CD44 and CD24. The cytomorphology of the leukemic cells was lymhoblastic (Figure 3a, b). Importantly, molecular analysis of these
four leukemias by ligation-mediated PCR (LMPCR) and Southern blot revealed the same leukemic clone
(as characterized by the insertion site) in all four mice, indicating that the founder cell had undergone a
self-renewal division already during the short culture phase prior to the first BMT. This lymphoblastic
leukemic clone contained its single insertion 5,100 bp upstream of the fourth exon of Evi1 that harbours
the translational start site.
Myeloid leukemia with maturation (M34-2)
A fifth leukemia case had a myeloid phenotype by FACS (Gr1+ and CD11b+) and cytology (exp.III/M34-2,
Figure 3a). There was no strong involvement of the lymph nodes, but the spleen was enlarged and the
liver infiltrated by leukemic cells. Southern blot analysis identified four retroviral vector copies in the
leukemic clone and therefore this leukemia was distinctly different to the B220 positive leukemia detected
in the four other mice (Figure 3c). This leukemic clone contained a vector insertion -69,696 bp upstream
of the second exon of Evi1. The three further insertions of this clone were close by the genes Lcp2,
Snx10 and Mrpl39 (suppl. Table 1).
MPD-like myeloid leukaemia (M31-1 and M31-2)
Two further mice (exp.III/M31-1 and M31-2) transplanted from the same primary donor were diagnosed
leukemic during necropsy despite not showing any obvious symptoms while being alive. Spleen and bone
marrow were infiltrated by CD11b/Gr1 positive myeloid cells that were well differentiated to granulocytes.
The leukemia was therefore phenotyped as a MPD-like myeloid leukemia. Southern blot analysis
detected the same single vector insertion in both mice (Figure 3c) that was located upstream of the
second exon of Evi1 (-109 689 bp),
Myeloid leukemias induced by LTR vectors
M3-1 (exp. I): This mouse presented with massively enlarged spleen and liver and no involvement of the
thymus. The BM was pale. There was ten times increase of leukocyte counts. The leukemic cells were
positive for the surface marker Gr1 and CD11b as shown in the suppl. figure 3.
M(48-50)-1 and M(48-50)-3 and M(48-50)-2 and M(48-50)-4 (exp. III): All mice has enlarged spleens and
liver (see table2) and leukocytosis. Leukemic cells were positive for Gr1, CD11b and ckit (see suppl.
figure 3).
Supplementary Materials and Methods
Virus production
Cell–free supernatants were generated by calcium phosphate transfection of 293T packaging cells or
Phoenix-gp cells (kindly provided by G. Nolan, Stanford, USA) by co-transfection of a gag/pol construct
(MLV or HIV gag/pol) and the ecotropic envelope construct (kindly provided by T. Kitamura, Tokyo,
Japan). In addition, for lentiviral vectors, a plasmid encoding Rev was co-transfected. Virus titrations were
performed on SC1 fibroblasts. Viral titres were in the range of 1-10x106 t.u./ml.
BM cell purification and transduction
Briefly, Lin- cells were isolated from complete BM by magnetic sorting using lineage- specific antibodies
(Gr1, CD11b, CD45R/B220, CD3e, TER-119; Miltenyi Biotech, Bergisch Gladbach, Germany). Prior to
retroviral transduction lin. neg. BM cells were prestimulated for 2 days in StemSpan HS2000 medium
(CellSystems, St. Katharinen, Germany), containing 50 ng/ml mSCF, 100 ng/ml hFlt-3 ligand, 100 ng/ml
hIL-11, 10 ng/ml mIL-3, 1% penicillin/streptomycin, 2 mM glutamine. On day 3, cells were infected on
virus preloaded Retronectin (TaKaRa, Otsu, Japan) plates. For preparation of plates, the bottom surface
of the wells were coated with 10 µg/cm 2 Retronectin and virus loaded by centrifugation of viral
supernatants by 900-1000g for 30 minutes at 4C. Virus preloading was repeated up to three times to
enrich low titre supernatants (3). Lin- cells were infected on two following days (day 3 and 4).
Transduction efficiency was measured before BMT (day 5) by FACS, and after BMT by PCR in genomic
DNA of blood leukocytes.
