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ssBioMed CentBMC Biotechnology
Open AcceResearch article
PhiC31 integrase induces a DNA damage response and
chromosomal rearrangements in human adult fibroblasts
Jian Liu†1,2, Tina Skjørringe†1, Torben Gjetting3 and Thomas G Jensen*1,4
Address: 1The Kennedy Center, Gl. Landevej 7, 2600 Glostrup, Denmark, 2Department of Laboratory Medicine, Clinical Research Center, Malmö
University Hospital, 20502 Malmö, Sweden, 3Department of Radiation Biology 6321, Finsen Centre, Rigshospitalet, 2100 Copenhagen Ø,
Denmark and 4Institute of Human Genetics, University of Aarhus, Aarhus, Denmark
Email: Jian Liu - jian.liu@med.lu.se; Tina Skjørringe - tinaskj@gmail.com; Torben Gjetting - torben.gjetting@rh.regionh.dk;
Thomas G Jensen* - thomas@humgen.au.dk
* Corresponding author †Equal contributors
Abstract
Background: PhiC31 integrase facilitates efficient integration of transgenes into human and mouse
genomes and is considered for clinical gene therapy. However recent studies have shown that the
enzyme can induce various chromosomal abnormalities in primary human embryonic cells and
mammalian cell lines. The mechanisms involved are unknown, but it has been proposed that PhiC31
attachment sites in the host genome recombine leading to chromosomal translocations.
Results: We have studied possible effects of the PhiC31 integrase expression in human adult
fibroblasts by karyotyping. All control cells were cytogenetically normal, whereas cells expressing
PhiC31 integrase show chromosomal abnormalities confirming our previous results using primary
embryonic fibroblasts. In order to study the early mechanisms involved we measured H2AX
phosphorylation – a primary event in the response to DNA double-strand-breaks. Transient
transfection with PhiC31 integrase encoding plasmids lead to an elevated number of cells positive
for H2AX phosphorylation detected by immunofluorescence. Western blot analysis confirmed the
upregulated H2AX phosphorylation, whereas markers for apoptosis as well as p53 and p21 were
not induced. Cells transfected with plasmids encoding the Sleeping Beauty transposase remained
cytogenetically normal, and in these cells less upregulation of H2AX phosphorylation could be
detected.
Conclusion: In primary human fibroblasts expression of PhiC31 integrase leads to a DNA damage
response and chromosomal aberrations.
Background
PhiC31 integrase, originally isolated from Streptomyces
lividans, is widely used for non-viral gene delivery and vec-
tor integration [1]. PhiC31 integrase mediated integration
leads to prolonged gene expression and has been used for
tions, chromosomal rearrangements and chromosomal
instability [4-8]. In mouse cells it was recently shown that
the integrase does lead to imprecise deletion of self exci-
sion cassettes [9]. In cell lines, PhiC31-mediated integra-
tion of plasmid DNA may be accompanied by
Published: 2 April 2009
BMC Biotechnology 2009, 9:31 doi:10.1186/1472-6750-9-31
Received: 15 October 2008
Accepted: 2 April 2009
This article is available from: http://www.biomedcentral.com/1472-6750/9/31
© 2009 Liu et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 8
(page number not for citation purposes)
correction of disease models [2,3]. However, it has been
reported that PhiC31 integrase can lead to genomic dele-
chromosomal rearrangements in the mammalian host
genome with a frequency up to 15% [10]. The mecha-

