Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran
Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR,
Department of Biology, Facualty of Sciences, Uneversity of Isfahan, Isfahan, Iran
Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
P.O. BOX: 8165131378
Department of Reproductive Biotechnology
Reproductive Biomedicine Research Center
Royan Institute for Biotechnology
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Sekhavati Mohammad Hadi,
Hosseini Sayed Morteza,
Nasr-Esfahani Mohammad Hossain.
PhiC31-based Site-Specific Transgenesis System for Production of
Transgenic Bovine Embryos by Somatic Cell Nuclear Transfer
and Intracytoplasmic Sperm Injection.
The Streptomyces phage phiC31 integrase offers a sequence-specific method of transgenesis with a
robust long-term gene expression. PhiC31 has been successfully developed in a variety of tissues and organs for
purpose of in vivo gene therapy. The objective of the present experiment was to evaluate PhiC31-based site-specific
transgenesis system for production of transgenic bovine embryos by somatic cell nuclear transfer and intracytoplasmic
Materials and Methods
In this experimental study, the application of phiC31 integrase system was evaluated for
generating transgenic bovine embryos by somatic cell nuclear transfer (SCNT) and sperm mediated gene transfer
PhiC31 integrase mRNA and protein was produced in vitro and their functionality was confirmed. Seven
phiC31 recognizable bovine pseudo attachment sites of phage (attP) sites were considered for evaluation of site specific
recombination. The accuracy of these sites was validated in phic31 targeted bovine fibroblasts using polymerase chain
reaction (PCR) and sequencing. The efficiency and site-specificity of phiC31 integrase system was also confirmed in
generated transgenic bovine embryo which successfully obtained using SCNT and SMGT technique.
The results showed that both SMGT and SCNT-derived embryos were enhanced green fluorescent
protein (EGFP) positive and phiC31 integrase could recombine the reporter gene in a site specific manner. These
results demonstrate that attP site can be used as a proper location to conduct site directed transgenesis in both
mammalian cells and embryos in phiC31 integrase system when even combinaed to SCNT and intracytoplasmic sperm
injection (ICSI) method.
Genetically engineered (transgenic) animals hold
promising applications in biomedicine and agriculture.
Recently, transgenic animal models have become a key
tool in functional genomics to understand the initiation
and perpetuation of human diseases. Moreover, they
are an invaluable system for large scale production of
therapeutic proteins (1, 2). The contemporary methods
that are used for production of transgenic animals include
intra-pronuclear zygotic DNA microinjection and somatic
cell nuclear transfer (SCNT). DNA microinjection into the
male pronucleus of a zygote is well-established in rodents
(3), SCNT is an obvious choice of transgene delivery
method in farms mammalian species because their
zygotes are optically opaque, due to the presence of lipid
granules in the cytoplasm; which makes the pronuclear
microinjection difficult and inefficient (4, 5). Recent
developments in studies of sperm-mediated gene transfer
(SMGT) suggested that sperm cells can be considered
vectors to transfer DNA into the oocyte during in vitro
fertilization (IVF) or intra cytoplasmic sperm injection
(ICSI) (6-8), but also suggests that the final fate of the
exogenous sequences transferred by sperm is not always
predictable (6). Since the highly condensed structure
of sperm chromatin makes it virtually inaccessible to
foreign molecules, we previously showed that in vitro
decondensation of bovine sperm with heparin and
glutathione (GSH) not only remarkably increase the
efficiency of ICSI, but also provided new insights for in
vitro transfection of sperm cells before being used for
Classical methods for generating of transgenic animals
usually integrates an uncontrolled number of transgene
copies into random genomic sites (10). Transgenes which
generated by this method are additionally susceptible to
transgene silencing due to position site-dependent effects
or tumor activation which caused by transgenesis near to
oncogenes (11, 12). Although retroviruses and transposons
would improve the efficiency of single-copy transgenesis,
uncontrollability of the integration copy number is still a
major limitation (13). Homologous recombination targets
the transgene to a specific genomic site, but targeting loci
by homologous recombination in technically demanding
and time consuming (14) and the final efficiency is
presently extremely low in mammalian cells (15). These
problems can be overcome by recently developed hybrid
nuclease technologies including zinc-finger (ZFN),
transcription activator-like effector nuclease (TALEN)
and CRISPR associated protein 9 (Cas9), but it is still
challenging to screen nucleases with high affinity and
specificity (16). Therefore, developing of an alternative
molecular tools which introduce site-specific transgene
integration with robust gene expression are still a problem
for site-directed transgenesis.