Analysis of leukemic mice
Mice were sacrificed when symptomatic and macroscopically examined for pathological abnormalities
during dissection. Enlarged organs were weighted. A panel of tissues was collected, fixed in 4% formalin,
and paraffin-embedded for histological examinations. Cells from BM, spleen, thymus (if enlarged) and
blood were subjected to flow cytometry using a FACS calibur. After red blood cell lysis, cells were stained
with lineage-specific antibodies against Gr1, CD11b (myeloid cells), CD19, B220 (B-cells), Ter119
(erythroid cells), CD3, CD4, CD8, CD25 (T-cells), CD41 (megakaryocytes), Sca1 and c-Kit (Pharmingen,
Hamburg, Germany). Antibodies were usually directly linked to FITC, PE or APC fluorochromes. Dead
cells were excluded by propidium iodide staining. Leukocyte morphology was also evaluated in Giemsa
stained blood smears and cytospins of BM and spleen cells. Blood cell counts were collected by
microscopic counting or by using an automatic analyzer (SCIL Vet abcTM blood counter). Mice that did not
develop leukemia were analyzed according this protocol after an observation period of 6-11 months.
LM PCR
To amplify the 5’LTR junction of transduced BM cells, DNA was digested with 2.5 U of restriction enzyme
Tsp5091 (New England BioLabs, UK) per microgram of DNA for 2 h at 65° C. For primer extension 0.25
pmol of biotinylated retroviral primer LTR1 (5’bioTGCGGTGACCATCTGTTCTTGGCCCCG3’) was used.
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 by using Extensor Hi-Fidelity PCR Master Mix (AB gene), retroviral primer LTR2
(5’GACCTTGATCTGAACTTCTC3’) and linker specific primer OCI (4). The nested PCR was performed
under identical conditions, but using retroviral primer LTR3 (5’TCCATGCCTTGCAAAATGGCG3’) and
linker specific primer OCII.
For the detection of gammaretroviral SIN vector insertions a wPre specific primer was used for the linear
extension (SINPRE: 5’bioGCACTGATAATTCCG TGGTGTTGTC). For the exponential PCRs LTR
primers were used as follows:
SIN LTR2: 5’AGCGATATCGAATTCACAACC3’, SIN LTR3:
5’CCCAATAAAGCCTCTTGCTGT3’. In the case of amplification of gammaretroviral SIN vector insertion
sites in the leukemic clone of mouse M34-2 the SIN LTR2 primer was used as biotinylated primer for the
linear extension. The exponential PCR was performed with and additional primer SIN LTR2b:
5’GTCCTCCGATTGACTGCGTCG3’ and SIN LTR3.
For the detection of lentiviral insertion sites the primer were used as published in (4). lvLTR1:
5’bioGAACCCACTGCTTAAGCCTCA3’,
lvLTR2
5’AGCTTGCCTTGAGTGCTTCA3’.
lvLTR3
5’AGTAGTGTGTGCCCGTCTGT3’.
As reverse primers for the exponential PCR in the gammaretroviral SIN LMPCR and lentiviral LMPCR the
linker specific primer OCI and OCII were used.
PCR products were isolated after gel electrophoresis using QIA quick Gel Extraction Kit (QIAGEN, Hilden
Germany), subcloned into the pCR2.1 TA vector (Invitrogen, Karlsruhe, Germany) and sequenced using
M13 primers.
Quantitative PCR
Quantitative PCR for the PRE and IL2RG was performed on a 7300 Real Time PCR System (Applied
Biosystems) using QuantiTect SYBR Green (Qiagen). For quantification of vector copy numbers a qPCR
was used detecting a 94 bp wPRE specific sequence (wPRE specific primers forward 5’GAGGAGTTGTGGCCCGTTGT-3’ and reverse 5’-TGACAGGTGGTGGCAATGCC-3’). The PRE specific
signal was normalized by the signal of a housekeeping gene (flk-1 intron enhancer (AF061804, bases
352-459),
forward
5'-GTGAATTGCAGAGCTGTGTGTTG-3'
and
reverse
5'-
ATTCATTGTATAAAGGTGGGATTG-3'). The primers have equal efficiencies. Results were quantified
using the comparative CT method using a murine hematopoietic cell clones with 9 retroviral genome
copies as a standard. The copy number of IL2RG integrated genomes was quantified with IL2RG specific
primers
(IL2RG
forward:
5’-TGCTAAAACTGCAGAATCTGGT-3’;
IL2RG
reverse
5’-
AGCTGGGATTCACTCAGTTTG-3’).