BMC Biotechnology 2009, 9:31 http://www.biomedcentral.com/1472-6750/9/31
nisms involved are unknown, but it has been speculated
that cryptic PhiC31 attachment sites recombine leading to
chromosomal translocations [10]. In contrast to this, the
system for transposon-directed genomic integration facil-
itated by the transposase Sleeping Beauty apparently does
not cause chromosomal aberrations [8]. In order to assess
the mechanistic and potential harmful effects of cellular
expression of PhiC31 integrase and Sleeping Beauty we
have studied the immediate DNA damage responses and
the long-term effect of induction of genomic rearrange-
ments.
Results and discussion
Generation of primary human fibroblasts expressing
PhiC31 integrase
In gene transfer applications, the PhiC31 integrase medi-
ates the integration of plasmids bearing an attB site into
sequences with partial sequence identity to attP (pseudo-
attP sites). Primary adult human fibroblasts were co-trans-
fected with the plasmids pBabepuro or pBabepuroatt,
containing the 285-base pair attB sequence, in combina-
tion with the plasmid pCMV-Int encoding the PhiC31
integrase. Transfections were performed in combination
with a 3:1 molar excess of pCMV-Int compared to the pBa-
bepuro/pBabepuroatt plasmids in order to increase the
likelihood that pCMV-Int plasmids were integrated in the
genome. After puromycin selection, DNA and RNA were
isolated for PCR analysis using primers specific for the
integrase gene. In this analysis clear signals were detected
from puromycin selected cells transfected the pCMV-Int
plasmid showing that the integrase is exclusively present
and expressed in cells transfected with pCMV-Int (data
not shown).
Cytogenetic analysis
The karyotypes of puromycin selected cells are shown in
Table 1 (left column, transfection experiment 1). Cells
transfected with pBabepuro or pBabepuroatt alone had
normal karyotypes. In contrast, abnormal karyotypes
including aneuploidy and deletions were found in cells
co-transfected with the pCMV-Int plasmids confirming
our previous results with primary embryonic cells [6]. The
data in Table 1, right column show evaluation of effect of
expression of the Sleeping Beauty transposase, as will be
described below.
Analysis of histone H2AX phosphorylation
The histone H2AX is a key protein of the cellular response
to DNA damage and becomes rapidly phosphorylated in
response to DNA damage [11,12]. Phosphorylated H2AX
can be detected using immunofluorescence leading to
clear and distinct nuclear staining (Fig. 1a). Normal
Human Dermal Fibroblasts (NHDF) treated with col-
cemide, serving as a positive control [13], or transfected
with pBabepuro or pBabepuro+pCMV-Int were analysed
for H2AX phosphorylation at various times after transfec-
tion. As seen in Fig. 1b, cells transfected with pBa-
bepuro+pCMV-Int show an approx. 4 fold increase in the
number of nuclei positive for H2AX phosphorylation
three days after the treatment. This is comparable to the
number of stained nuclei resulting from colcemide treat-
ment. Cells only transfected with the control plasmid pBa-
bepuro showed no increase in the fraction of cells positive
Table 1: Karyotypes of primary human fibroblasts
Cytogenetic analysis
Transfected constructs Transfection experiment 1 Transfection experiment 2
pBabepuro 46, XY (n = 10) 46, XY (n = 15)
pBabepuroatt 46, XY (n = 10) 46, XY (n = 18)
pBabepuro + pCMV-Int 46, XY (n = 6)
Extra chr. 10 (n = 1)
Del(1)(p11) (n = 1)
Add 17q (n = 1)
Chr. 2 loss (n = 1)
pBabepuroatt + pCMV-Int 46, XY (n = 6)
Extra chr. (n = 1)
Ring Chr. No. 7 (n = 2)
Del(13)(q21.1) (n = 1)
pBabepuro + pCMV-SB 46, XY (n = 17)
pBabepuro + pCMV-mSB 46, XY (n = 2)
pBabepuro-SB* 46, XY (n = 16)
pBabepuro-mSB* 46, XY (n = 15)
Number of cells analysed (n) are indicated in parentheses. *The expression cassettes for SB or mutant SB (mSB) were cloned into the pBabepuro Page 2 of 8
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plasmid. Metaphase chromosomes were analyzed by standard Q-banding techniques. The description of chromosome aberrations was based on the
recommendations of the International System for Human Cytogenetic Nomenclature (ISCN 1995).

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Page 3 of 8
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Analysis of H2AX phosphorylationFigure 1
Analysis of H2AX phosphorylation. a) Immunofluorescence analysis of NHDF cells transfected with pBabepuro alone
(control) or in combination with pCMV-Int (integrase). Blue color is DAPI-staining, visualizing nuclear DNA. Red color is stain-
ing of phosphorylated H2AX using anti-γ-H2AX antibodies. b) Quantitation of the percentage of cells with upregulated H2AX
phosphorylation. Each experiment was repeated 3 times, and 100 cells were counted in each sample. Error bars indicate stand-
ard error of the mean. Only in day 3 there is a significant difference between the three treatments (p < 0.05).
0
2
4
6
8
10
12
Day1 Day2 Day3
1.pBabepuro 2.pBabepuro +pCMV-Int 3.colcemide
a
b