The streptomyces phage PhiC31 integrase has been used
as a powerful tool to carry out irreversible and unidirectional
recombination between attachment sites of phage (attP) and
bacteria (attB) genomes (17). Interestingly, these prokaryotederived
integrases have been successfully used to target
transgene to specific sites in eukaryotic cells of several
species (2, 14, 18-21). This system can integrate the whole
plasmid harboring attB sequence into the preferred locations
in mammalian genome which so called pseudo attP reviewed
by Calos (22). Pseudo sites are naturally present in the region
of open chromatin (23). Transgene expression in these sites is
robust compared to random integration (24, 25). Importantly,
the number of pseudo attP sites is estimated in the range of
100-1000 sites in mammalian genome (26, 27). For example,
in bovine genome, 36 pseudo attP sites have been recognized
so far by phiC31 integrase system (28-31). As bovine is an
economically important farm animal, we introduced three
new attP site within bovine genome and then demonstrated
that these new pseudo attP sites are in favor of enhanced
green fluorescent protein (EGFP) transgene expression in
bovine embryos produced by SCNT and SMGT.
Materials and Methods
In this experimental study, unless otherwise specified,
all chemicals and media were obtained from Sigma
Chemical Co. (St. Louis, MO, USA) and Gibco (Grand
Island, NY, USA), respectively. All animal experiments
and procedures described in this study were approved
by the Royan Institute Animal Ethics Committee (No.
The pCMVInt and the pBCPB+ vectors were kindly
gifted by Professor M.P. Calos (Stanford University). The
pCMVInt contained a phiC31-cDNA site sequence and
pBCPB+ contained att site sequence. These vectors also
contained EGFP under the control of the cytomegalovirus
(CMV) promoter, the SV40 promoter driving the
neomycin (G418)-resistance marker, and the phiC31 attB
site. PhiC31 cDNA was cloned into a pET15b vector
(Novagen, USA) as follows: pCMVInt vector containing
phiC31 was linearized by KpnI and then digested product
blunted using klenow fragment (Thermo, USA). In the
second step, the cDNA of phiC31 excised from linearized
and blunted pCMVint by BamHI. In parallel, PET15b
plasmid (Novagen, CA, USA) was linearized and blunted
by NdeI and klenow fragment, respectively. Linearized
pET15b was digested by BamHI. Finally, linearzied and
blunted pET15b backbone and phiC31 open reading frame
were gel extracted and ligated using DNA Ligation Kit
(Takara, Japan). The pET-phiC31 expression plasmid was
amplified in a DH5α strain of E. coli. (Invitrogen, USA).
We confirmed PhiC31 integrase cDNA by sequencing
and also expressed and purified integrase protein in
the E.coli by using the Ni2+-agarose columns (Qiagen,
CA) as described previously (20). To construct the
pUC19phiC31polyA vector, phiC31 cDNA was amplified
by polymerase cjain reaction (PCR) from pCMVInt and
then cloned into the pUC19 vector. The PhiC31 integrase
cDNA was amplified from the pCMVInt using:
The procedure of PCR is as follows: 94°C for 5 minutes
as an initial denaturation step, followed by 27 repetitive
cycles at 94°C for 30 seconds, 65°C for 45 seconds,
and 72°C for 60 seconds. Final extension of 72°C for 5
minutes was performed at the last stage. PCR products
were subjected to electrophoresis on 1% (w/v) agarose
(CinnaGen, Iran), purified from agarose gel (Promega,
USA) and then cloned into the TA vector (InsTAcloneTM
PCR Cloning Kit, Thermo, USA). The pTZphiC31polyA
construct digested with XbaI enzyme and cloned into an
XbaI digested pUC19 (Promega, USA). All restriction
enzymes were purchased from Thermo (USA).