For the specific quantification of the Mds/Evi1 fusion and Evi1 transcript primers were designed as
follows:
Mds/Evi1 forward primer 5’GATTCCAGCTATGGATGGGAGATCTTAGATGAG-3’;
Evi1 exon 1/3 forward 5’-GACCTTCATCGAAAGAGGCACAGATCTTAGATGAG-3’,
Evi exon 2/3 forward 5’-GAGGCCGTAGAAATCGGAAGATCTTAGATGAG -3’,
Evi1 exon 3 reverse 5’-CTGGCATGCAACAAGGTTGTGCTGATC-3’.
Chromosome preparation and spectral karyotyping
Metaphase chromosomes were prepared by treating cells with colcemid (0.035µg/ml, 6 hours) followed
by incubation in 0.075M KCl for 20min at 37°C and fixation in a freshly prepared mixture of
methanol:acetic acid (3:1) at room temperature. Cell suspension was dropped onto glass slides in a
climate chamber (Polymer, Kassel, Germany) at 22°C and 48% humidity.
Spectral karyotyping (SKY) was performed as described previously (5) and according to the
manufacturer´s instructions (ASI; Applied Spectral Imaging, Ltd., Migdal HaEmek, Israel). Spectral
images were acquired using an epifluorescence microscope equipped with an interferometer
(SpectraCubeTM ASI), a custom-designed optical filter and the SkyViewTM software (ASI).
References
1.
Will E, Bailey J, Schuesler T, Modlich U, Balcik B, Burzynski B, Witte D, Layh-Schmitt G,
Rudolph C, Schlegelberger B, von Kalle C, Baum C, Sorrentino BP, Wagner LM, Kelly P, Reeves L,
Williams DA. Importance of murine study design for testing toxicity of retroviral vectors in support of
phase I trials. Mol Ther. 2007 Apr;15(4):782-791.
2.
Siapati EK, Bigger BW, Kashofer K, Themis M, Thrasher AJ, Bonnet D. Murine leukemia
following irradiation conditioning for transplantation of lentivirally-modified hematopoietic stem cells. Eur J
Haematol. 2007 Apr;78(4):303-313.
3.
Kustikova OS, Wahlers A, Kuhlcke K, Stahle B, Zander AR, Baum C, Fehse B. Dose finding
with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer
efficiency in a cell population. Blood. 2003 Dec 1;102(12):3934-3937.
4.
Schmidt M, Hoffmann G, Wissler M, Lemke N, Mussig A, Glimm H, Williams DA, Ragg S,
Hesemann CU, von Kalle C. Detection and direct genomic sequencing of multiple rare unknown flanking
DNA in highly complex samples. Hum Gene Ther. 2001 May 1;12(7):743-749.
5.
Frank O, Rudolph C, Heberlein C, von Neuhoff N, Schrock E, Schambach A, Schlegelberger B,
Fehse B, Ostertag W, Stocking C, Baum C. Tumor cells escape suicide gene therapy by genetic and
epigenetic instability. Blood. 2004 Dec 1;104(12):3543-3549.
suppl. Table 1: Insertion sites recovered from retroviral SIN vector transplanted mice
nearest gene
2010208K18Rik
2310022M17Rik
4933400A11Rik
5830415L20Rik
6530401C20Rik
6720456B07Rik
Acox1
Ap1b1
Arpc2
Arrb1
B3gnt2
Bcl11a
Capzb
Ccdc50
Cstad
Dhx34
Dnajc5
Dscr3
Dusp2
EG433632
EG627782
Etv1
Etv6
Evi1
Evi1
Gpr85
Haao
Hipk1
Iqgap1
Krt80
Lcp2
Lmo2
Lrrfip1
Lsm4
Map4k5
Mbd6
Memo1
Ndufb6
Nos2
Osbpl3
Palld
Pdcd1lg2
Plekha5
Prdm16
Rasgrp4
Scd1
Sec14l1
Sema4b
Slc35e1
Smarca5
Snx10
Spnb2
Stat4
Strn3
Stx1a
Tbc1d2
Tex22
Tpd52-ps
Trmt5
Vps45
gene ID
chromosome
72096
69556
66747
68152
231842
101314
11430
11764
76709
109689
53625
14025
12345
67501
78617
71723
13002
13185
13537
433632
627782
14009
14011
14013
14013
64450
107766
15257
29875
74127
16822
16909
16978
50783
399510
110962
76890
230075
18126
71720
72333
58205
109135
70673
233046
20249
74136
20352
270066
93762
71982
20742
20849
94186
20907
381605
75671
107730
76357
22365
7F4
11A4
XF5
5A1
5G1
6E3
11E2
11A1
1C3