BMC Biotechnology 2009, 9:31 http://www.biomedcentral.com/1472-6750/9/31
for H2AX phosphorylation. The increase was detected in
cells transfected with both plasmids pCMV-Int and pBa-
bepuroInt, but not after transfection with mutant PhiC31
genes (Fig 2a). The upregulated H2AX phosphorylation
was confirmed by Western blotting analysis (Figure 2b).
Here, in cells transfected with pBabepuro in combination
with pCMV-Int (lane 2) or one plasmid containing gene
expression cassettes for both puromycin-resistance and
integrase (lane 3) clear bands representing phoshorylated
H2AX is detected. Band intensity is comparable to cells
treated with colcemide (lane 6), whereas cells transfected
with the control plasmids pBabepuro alone (lane 1) or
plasmids expressing an inactive mutant variant integrase,
pBabepuro+pCMV-mInt (lane 4) and pBabepuromInt
(lane 5) show only weak bands of gamma-H2AX.
Using immunofluorescence the degree of H2AX phospor-
ylation was scored by an arbitrary range ranking of stain-
ing intensities. Score 3 was defined as the very intensive
nuclear staining, score 2 intermediate, and score 1 being
the faint but yet defined nuclear staining (Fig. 3). NHDF
cells transfected with pCMV-Int had significantly more
cells positive for H2AX phosphorylation scored as 3 and 2
compared to non-transfected cells (P = 0.0015, Fisher's
exact test). Immunofluorescence analysis of transfected
cells did not reveal upregulation of p53 and p21, and early
markers for apoptosis were not significantly upregulated
(data not shown). Collectively, these results suggest that
active PhiC31 integrase leads to a DNA damage response
in transfected NHDF cells.
H2AX phosphorylation 3 days after transfection of NHDF cellsFigure 2
H2AX phosphorylation 3 days after transfection of NHDF cells. a) Immunofluorescence analysis. b) Western blotting
analysis. Lanes: 1) pBabepuro 2) pBabepuro+pCMV-Int 3) pBabepuroInt 4) pBabepuro+ pCMV-mInt 5) pBabepuromInt 6) Col-
0
2
4
6
8
10
12
14
1.p
Ba
be
pu
ro
2.p
Ba
be
pu
ro
+p
CM
V-I
nt
3.p
Ba
be
pu
ro
Int
4.p
Ba
be
pu
ro
+p
CM
V-m
Int
5.p
Ba
be
pu
ro
m
Int
6.C
olc
em
ide

(30
ng
/m
l)
1 2 3 4 5 6
J-H2AX
E-actin
a
bPage 4 of 8
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cemide control experiment causing DNA damage.

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Page 5 of 8
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Analysis of H2AX phosphorylation using co-transfection of plasmids encoding EGFP and PhiC31 integrase or Sleeping Beauty (SB) transposaseFigure 3
Analysis of H2AX phosphorylation using co-transfection of plasmids encoding EGFP and PhiC31 integrase or
Sleeping Beauty (SB) transposase. a) Cells co-transfected with plasmids encoding EGFP (green) and integrase were immu-
nostained using anti-γ-H2AX antibodies (red). b) H2AX immunostaining was quantified by ranking H2AX positive cells from 1
to 3, 3 being the very intensive nuclear staining and 1 being the vague but yet defined nuclear staining. Staining intensities were
quantified in GFP positive cells.
a




b

0
10
20
30
40
50
60
70
80
90
no transfection pCMV-Int pCMV-mInt pCMV-SB
Pe
rc
e
n
ta
ge

