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[1] So, C.W., Karsunky, H., Wong P, Weissman, I.L. & Cleary, M.L. Leukemic transformation of
hematopoietic progenitors by MLL-GAS7 in the absence of Hoxa7 or Hoxa9. Blood 103,
3192-3199 (2004).
[2] Yoshida, Y. et al. Leukemic transformation of hematopoietic progenitors by MLL-GAS7 in
the absence of Hoxa7 or Hoxa9. Cell 103, 1085-1097 (2000).
[3] Yuasa, H. et al. Oncogenic transcription factor Evi1 regulates hematopoietic stem cell
proliferation through GATA-2 expression. EMBO J. 24, 1976-1987 (2005).
[4] Martini, A. et al. Recurrent rearrangement of the Ewing's sarcoma gene, EWSR1, or its
homologue, TAF15, with the transcription factor CIZ/NMP4 in acute leukemia. Cancer Res.
62, 54080-5412 (2002).
[5] Huang, J.S. et al. Diverse cellular transformation capability of overexpressed genes in
human hepatocellular carcinoma. Biochem. Biophys. Res. Commun. 315, 950-958 (2004).
[6] Harada, J.N. et al. Identification of novel mammalian growth regulatory factors by genomescale quantitative image analysis. Genome Res. 15, 1136-1144 (2005).
[7] Paterlini-Brechot, P. et al. Hepatitis B virus-related insertional mutagenesis occurs
frequently in human liver cancers and recurrently targets human telomerase gene.
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[8] Ryu, S., Zhou, S., Ladurner, A.G. & Tjian, R. The transcriptional cofactor complex CRSP is
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normal growth and development and does not predispose to spontaneous tumorigenesis.
DNA Cell Biol. 25, 376-382 (2006).
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SUPPLEMENTARY METHODS
Integration site analysis
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Most vector integration sites from HT1080 cell clones were identified by inverse PCR as
previously described,1 with minor modifications. In short, genomic DNA was digested
individually with either PstI, Hind III, BglII, SphI, BamHI, XbaI, or a combination of BglII and
BamHI, all of which cut within the vector provirus. The restriction enzymes were inactivated,
and approximately 100 ng was ligated with 400 units T4 DNA ligase in 20 ul reaction volume.
Provirus-genomic junction fragments were then amplified by nested PCR using vector-specific
primers. First primer pair, 5’-CTAGAAACTGCTGAGGGCGG and 5’-CTGATCCTTGGGAGGGT;
nested primer pair, 5’-TCCTAACCTTGATCTGA and 5’-CAGATTGATTGACTGCC. The
resulting PCR products were separated by gel electrophoresis, excised, and column purified
with the Qiaquick gel extraction kit (Quigen). Junction fragments were then sequenced either
directly using the PCR fragments as template and the nested PCR oligos as primers, or after
subcloning into TOPO TA cloning vectors (Invitrogen Corp., Carlsbad, CA).
Some vector integration sites from HT1080 cell clones were identified by a directed DNA
library screening approach as previously described,2 with modifications. First, genomic DNA
was digested with XbaI (which cuts once in each vector LTR), and half of the digested product
was subjected to Southern analysis essentially as described above using probes for the vector
LTR that are located either proximally (5' LTR) or distally (3' LTR) to the Xba I site in order to
identify band sizes for individual provirus junction fragments. The remaining digested DNA
products were then separated by gel electrophoresis under identical conditions as those used
for the Southern analysis, and band-specific regions were excised and column purified.
Extracted DNA was then subcloned into pUC19 cloning vectors, and the resulting libraries were
screened by conventional colony lifts and hybridization using the same proximal LTR probe.
Plasmid inserts from positive colonies were subsequently sequenced.
Some vector integration sites in HT1080 cell clones, and all vector integration sites in
32D cell clones, were identified by linear amplification-mediated polymerase chain reaction
(LAM-PCR) essentially as previously described.3 In short, single-stranded copies of the viral 5'
Li, Stamatoyannopoulos & Emery
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LTR junction fragments were first generated by 100 cycles of linear amplification using
biotinylated primers specific for the proximal region of the vector LTR's (vector MGPN2, 5'biotinTTCTCTAGAAACTGCTGAGG; vector INS4(+), 5'biotin-ATTCTAAATCTCTCTTTCAGCC). The
products were then isolated using streptavidin-coated magnetic M280 Dynabeads (Dynal
Biotech, Oslo, Norway), converted to double-stranded DNA using random hexamers and
Klenow, digested with either Tsp509I, HaeIII, or RsaI (which cut in the genomic sequences),
and capped with anchor primers compatible with the restricted ends. The vector-genomic
junction fragments were eluted from the Dynabead matrix and amplified by two additional
rounds of nested PCR using primers specific to the vector LTR and anchor primer sequences.
The resulting LAM-PCR products were separated by gel electrophoresis, excised, and column
purified. Junction fragments were then sequenced either directly using the PCR fragments as
template and the nested PCR oligos as primers, or after subcloning in TOPO TA cloning vectors.
Sequences were BLAST searched against either the human genome (March 2006
assembly) or the mouse genome (February 2006 assembly) using the UCSC Genome Browser
(http://genome.ucsc.edu/) as previously described.4 Insertion sites were considered authentic if
they contained adjoining retroviral sequences and gave a unique best match with better than
90% identity. Analysis of integration sites relative to flanking transcription units were also
performed using the UCSC Genome Browser and included all known genes (UCSC known
genes based on UniProt, RefSeq, and GeneBank mRNA). Simulated random integration
datasets were generated essentially as described.4 In short, random sites in the human or
mouse genomes were chosen using a random number generator. Sequences of lengths about
the same size as the experimental data (50 bp) were then identified adjacent to these sites and
BLAST searched using the criteria used for the experimental datasets described above.