7F1
11A3.2
11A3.3
4D3
16B2
2B
7A2
2H4
16C4
2F2
3F3
XA1.2
12B3
6G1
3A3
3A3
6A2
17E4
3F3
7E1
15F3
11A5
2E2
1D
8C2
12C3
10D3
17E2
4A5
11B5
6
8B3.3
19C2
6G1
4E2
7B1
19D1
11E2
7E1
8C2
8C3
6B3
11A4
1C1
12C1
5G1
4B2
12F2
8
12D1
3F2
RTCGD
×
×
×
CIS
×
CIS
×
×
CIS
×
CIS
CIS
CIS
×
×
CIS
×
CIS
×
CIS
×
×
×
×
×
description
RIKEN cDNA 2010208K18 gene
RIKEN cDNA 2310022M17 gene
RIKEN cDNA 4933400A11 gene
RIKEN cDNA 5830415L20 gene
RIKEN cDNA 6530401C20 gene
RIKEN cDNA 6720456B07 gene
acyl-Coenzyme A oxidase 1, palmitoyl
adaptor protein complex AP-1, beta 1 subunit
actin related protein 2/3 complex, subunit 2
arrestin, beta 1
UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2
B-cell CLL/lymphoma 11A (zinc finger protein)
capping protein (actin filament) muscle Z-line, beta
coiled-coil domain containing 50
CSA-conditional, T cell activation-dependent protein
DEAH (Asp-Glu-Ala-His) box polypeptide 34
DnaJ (Hsp40) homolog, subfamily C, member 5
Down syndrome critical region gene 3
dual specificity phosphatase 2
13 days embryo lung cDNA, RIKEN full-length enriched library
similar to proteasome (prosome, macropain) subunit
ets variant gene 1
ets variant gene 6 (TEL oncogene)
ecotropic viral integration site 1
ecotropic viral integration site 1
G protein-coupled receptor 85
3-hydroxyanthranilate 3,4-dioxygenase
homeodomain interacting protein kinase 1
IQ motif containing GTPase activating protein 1
keratin 80
lymphocyte cytosolic protein 2
LIM domain only 2
leucine rich repeat (in FLII) interacting protein 1
LSM4 homolog, U6 small nuclear RNA associated
mitogen-activated protein kinase kinase kinase kinase 5
methyl-CpG binding domain protein 6
RIKEN cDNA 0610016J10 gene
NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6
nitric oxide synthase 2, inducible, macrophage
oxysterol binding protein-like 3
palladin, cytoskeletal associated protein
programmed cell death 1 ligand 2
pleckstrin homology domain containing, family A member 5
PR domain containing 16
RAS guanyl releasing protein 4
stearoyl-Coenzyme A desaturase 1
SEC14-like 1 (S. cerevisiae)
semaphorin 4B
solute carrier family 35, member E1
SWI/SNF related, actin dependent regulator of chromatin
sorting nexin 10
spectrin beta 2
signal transducer and activator of transcription 4
striatin, calmodulin binding protein 3
syntaxin 1A (brain)
TBC1 domain family, member 2
testis expressed gene 22
Tpd52-ps tumor protein D52, pseudogene
TRM5 tRNA methyltransferase 5 homolog (S. cerevisiae)
vacuolar protein sorting 45 (yeast)
position to
orientation
TSS
(F, Forward; R, mouse ID
(i, intron; e,
Reverse)
exon)
11184, i
F
M(53-55)-3
-60784
F
M60
-219062
R
M60
-55662
F
M28
11392, i
F
M25, M25-1
-725
F
M60
677, e
F
M25-1
25047, i
F
M14
13372, i
F
M34
2249, i
F
M60
-6487
F
M26
163769
F
M25
3035, i
F
M56
28831, i
R
M29
-7471
R
M57
950, i
F
M29
-442
F
M26
-1288
R
M57, M(56+57+60)1/2
-2981
F
M56
35477
F
M35
342, i
R
M25, M25-1
127291
R
M56
966, i
R
M53, M(53-55)-2
-105562
F
M30
-109689
F
M31
-30981
R
M26
-56356
R
M53
2057, i
R
M34
24870, i
F
M55, M(53-55)-1/2/3
23616
F
M25
-16909
R
M34
-36395
F
M26
9889, i
R
M15
1561, i
R
M60
1047, i
R
M53
-742
R
M56
-8908
R
M31
-50342
F
M53
-34585
R
M56
3021, i
R
M16
-7342
R
M29
22162, i
F
M25
-87020
F
M15
50160, i
F
M16
397, i
F
M16
31, e
F
M15
31610, i
F
M29
10578, i
F
M60
2788, i
F
M54
-600
R
M29
90129
F
M34
29913, i
R
M25
4527, i
R
M54
-136910
R
M57
2275, i
R
M30
6057, i
F
M60
-26985