o
f H
2A
X
po
si
tiv
e
c
e
lls
3
2
1
0

BMC Biotechnology 2009, 9:31 http://www.biomedcentral.com/1472-6750/9/31
Analysis of possible effects of expression of SB transposase
Plasmids encoding wildtype and mutant SB transposase
were co-transfected with pBabepuro and the cells subse-
quently selected in puromycin. After drug selection the
chromosomes were analysed using G-banding. As seen in
Table 1 (right column) all puromycin selected cells had
normal chromosomes. After transient transfection the SB
transposase resulted in lower levels of H2AX phosphor-
ylation compared to cells transfected with the PhiC31
integrase plasmids measured by immunofluorescence
(Fig. 3).
Altogether we have shown that the PhiC31 integrase
induces chromosome rearrangements in primary human
adult cells confirming our previous results using primary
human embryonic fibroblasts [6]. The observed effects
take place even in the absence of donor plasmids contain-
ing attB sequences indicating that the effects are caused by
the integrase enzyme directly. Furthermore we show that
the PhiC31 integrase, in contrast to the SB transposase,
leads to significant up-regulation of H2AX phosphoryla-
tion. Interestingly, immunofluorescence staining reveal
numerous bright spots in the nucleus in a pattern similar
to that observed when cells are treated with colcemide,
which causes DNA damage in multiple loci. H2AX phos-
phorylation leads to recruitment of repair factors to dam-
aged DNA [14] and efficient homologous
recombinational repair of chromosomal double-strand
breaks [15] independent of p53 [16].
H2AX phosphorylation was observed already 3 days after
transfection, whereas the abnormal karyotypes were
measured after drug selection (15–17 days after transfec-
tion). The inducible PhiC31 integrase system described
recently may be used to establish whether the two events,
DNA damage and chromosome rearrangements, are caus-
ally linked [17]. Moreover, the dosage dependent effects
of integrase are still unknown and need to be further clar-
ified. Our findings highlight the importance of screening
for chromosomal rearrangements in mammalian cells
and investigation of the genetic and phenotypic conse-
quences.
Conclusion
In primary human adult fibroblasts PhiC31 integrase
leads to a DNA damage response and chromosomal aber-
rations. Our findings have important implications for
gene therapy approaches based on PhiC31 emphasising
that safety issues for gene therapy approaches need to be
carefully addressed.
Methods
Plasmid constructs and cell lines
bepuroatt contains the specific 285 bp bacterial attach-
ment site for PhiC31 integrase [6]. Plasmid pCMV-Int
harbouring the PhiC31 integrase gene driven from a CMV
promoter was a generous gift from Dr. Calos, Stanford
University, CA, USA. The coding sequence of PhiC31 inte-
grase was excised from pCMV-Int by PstI and BamHI
digestion and ligated into pBabepuro to create the plas-
mid pBabepuroInt. The plasmids pCMV-mInt and pBa-
bepuromInt were developed by deleting a 537 bp in-
frame fragment in the integrase gene using the restriction
enzyme PmlI (New England Biolabs, USA). The deletion
reduces the polypeptide length from 613 amino acid resi-
dues (68 kDa) to 434 amino acid residues (48 kDa) and
encompasses the entire DNA binding region of the pro-
tein rendering it inactive. The plasmid pCMV-SB, express-
ing the SB transposase (SB10) from the CMV immediate-
early promoter, and pCMV-mSB expressing an inactive
variant of the SB transposase have been described previ-
ously [19]. The pEGFP-N1 plasmid contains a variant of
GFP, EGFP, driven by a CMV immediate-early promotor
(CLONTECH Laboratories, Inc., USA).
NHDF were purchased from Cambrex Corporation. The
cells were cultivated in Dulbecco' s modified Eagle
medium (Invitrogen) supplemented with 10% fetal
bovine serum (GIBCO) and 1% Penicillin-streptomycin
(GIBCO).
Transfections
Cells that had reached 50–80% confluency in 25 cm2 flask
were transfected with plasmids pBabepuroInt and pBa-
bepuromInt alone, or pBabepuro and pBabepuroatt con-
taining integrase and/or the recognition site by using
FuGene 6 transfection reagent (Roche) at a ratio of 3 μl of
FuGene 6 transfection reagent per μg of DNA. For stable
transfection cells were subjected to puromycin selection
(5 μg/ml) 24 hrs after transfection.
PCR and RT-PCR analysis of integrase
Genomic DNAs were extracted from transfected cells
using a Genomic DNA purification kit (Gentra Systems).
RNA was extracted using RNeasy Mini Kit (Qiagen). DNA
was analyzed by polymerase chain reaction using primers
for the integrase gene, forward primer 5'-ACTCGACCACT-
TCCCTTACC-3' and reverse primer 5'-ACCACGCCT-
GAAGCTCATAC-3'. RNA was isolated by an RNeasy kit
(Qiagen) and reverse transcribed to cDNA using first
strand cDNA synthesis kit for RT-PCR (AMV) (Roche).
Subsequently, the cDNA was subjected to PCR analysis
using the primers mentioned above.
Cytogenetic analysis
Metaphase chromosomes were analyzed by standard Q-Page 6 of 8
(page number not for citation purposes)
The plasmid pBabepuro containing a puromycin selection
marker has been described before [18]. The plasmid pBa-
banding techniques 15–17 days after transfection. The
description of chromosome aberrations was based on the