It has been suggested that CMV promoter may be prone
to silencing mediated by de novo methylation during
zygote genome activation in early embryos (32). To
overcome this possible problem, a new vector containing
eukaryotic elongation factor 1 alpha driven EGFP
(EGFPEF1 alpha vector) was constructed by replacing
the CMV promoter with EF1 alpha promoter in the pDB2
vector. Briefly, EF1 alpha promoter was amplified using
five prime linked primers (Table 1,, Table EF1 and EF2 primers,)
with AseI and NheI restriction enzymes when pBudCE4.1
plasmid (Invitrogen, USA) was used as template in PCR
reaction. Subsequently, the full length of EF1 alpha
promoter was ligated into the pDB2 plasmid digested by
AseI and NheI.
Assessment of the phiC31 protein and mRNA
To assess the phiC31 protein functionality, 1 µg of pB-
CBP+ plasmid was incubated with 1 µg purified phiC31
integrase protein in a reaction buffer (pH=8.5, was comprised
of 20 mM HEPES 100 mM KCl, 10 mM dithiothreitol,
0.01% bovine serum albumin (BSA) at 30°C for 1
hour. Subsequently, PCR was conducted for screening of
site-specific recombination junction using:
primers. In parallel, for assessment of the phiC31 mRNA activity,
PhiC31 RNA and pBCBP+ vector were diluted in microinjection
TE buffer to a final concentration of 10:100 ng/µland micro injected into bovine in vitro matured (MII) oocytes(n=50) which were prepared as described later in this manuscript.
For positive control, pCMVInt was replaced to phiC31mRNA in a parallel micro injection experiment. Injected oocytes
were chemically activated and cultured for 48 hours in
vitro as described later in this manuscript. Two cell embryos
were lysed using CelLytic M (Sigma, USA) and screened forsite-specific recombination using PCR. The process of spermchromatin in vitro decondensation was as described previously
DNA labeling and incubation of sperm cells with
plasmid DNA and in vitro decondensation of sperm
In order to track the uptake and localization of the DNA bysperm cells, pDB2 plasmid was labeled by CX-Rhodamineusing IT® Tracker™ Intracellular Nucleic Acid Localization
Kit (Mirus, USA) according to manufacturer’s guideline
(Fig .1,). In brief, commercial frozen sperm from three different
bulls was thawed and pooled together. Completely motile
bovine sperm were obtained by centrifugation of thawed and
washed semen over discontinuous layers of PureSperm®
gradients. Motile sperm were washed with tissue culture
medium 199 (TCM-199) (10 minutes, 1000×g), the sperm
pellet resuspended in 30 µl TCM-199 and then a population of
1×106 sperm cells were incubated with 200 ng labeled plasmid
for 30 minutes at room temperature (RT). Labeled sperm
cells were co-incubated in a new tube containing heparin (80
mM) and GSH (15 mM) for 7 hours at 39°C, 20% O2 and
6% CO2. DNA-uptake by sperm cells was assessed at ×100
magnification of a fluorescent microscope (Olympus BX51,
Japan). Upon exposure to UV light (excitation and emission
are needed), a digital image of each sample was taken with a
high sensitive camera (Olympus DP-72) operated on DP2BSW
- Head sperm DNA uptake, sperm mediated gene transfer and screening
for EGFP expression in SMGT derived embryos. A. Sperm decondensation
and exogenous DNA uptake, I. Phase contrast microscopy observation for
decondensedsperm, II. Hoechst staining for decondensedsperm, III. The
pattern of DNA uptake by decondensedsperm. This pattern depicted by arrowseither over acrosomal ridge or in the post-acrosomal region, B. SMGT derived
embryos, I. Blastocysts formation by injection of sperm which incubatedwith 200 ng pDB2 for 30 minutes at RT, II. Blastocysts formation by injectionof sperm which incubated with 1000 ng pDB2 for 30 minutes at RT, and C.