R
M54
1897
R
M15
-83911
F
M57
-12568
R
M56
vector
outcome
DsRED
DsRED
DsRED
DsRED
DsRED
DsRED
DsRED
IL2RG
IL2RG
DsRED
DsRED
DsRED
DsRED
DsRED
DsRED
DsRED
DsRED
DsRED
DsRED
IL2RG
DsRED
DsRED
DsRED
IL2RG
IL2RG
DsRED
DsRED
IL2RG
DsRED
DsRED
IL2RG
DsRED
IL2RG
DsRED
DsRED
DsRED
IL2RG
DsRED
DsRED
IL2RG
DsRED
DsRED
IL2RG
IL2RG
IL2RG
IL2RG
DsRED
DsRED
DsRED
DsRED
IL2RG
DsRED
DsRED
DsRED
DsRED
DsRED
DsRED
IL2RG
DsRED
DsRED
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
Insertion sites recovered from lentiviral vector transplanted mice (RRL.PPT.IL2RG.pre)
nearest gene
gene ID
chromosome
RTCGD description
2210404O07Rik
72273
10
2310005P05Rik
67484
9A4
RIKEN cDNA 2310005P05 gene
B3gntl1
210004
11E2
UDP-GlcNAc:betaGal beta-1
Ccdc53
67282
10C2
coiled-coil domain containing 53
E030018B13Rik
381994
7
Fras1
231470
5E3
Fraser syndrome 1 homolog (human)
Ibrdc3
75234
4D2.2
IBR domain containing 3
Klrb1f
232408
6
CIS
killer cell lectin-like receptor subfamily B member 1F
Lap3
66988
5B3
leucine aminopeptidase 3
LOC100039599
100039599
15
LOC100043572
100043572
3
P2ry1
18441
3E1
purinergic receptor P2Y
Pgr
18667
9A1
progesterone receptor
Rasa1
218397
13C3
CIS
RAS p21 protein activator 1
Rbm17
76938
2A1
RNA binding motif protein 17
Rshl3
212892
10B1
radial spokehead-like 3
Zfp26
22688
9A3
zinc finger protein 26
position to TSS orientation
7285
R
43446
R
19581
R
16305
R
-16980
F
56034
R
18129
R
-12858
F
-7609
F
87996
F
6636
F
-51108
F
4263
R
-30932
R
-9274
R
24991
F
20567
F
Mouse ID
M37-1
M37-1
M39
M38
M40-2
M40-2
M38
M39
M36
M39
M37-1
M23-2
M36
M38
M38
M23-2
M40-2
Vector
IL2RG
IL2RG
IL2RG
IL2RG
IL2RG
IL2RG
IL2RG
IL2RG
IL2RG
IL2RG
IL2RG
IL2RG
IL2RG
IL2RG
IL2RG
IL2RG
IL2RG
outcome
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
Insertion sites recovered from SIN leukemias
nearest gene
gene ID
chromosome
Evi1
14013
3A3
Evi1
14013
3A3
Evi1
14013
3A3
Lcp2
16822
11A5
Mrpl39
27393
16C3.3
Snx10
71982
6B3
position to TSS orientation
-109689
F
-5100
R
-69696
R
-16909
R
153095
R
90129
F
Mouse ID
M31-1
M32-2
M34-2
M34-2
M34-2
M34-2
Vector
IL2RG
IL2RG
IL2RG
IL2RG
IL2RG
IL2RG
outcome
MPD-ML
LL
ML
ML
ML
ML
RTCGD
CIS
CIS
CIS
×
description
ecotropic viral integration site 1
ecotropic viral integration site 1
ecotropic viral integration site 1
lymphocyte cytosolic protein 2
mitochondrial ribosomal protein L39
sorting nexin 10
Insertion sites recovered from LTR leukemias
nearest gene
gene ID
chromosome
RTCGD description
position to TSS orientation
Mouse ID
Vector
outcome
Prdm16
70673
4 E2
CIS
PR domain containing 16
-1327
R
M3-1
dsRED
ML
Evi1
14013
3A3
CIS
ecotropic viral integration site 1
-71264
R
M(48-50)-1
dsRED
ML
Bcl2l1
12048
2H1
CIS
Bcl2-like 1
43444, i
R
M(48-50)-1
dsRED
ML
Bmx
12169
XF
BMX non-receptor tyrosine kinase
-61
F
M(48-50)-1
dsRED
ML
Evi1
14013
3A3
CIS
ecotropic viral integration site 1
-109564
R
M(48-50)-2
dsRED
ML
9530008L14Rik
109254
13 A4
×
RIKEN cDNA 9530008L14 gene
59566, i
R
M(48-50)-2
dsRED
ML
Paqr7
71904
4 D3
×
progestin and adipoQ receptor family member VII
1270
R
M(48-50)-2
dsRED
ML
× ( inserton site listed in the RTCGD only once), ML (myloid leukemia), MPD-ML (MPD like myeloid leukemia), LL (lymphatic leukemia), RTCGD (retrovirus-tagged cancer gene database), CIS (common insertion site),
TSS (transcriptional start site)
All insertion site were amplified in DNA of blood or BM samples taken at the day of sacrifice
Figure legend
Suppl. figure 1