BMC Biotechnology 2009, 9:31 http://www.biomedcentral.com/1472-6750/9/31
recommendations of the International System for Human
Cytogenetic Nomenclature (ISCN 1995) [20].
Immunofluorescence analysis
Cells were grown on 12 mm glass coverslips in 6 well
plates. After transient transfection the coverslips were
briefly washed in cold PBS, fixed for 10 min at room tem-
perature with ice cold ethanol/methanol (1:1, v:v). After
drying, the coverslips were stained using anti-γ-H2AX
antibodies (Abcam, Cambridge, MA, USA) and Texas red-
anti-rabbit secondary antibodies (Abcam) with 10 min
PBS washing steps and finally evaluated by epifluores-
cence microscopy.
Alternatively, when detection of fluorescent proteins in
transfected cells was needed, a paraformaldehyde fixation
method was used. Briefly, cells for ranking of γ-H2AX
expression were grown in 4-chamber Labtech glass slides
(NUNC, Denmark). Three days after transfection with
pIntegrase+pEGFP-N1+pBabepuroattB, pIntegrase-
mut+pEGFP-N1+pBabepuroattB, and pCMV-SB+pEGFP-
N1+pBabepuroattB, respectively (2:2:1), the cells were
briefly washed in PBS and fixed for 15 minutes in cold 2%
paraformaldehyde. Cells were permeabilised using 0.2%
saponin in the blocking buffer (PBS, 1% BSA). Staining
was done using anti-γ-H2AX antibodies (Abcam) 1:1000
and secondary anti-rabbit Alexafluor 594 (Invitrogen
Inc.) diluted 1:1000. Slides were mounted in Vectashield
and counterstained with 4', 6-diamidino-2-phenylindole
(DAPI; Vector Laboratories, Burlingame, CA) and ana-
lysed using a Fluoview FV1000 confocal microscope
(Olympus). Empirical ranking of staining intensities were
scored as 1 to 3, 3 being the very intensive nuclear stain-
ing, 1 being the vague but yet defined nuclear staining.
Cells were ranked in 48 to 68 independent windows. In
total 115 (pCMV-Int), 182 (pCMV-SB) 75 (pCMV-mInt)
and 1174 (no transfection) cells were monitored and
scored.
p53 and p21 were measured using immunofluorescence
(Abcam, Cambridge, UK) and apoptosis detected using an
In Situ Cell Death Detection Kit (Roche).
Western blot analysis
At day 3 after transfection, cells were washed twice in PBS
and lysed using Laemmli sample buffer as described by
Harlow and Lane, Antibodies, 2nd ed. Briefly, aliquots
were boiled at 95°C for 10 min and loaded onto 12% acr-
ylamide gels. After eletrophoresis, proteins were trans-
ferred to HybondECL membranes (GE Healthcare) by
semidry transfer. The membrane was blocked with PBST
(PBS containing 0.1% Tween 20) containing 5% nonfat
milk for 60 minutes before incubation with 50 ng/mL
was washed in PBST and then incubated with horseradish
peroxidase-conjugated anti-rabbit antibody (1/1000)
dilution for 1 hr at the room temperature and visualized
by chemiluminescence using ECL Plus Western Blotting
Detection System (GE Healthcare).
Authors' contributions
JL performed the plasmid constructions, most transfec-
tions, western blotting and immunofluorescence. TS per-
formed the experiments with co-transfection with EGFP
including analysis of H2AX phosphorylation in these
experiments. TG supervised JL and TS and performed the
statistical analysis. JL drafted the manuscript along with
TS and TGJ. TGJ participated in experimental designs and
finalised the manuscript. All authors read and approved
the final manuscript.
Acknowledgements
Iben Jeppesen and Kate Nielsen are thanked for analysis of the karyotypes.
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