Supplementary Materials and Methods
Transposon insertion site analysis. Three weeks post-transfection, G418-resistant HeLa
colonies were picked and transferred to 96-well plates. When the cells reached confluency
they were washed in PBS and frozen. To isolate genomic DNA, cells were lysed in 40 l
Lysis buffer per well [10 mM Tris (Roth)/HCl, pH 7.5; 10 mM EDTA (Merck); 10 mM NaCl
(Merck); 0.5% SDS (Roth); freshly add 1 mg/ml proteinase K (Merck)] for 2 hours at 60°C.
After centrifugation (Eppendorf, model 5810 R) of the 96-well plates for 2 minutes at 4000
rpm and 20°C, 80 l per well of cold 75 mM NaCl/ethanol solution were added and plates
were incubated for 1 hour at room temperature. Plates were washed three times with 150 l
70% ethanol for 2 minutes at 4000 rpm, and dried for 10 minutes using a vacuum centrifuge
(Eppendorf concentrator 5301). Finally, 40 l 5mM Tris/HCl, pH 8.0 per well were added
and plates were incubated overnight at room temperature.
The splinkerette adaptor was prepared as follows (for one well): 0.15 l 10 M
primer 40_SpAa.1, 0.15 l 10 M primer 41_SpBb.1, 0.05 l 10x SuRE buffer M (Roche)
and 0.65 l bidestilled water were mixed, heated to 97 °C for 4 minutes and slowly cooled
down to room temperature.
To prepare the plates for sequencing, a PCR protocol was performed in 10 l
reactions per well including 1 l second step PCR product, 1 l Big Dye Terminator mix
(MPI for Molecular Genetics, Sequencing Group, Berlin), 1 l 10 µM primer, and 7 l PCRgrade water. The temperature profile was 1 minute at 96 °C, 25 cycles at 96 °C for 10
seconds, 54 °C for 5 seconds, and 60 °C for 4 minutes. Products were precipitated by adding
22 l per well of a 3 mol/L Sodium acetate/96% ethanol solution. 96-well plates were
incubated for 10 minutes at room temperature on a shaker and afterwards centrifuged at 4000
rpm for 50 minutes. The supernatant was decanted and the plates were washed twice with
100 l per well 70 % ethanol at 4000 rpm for 15 minutes.
Western blotting. 48 hours post transfection, HeLa cells were lysed in RIPA buffer (150
mM NaCl, 1.0% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0)
supplemented with protease inhibitor cocktail (Complete Mini, Roche, Basel, Switzerland).
Total protein was quantified using BCA Protein Assay Kit (Pierce, Rockford, IL). Proteins
(10 g per lane) were loaded onto 10% polyacrylamide gel and subjected to sodium dodecyl
sulfate–polyacrylamide gel electrophoresis. Gels were transferred to nitrocellulose membrane
(Hybond–C Extra, Amersham Bioscience) and immunoblotting was performed according to
standard procedures. Proteins were detected with rat monoclonal anti-HA antibody (Roche,
Basel, Switzerland) dilution 1:2000, or mouse monoclonal anti-actin antibody (Dianova,
Hamburg, Germany) dilution 1:800, and chemiluminescence using ECL Plus Western
Blotting Detection Kit (Amersham Bioscience).
Clonogenic assays. Colony assays were performed by adding 50 l of Stemline medium
(Sigma-Aldrich, USA) supplemented with 100 ng/ml SCF, 20 ng/ml IL-6, 100 ng/ml IL-3, 20
ng/ml Flt3-L and 100 ng/ml TPO to the 100 l of electroporated CD34+ cell suspension.
Granulocyte/monocyte/macrophage clonogenic assays (CFU-GM) were performed by adding
30 l of the final cell suspension to 270 l of granulocyte/monocyte/macrophage
differentiation medium corresponding to semi-solid Methocult GF H4534 (Stemcell
Technologies, Vancouver Canada) composed of 1% methylcellulose (4000 cps), 30% fetal
bovine serum, 1% bovine serum albumin, 10-4 M 2-mercaptoethanol, 2 mM L-glutamine, 50
ng/ml rhSCF, 10 ng/ml rhGM-CSF, 10 ng/ml rhIL-3 in Iscove’s Modified Dulbecco’s
Medium (MDM). The cell suspensions were seeded over 3 wells in a 24-well plate at a
concentration of 5x104 cells per well. At day 14, GFP+ and total CFU-GM colonies were
counted using a fluorescence inverted microscope (Olympus, Japan). GFP expression and
scoring of CFU-Mk colonies was monitored using the Olympus fluorescence microscope. In
addition confocal microscopy was conducted at 488 nm with an Axiovert 100M, LSM510,
Zeiss using the AxioPlan 2 LSM 510 version 2.8 software.