DsRED2 transgene expression in mice of exp. III in primary and secondary recipients. SIN vectors and
LTR vectors expressing DsRED showed similar long-term expression with the exception of two cases: In
the SIN vector group, one mouse died due to bone marrow failure and one other mouse had no
transgene positive cells in the bone marrow at necropsy despite an initial high gene marking in the blood
at 9 weeks post BMT. The initial lower transduction rate of the SIN.SF transduced cells is apparent also in
vivo (blue lines) compared to the LTR transduced cells (red lines).
Suppl. figure 2
(a) The sensitivity of the LM PCR that was developed to detect insertions sites of gammaretroviral SIN
vectors was tested in defined DNA samples: DNA of a single clone, containing a single integration, was
mixed into a mass culture at different percentages. Starting with 20% input clone DNA the single clone
became already apparent.
(b) The absolute sensitivity of the LM PCR for gammaretroviral SIN vectors was tested by titration of the
amount of input DNA. DNA of one single clone with multiple vector copies was used as test DNA.
(c) LM PCR amplified in DNA of BM, spleen and peripheral blood cells of one healthy mouse. The
pattern was in all samples almost identical, indicating that indeed dominant hematopoietic clones were
identified by LM PCR.
Suppl. figure 3
FACS analysis of the three leukemic clones that developed after gene transfer with the SF91 LTR vector.
All three leukemias had a similar myeloid phenotype.
Suppl. Figure 4
LMPCR analysis of mice that received BM after prolonged culture time showed stronger selection for
single clones (M14-M16) compared to mice that received BM cells directly after the transduction (M2833). Right hand side picture shows the selection of dominant clones after secondary transplantation
including the leukemic mice M31-1 and M31-2.
Suppl. figure 5:
Spectral karyotyping (SKY) analysis of mouse M3-1. No chromosomal alterations were detected.
Suppl. figure 6:
The leukemic clone of M34-2 contained four insertion sites. To verify that the insertions were contained in
the same cell, leukemic cells were plated in semisolid methylcellulose medium. After a week clones
derived from single cells were picked and expanded in suspension culture for further three weeks. DNA
was extracted and LMPCR performed. The detection of the indentical insertion pattern in all four clones
derived from the leukemic mouse M34-2 clearly shows that the insertions were within the same cell. (The
LMPCR was performed using a biotinylated first primer that spanned the deletion in the U3 region for
primer extension. Therefore, an internal band was amplified, as indicated.)
Suppl. figure 7:
Enlarged view of figure 5c: The number of genes with forward orientation was divided by the number of genes with
reverse orientation and the resulting ratio was plotted. Here, the data is plotted to a window size of 10 kb giving a
better resolution of the distribution around the TSS. Red line: gammaretroviral SIN vectors, blue line: gammaretroviral
LTR vectors.
Suppl. figure 8:
LMPCR analysis of mice transplanted with lentiviral transduced bone marrow cells. LMPCR was
performed on DNA extracted from peripheral blood of primary and secondary mice taken on the day of
sacrifice. Mouse M23-2 was transplanted with BM cell that were expanded in vitro for 5 days prior to
transplantation.