Expression microarray analysis
Li, Stamatoyannopoulos & Emery
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The transduced HT1080 cell clones were screened for dysregulated cellular genes using
Codelink UniSet Human 20K I Bioarrays and gene expression system (Amersham / GE
Healthcare Bio-Sciences Corp., Piscataway, NJ) following the manufacturer's directions. These
arrays include approximately 20,000 human genes. Total RNA from HT1080 cell clones and
two independent aliquots of untransduced HT1080 cells was prepared by column purification
(RNeasy Mini kit, Qiagen), and used as template to prepare biotin-labeled cRNA target by linear
amplification. Labeled target was then fragmented and hybridized to individual bioarrays (one
array per clone or control). The hybridized arrays were then washed, stained with Cy5streptavidin, and scanned using a GenePix 4000A analyzer (Axon Instruments /
MolecularDevices, Sunnyvale, CA). Expression levels were first analyzed using the
manufacturer's software (CodeLink EXP v4.1) in order to assess the overall signal quality and to
establish minimum thresholds for signal reliability. Pair-wise comparisons between each of the
individual arrays versus all of the remaining arrays (two untransduced controls and 86
transduced clones) were then performed using GeneSifter software (VizX Lab LLC, Seattle,
WA). For this purpose we normalized signals to array means, and excluded individual spots if
they were background-contaminated, irregularly shaped, near background, or saturated;
otherwise, no additional transformations or corrections were made. A gene was considered to
be dysregulated if the intensity of that gene's signal within any one cell clone was either 5-fold
higher or 5-fold lower than the mean signal intensity for the remaining cell clones and
untransduced controls, that gene's signal was considered reliable by the manufacturer's criteria,
and that gene was not found to be dysregulated in more than one clone.
Statistical analysis
Li, Stamatoyannopoulos & Emery
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Most comparisons between discrete datasets were performed using the KolmogorovSmirnov (KS) test.5 This is a non-parametric and distribution free method that does not require
the datasets to be normally distributed. In cases where comparisons were performed between
the means of small matched datasets with apparent normal distributions, we used the paired,
two-tailed Student's t-test. In cases where comparisons were made between two discrete
proportions (frequencies), we used the Z-test for two proportions. Kaplan-Meier survival curves
were analyzed using the logrank test and chi-squared distribution.
In order to estimate the frequency of vector-mediated tumor formation (Fig. 5a), we first
estimated the number of independent transformation events based on the fraction of tumor-free
animals at 130 days using the Poisson distribution: vector MGPN2, 1 of 10 animals surviving
indicating 23 independent transformation events; vector INS4(+), 4 of 10 animals surviving
indicating 9 independent transformation events. We then divided the estimated number of
transforming events by the estimated number of cells that were transduced during the original
transduction culture (a total of 37,700 for vector MGPN2 and 89,680 for vector INS4(+),
Supplementary Table 3, experiments D and E). These ratios were then compared by the 1sided Z-test for two proportions.
In order to estimate the number of simulated random integration events found +/- 40 Mb
of dysregulated genes (Table 1), we first mapped 100 simulated random integration sites
relative to the dysregulated genes. This analysis revealed 29 cases (out of 32 dysregulated
genes) where unique simulated random integration sites were located within a 40 Mb window of
unique dysregulated genes, for an overall risk of 1 in 100 for each of these 29 genes (and 0 for
the remaining 3 genes). We then calculated the relative risk for each of the cell clones by
multiplying the risk for the dysregulated genes present in that clone (either 0 or 0.01) by the
number of authentic vector provirus present within that clone. Finally, we calculated the
cumulative risk for all such occurrences by summing over all clones for each vector panel.
Li, Stamatoyannopoulos & Emery
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Since none of the simulated random integration sites were found to be located within the body
of dysregulated genes, we calculated this risk to be 0.
References
1. Nolta JA, Dao MA, Wells S, Smogorzewska EM, Kohn DB. Transduction of pluripotent
human hematopoietic stem cells demonstrated by clonal analysis after engraftment in
immune-deficient mice. Proc Natl Acad Sci USA. 1996;93:2414-2419.
2. Li CL, Coullin P, Bernheim A, Joliot V, Auffray C, Zoroob R, Perbal B. Integration of
Myeloblastosis Associated Virus proviral sequences occurs in the vicinity of genes encoding
signaling proteins and regulators of cell proliferation. Cell Commun Signal. 2006;4:1-15.
3. Harkey MA, Kaul R, Jacobs MA, et al. Multiarm high-throughput integration site detection:
limitations of LAM-PCR technology and optimization for clonal analysis. Stem Cells Dev.
2007;16;381-392.
4. Aker M, Tubb J, Miller DG, Stamatoyannopoulos G, Emery DW Integration bias of
gammaretrovirus vectors following transduction and growth of primary mouse hematopoietic
progenitor cells with and without selection. Mol Ther. 2006;4;226-235.
5. Horn SD. Goodness-of-fit tests for discrete data: a review and an application to a health
impairment scale. Biometrics. 1977;33:237-247.
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