RT-PCR for detection of EGFP expressionin SMGT derived embryos. Lane 1:
DNA marker, I. SMGT derived embryos obtained by injection of sperm whichincubated by 200 ng pDB2 for 30 minutes at RT, and II. SMGT derived embryosobtained by injection of sperm which incubated by 1000 ng pDB2 for 30
minutes at RT (scale bars: 15 µm).
SMGT; Sperm mediated gene transfer and RT-PCR; Real time-polymerase chain reaction.
Oocyte preparation and in vitro maturation
The procedure of in vitro maturation (IVM) was performed
as described previously (33). In brief, cumulus-oocyte
complexes (COCs) were aspirated from antral follicles (2-8mm) of abattoir-derived ovaries using 18-gauge needlesattached to a vacuum pump (80 mmHg). COCs with
homogeneous cytoplasm and more than three layers ofcumulus cells were then incubated for 24 hours in maturation
medium [TCM-199 supplemented with 2.5 mM sodium
pyruvate, 100 IU/mL penicillin, 100 µg/mL streptomycin,
1 mg/mL estradiol-17ß, 10 µg/mL follicle-stimulating
hormone (FSH), 10 µg/mL luteinizing hormone (LH), 100ng/mL epidermal growth factor (EGF), 0.1 mM cysteamine,
and 10% fetal calf serum (FCS)] at 38.5°C in a humidified
atmosphere of 6% CO2 in air.
Intracytoplasmic sperm injection and artificial oocyte
Labeld and decondensed sperm cells were used for
ICSI according to Sekhavati et al. (9). Artificial activation
of injected oocytes was performed according to Nasr-
Esfahani et al. (34) with minor modifications. Briefly, 20
minutes after ICSI, oocytes were activated using calciumionophore
(5 µM for 5 minutes) prepared in HEPES-tissue
culture medium 199 (H-TCM99) plus 1 mg/ml BSA in
the dark, followed by washing in H-TCM199 plus 3 mg/
ml BSA. Activated oocytes were incubated in a modified
formulation of synthetic oviduct fluid (mSOF) left to rest
for 3 hours before being incubated in 2 mM 6-dimethyl
aminopurine (6-DMAP) for 4 hours. Oocytes cultured in
mSOF medium at 38.5°C, 6% CO2, 5% O2, and maximum
humidity for 8 days.
In vitro RNA production and microinjection into the
Capped phiC31 integrase RNA was generated by
transcription of pUC19-phiC31polyA vector using the
Transcript AidTM T7 High yield kit (Thermo, USA) and
m7G(5´)ppp(5´)G RNA Cap (Biolabs, UK). The integrity
of the RNA was assessed by electrophoresis on a 1%
agarose gel. Before loading on the gel, the RNA was
denatured by using the loading buffer provided in the
Thermo kit according to the manufacturer’s instruction.
PhiC31 RNA was diluted in microinjection TE buffer
(10 mM Tris and 0.1 mM EDTA, pH=7.4) to a final
concentration of 10 ng/µl. For microinjection, two rounds
of oocyte microinjection were conducted. In first round,
phiC31 RNA was injected into the cytoplasm of each MII
oocyte. In the second round, completely decondensed
sperm cells were incubated with pDB2 plasmidand used
for microinjection into the oocyte. Microinjected oocytes
were artificially activated and cultured for embryodevelopment as described above for ICSI oocytes. In this
study, ICSI with non-transfected decondensed sperm cells
was considered as control.