Erythroid clonogenic assays (CFU-E) were performed by adding 30 l of the final
cell suspension to 270 l of erythoid differentiation medium corresponding to semi-solid
Methocult SFBIT H4436 (Stemcell Technologies, Vancouver, Canada) composed of
methylcellulose, fetal bovine serum, bovine serum albumin, 2-mercaptoethanol, L-glutamine,
rhSCF, rhGM-CSF, rhIL-3, rhIL-6, rhG-CSF, rh Epo in Iscove’s MDM. The cell suspensions
were seeded over 3 wells in a 24-well plate, hence containing 5x104 cells per well. At day 7,
colonies were counted which typically contained about 70% glycophorin A+ cells, a
characteristic marker of erythroid cells. GFP+ CFU-E colonies were monitored as mentioned
above. Cytologic analysis was performed by Giemsa staining.
Oligonucleotides
Primer name
Length
(bp)
SB/XhoI/Kozak
41
Cloning of
pCSB100XNpA
SB/NotI
46
Cloning of
pCSB100XNpA
Tol2/XhoI/Kozak
39
Cloning of
pCTol2NpA
Tol2/NotI
40
Cloning of
pCTol2NpA
mPB/XhoI/Kozak
41
Cloning of
pCmPBNpA
mPB/NotI
40
Cloning of
pCmPBNpA
Usage
Sequence (5’ – 3’)
TACCACTCGAGCCACCA
TGGGAAAATCAAAAGAA
ATCAGCC
AGCAACTAGCGGCCGCC
TAGTATTTGGTAGCATTG
CCTTTAAATTG
TACCACTCGAGCCACCA
TGGAGGAAGTATGTGAT
TCATC
AGCAACTAGCGGCCGCC
TACTCAAAGTTGTAAAA
CCTCAG
TACCACTCGAGCCACCA
TGGGCAGCAGCCTGGAC
GACGAGC
AGCAACTAGCGGCCGCT
CAGAAACAGCTCTGGCA
CATGTC
Tm
(°C)
120
134
116
120
136
126
iPB/XhoI/Kozak
41
Cloning of
pCiPBNpA
iPB/NotI
40
Cloning of
pCiPBNpA
Cloning of
transposase
constructs
Cloning of
transposase
constructs
poly-A/NotI
36
poly-A/SacI
36
SB-HA/NotI
100
Cloning of
pCSB100XHApA
Tol2-HA/NotI
94
Cloning of
pCTol2HApA
mPB-HA/NotI
101
Cloning of
pCmPBHApA
SB
34
Cloning of
pUC19SBneo
Tol2L
40
Cloning of
pUC19Tol2 neo
Tol2R
41
Cloning of
pUC19Tol2 neo
PBL
32
PBR
28
40_SpAa.1
61
Splinkerette
adaptor
48
Splinkerette
adaptor with BstYI
recognition
sequence
41_SpBb.1
Cloning of
pUC19PBneo
Cloning of
pUC19PBneo
TACCACTCGAGCCACCA
TGGGTAGTTCTTTAGAC
GATGAGC
AGCAACTAGCGGCCGCT
CAGAAACAACTTTGGCA
CATATC
AGCAACTTGCGGCCGCG
TTGTTAACTTGTTTATTG
C
AACGTACCGAGCTCGAT
TTAACAAAAATT
TAACGCG
AGCAACTAGCGGCCGCT
TAGGCATAATCCGGTAC
ATCATAAGGGTAGGCAT
AATCCGGTACATCATAA
GGGTAGTATTTGGTAGC
ATTGCCTTTAAATTG
AGCAACTAGCGGCCGCT
TAGGCATAATCCGGTAC
ATCATAAGGGTAGGCAT
AATCCGGTACATCATAA
GGGTACTCAAAGTTGTA
AAACCTCAG
AGCAACTAGCGGCCGCT
TAGGCATAATCCGGTAC
ATCATAAGGGTAGGCAT
AATCCGGTACATCATAA
GGGTAGAAACAGCTCTG
GCACATGTCGATGTTG
TATCTTTACAGTTGAAGT
CGGAAGTTTACATACA
TATCTTTACAGAGGTGT
AAAGTACTTGAGTAATT
TTACTT
TATCTTTACAGAGGTGT
AAAAAGTACTCAAAAAT
TTTACTC
TTAACCCTAGAAAGATA
ATCATATTGTGACGT
TTAACCCTAGAAAGATA
GTCTGCGTAAA
CGAAGAGTAACCGTTGC
TAGGAGAGACCGTGGCT
GAATGAGACTGGTGTCG
ACACTAGTGG
GATCCCACTAGTGTCGA
CACCAGTCTCTAATTTTT
TTTTTCAAAAAAA
124
120
106
100
288
274
300
90
102
104
84
76
-
-
Splinkerette
adaptor with NspI
52_SpNspI
48
recognition
sequence
First round adaptor
38_Sp0F.2
28
primer
Second round
39_Sp1F.2
25
adaptor primer
First nested primer
72
24
for 3' site of SB
insertions
First nested primer
73
27
for 5' site of SB
insertions
Second
nested
primer for 5' and 3'
T-Bal
27
site
of
SB
insertions
and
sequencing primer
First nested primer
70
18
for 5' site of mPB
insertions
First nested primer
71
21
for 3' site of mPB
insertions
Second
nested
primer for 5' site of
XLfor
25
mPB insertions and
sequencing primer
Second
nested
primer for 3' site of
XLrev
22
mPB insertions and
sequencing primer
First nested primer
74
22
for 5' site of Tol2
insertions
First nested primer
81
24
for 3' site of Tol2
insertions
Second
nested
primer for 5' site of
61
25
Tol2 insertions and
sequencing primer
Second
nested
primer for 3' site of
75
24
Tol2 insertions and
sequencing primer
Tm, melting temperature of the primers in °C
CATGCCACTAGTGTCGA
CACCAGTCTCTAATTTTT
TTTTTCAAAAAAA