Somatic cell preparation and transgenesis
Primary bovine fetal fibroblast (BFF) cell line was
established from a 65-day old female fetus conceived by
natural mating as follows: primary bovine fetal fibroblast
culture derived from a natural mating was established
by isolating the cell from a 65-day old fetus. The skin of
the fetus was extensively washed in Ca2+ and Mg2+ free
phosphate buffer solution (PBS) containing 1% (v/v) of
a cocktail of penicillin-streptomycin and amphotericin B.
Then, the sample was cut into 2-3 mm pieces. The explants
were cultured in Dulbecco’s modified Eagle medium
F-12 (DMEM/F-12) containing 10% FCS, 1% penicillin-
streptomycin and amphotericin B at 37°C in a humidified
atmosphere of 5% CO2 until reaching 90-95% confluence.
Cells were passaged twice, and then frozen, thawed and
passaged in liquid nitrogen prior to transfection.
The procedure of primary cell culture was as described
previously (35). In brief, A population of 2×105 BFF was
cultured in 6-well tissue culture plates (Orange Scientific,
Switzerland) containing DMEM/F-12 enriched with 10%
FCS. BFFs at 60-70% confluence were co-transfected
by 1 µg pDB2 and 1 µg EGFP-EF1 alpha plasmids with
3 µg pCMVInt (1:3 ratio) using Lipofectamine2000
(Invitrogen, USA) according to the manufacturer’s
instruction. Six hours following transfection, the medium
was changed with fresh culture medium for 24 hours
before being distributed colony selection culture dishes
(Falcon, 1005, Germany). Forty eight hours post culture,
cells were treated with 400 µg/ml G418 for 21 days
and developed colonies were isolated for subculture
asdescribed previously. An established BFF line which
was previously obtained by transfection of EGFP-OCT4
plasmid (www.royaninstitute.org) containing a neomycin
resistance gene, via lipofectione2000 according to
Jafarpuor et al. (35). To assess the expression of EGFP in
colony cells derived following G418 treatment, cells were
visualized and observed under a fluorescent microscope
(Olympus BX51, Japan). To detect the nuclei, cell were
stained with Hoechst 33342 before observation. The long
term ectopic expression of EGFP in the first two cell lines
was evaluated during 5 weeks following colony selection
using fluorescence microscopy observation. These clones
were trypsinized and subcultured to prepare a monolayer
of stably transfected cells.
Identification of pseudo attP sites and polymerase
chain reaction screening for site specific integration in
To find possible new pseudo attP sites that could be
recognized by phiC31 integrase, an inverse PCR (IPCR)
approach was implemented as described. In brief, stably
transfected bovine fibroblasts were harvested and the
genomic DNA extracted by DNeasy Blood < Tissue
Kit (Qiagen, Germany). Five µg of genomic DNA was
digested with a couple of compatible enzymes, BglII
and BamHI. The aforementioned enzymes recognize
two different sites but produce similar cohesive ends.
Both enzymes cut at least one site in PDB2. The digested
fragments were extracted with phenol/chloroform and
precipitated with ethanol. It was important to use low
amounts of DNA for appropriate self-circulation of
digested DNA in the ligation reaction for efficient inverse
PCR. Thus, various amounts of DNA (0.5 to 5 ng) were
prepared and used for ligation using DNA Ligation Kit
(Takara, Japan) as describe in manufacturer protocol. The
half-nested PCR was performed across the left junction
of assumed recombination site. The circulated DNA was
used as a template for the first round of PCR utilizing
attB-F3: 5´-GTAGGTCACGGTCTCGAAGC-3´ primers.