CGAAGAGTAACCGTTGC
TAGGAGAGACC
GTGGCTGAATGAGACTG
GTGTCGAC
-
64
63
CACCCTAACTGACACAC
ATTCCAC
60
CTCATCAATGTATCTTAT
CATGTCTGG
57
CTTGTGTCATGCACAAA
GTAGATGTCC
60
CCGGGCTGCAGGAATTC
G
60
GACAAGCACGCCTCACG
GGAG
64
TACAGACCGATAAAACA
CATGCGTC
58
CCAAGCGGCGACTGAGA
TGTCC
63
CATGTCTGGATCGACTA
CTAGG
58
GTATCTTATCATGTCTGG
ATCGAC
56
CTCAAGTAAGATTCTAG
CCAGATAC
56
GACTGTAAATAAAATTG
TAAGGAG
51
Supplementary Figure Legends
Supplementary Figure 1. Levels of SB100XHA, Tol2HA and mPBHA transposase
expression in HeLa cells. (a) Schematic representation of HA-tagged transposase constructs.
CAGGS, CAGGS promoter; pA, polyadenylation sequence; SB100X, hyperactive SB
transposase; Tol2, Tol2 transposase; mPB, mouse codon-optimized PB transposase, HA,
double HA-tag added to the C-terminus of each transposase. (b) Western blot analysis of
SB100XHA, Tol2HA and mPBHA transposase expression. The transfected amounts of
transposase expression plasmids correspond to those and higher than required for the peak
activity of each system (i. e., ≥5 and 50 ng for SB100X, ≥125 and 250 ng for Tol2 and ≥50
and 250 ng for mPB).
Supplementary Figure 2. Relative transposition efficiencies of SB100X, Tol2 and PB
transposons in “low” and “high” transposon DNA conditions. Transposition activity was
measured at the peak conditions for both the low (10 ng) (a) and the high (500 ng) (b)
dosages of transfected transposon plasmids by determining the numbers of antibiotic-resistant
colonies per transfected cell population multipled by the average numbers of transposon
insertions per colony for each of the three transposon systems. The graphs show average
numbers of transposon insertions per 100 transfected cells. Error bars represent standard error
of the mean (SEM).
Supplementary Figure 3. Persistence of transposition-mediated gene expression in
human HeLa cells. Photographs of representative, exemplary cell colonies stably transfected
with Venus-tagged transposon vectors are shown. (a) Uniformly expressing colony showing
no signs of transgene silencing; (b) uniformly silenced colony that lost Venus expression; (c)
mosaic colony showing sectors with cells that lost Venus expression; (d) uniformly silenced
colony with attenuated Venus expression.
Supplementary Figure 4. Sequence logo analysis of SB100X, Tol2 and PB integration
sites. Web logo analyses and nucleotide probability plots of 46 SB100X (a), 113 Tol2 (b) and
46 PB (c) integration sites in HeLa cells.
Supplementary Table 1. Distribution of SB, PB and Tol2 integration sites in vertebrate
genomes. The table summarizes results produced by this study on the distribution of Tol2
transposon insertions in human HeLa cells in the context of transposon mapping data
obtained by others. Values typed in bold are significantly different from random datasets.
b
Insertions mapped in this study were obtained with a Tol2 transposon-based enhancer trap
cassette remobilized in two transgenic donor lines. cValues for insertions only upstream of
genes were determined. ND – not determined. References: 1. Yant SR, Wu X, Huang Y,
Garrison B, Burgess SM, Kay MA (2005). High-resolution genome-wide mapping of
transposon integration in mammals. Mol Cell Biol; 25: 2085-2094.; 2. Wilson MH, Coates
CJ, George AL, Jr. (2007). PiggyBac transposon-mediated gene transfer in human cells. Mol
Ther; 15: 139-145.; 3. Galvan DL, Nakazawa Y, Kaja A, Kettlun C, Cooper LJ, Rooney CM,
et al. (2009). Genome-wide mapping of piggyBac transposon integrations in primary human
T cells. J Immunother. 32: 837-844.; 4. Kondrychyn I, Garcia-Lecea M, Emelyanov A,
Parinov S, Korzh V (2009). Genome-wide analysis of Tol2 transposon reintegration in
zebrafish. BMC Genomics; 10: 418.