1 µl of the first round PCR product was used in the
second round of PCR utilizing attB-F3 and EGFP-F
primers. The PCR steps were conducted as follows: 94°C
for 10 minutes as an initial denaturation step, followed
by 35 repetitive cycles at 94°C for 30 seconds, 56°C for
4 minutes, and 72°C for 2 minutes. Final extension of
72°C for 5 minutes was performed at last stage of PCR
and PCR products subjected to 1% (w/v) agarose. The
obtained bands from IPCR were purified and ligated into
T-vector and sequenced. To determine genomic location
of pseudo attP, the obtained sequences were analyzed
by BLAST search against bovine genome in various
databases. PCR screening for detection of site specific
recombination junction was carried out for 7 sites. Five
sites were those previously reported (28-30) and two
unknown sites which were detectedin the present study
using IPCR. Nested primers were designed (Table 1,).
Approximately 103 transfected cells were lysed by
freezing/thawing and a nested PCR was performed
for detection of site specific junction in transfected
fibroblasts and colonies selected under antibiotic
therapy. The first round of PCR (PCRI) program
was as follows: 94°C for 5 minutes as initial step of
denaturation, followed by repetitive 35 cycles of 94°C
for 30 seconds; 55°C for 30 seconds; and 72°C for 20
seconds, and a final extension period of 72°C for 5
minutes. PCRI products were used as template for the
second round of PCR (PCRII) with program as followed:
94°C for 5 minutes as initial step of denaturation,
followed by repetitive 35 cycles of 94°C for 30 seconds;
62°C for 30 seconds; and 72°C for 20 seconds, and a final
extension period of 72°C for 5 minutes. Direct sequencing
was performed using automatic DNA sequencing method
utilizing the same primers.
Somatic cell nuclear transfer
The process of zona-free SCNT was as described by
Oback et al. (36) with minor modifications. In brief,
denuded IVM oocytes were released from their zona
pelucida by brief incubation (up to 45 seconds) in 5 mg/
ml pronase dissolved in HTCM199 containing 10% FCS.
Enucleation of the oocytes were performed in phosphate
buffer saline free of Ca2+ and Mg2+ (PBS) supplemented
with 20% FCS, Na-pyruvate (2 mg/ml), BSA (1 mg/
ml), polyvinyl alcohol (PVA, 1 mg/ml) and glucose
(0.036 mg/ml). Zona-free oocytes were incubated in
enucleationmedium containing 5 µg/ml H33342 for 5
minutes before enucleation. Enucleation was carried
out at ×100 magnification of a pre-warmed microscopic
stage (Olympus, IX71, Japan) under UV exposure with
the help of blunt perpendicular break enucleation pipettes
(15-20 µm inner diameter). Nuclear transfer was carried
out using three cell types: two cell lines in which EGFP
gene was stably integrated into pseudo sites detected in
this study and one EGFP-OCT4 cell line. For cell cycle
synchronization at G0/G1, cells were cultured in presence
of 0.5% FCS for 4-5 days. Immediately before nuclear
transfer, a low density of somatic cells was prepared in
a drop of HTCM199+0.5% FCS containing 10 µg/ml
phytoheamoglutinin. Then, a group of 5 to 10 enucleated
oocytes were added to the droplet and each oocyte was
gently pushed attP over a single cell of population of
cells synchronized in G0/G1 stage of cell cycle by serum
starvation. The oocyte-donor cell couplets were placed
between two electrodes (0.5 mm apart), overlaid with a
hypo-osmotic fusion medium (0.2 M mannitol, 100 µM
MgSO4, 50 µM CaCl2, 500 µM Hepes, 0.05% BSA) and
aligned first manually and then by application of AC
current (7 v/cm, 1000 kHz, for 10 seconds). Fusion was
induced by two successive DC currents (1.75 kv/cm, 30
µseconds with 100 µseconds interval) fused couplets
were kept in maturation medium containing 0.0 and 1.0
mM SAH for 1-2 hours. All fused embryos were further
activated, in brief, embryos were incubated with 5 µM
calcium-ionophore for 5 minutes followed by 4 hours
exposure to 2 mM 6-dimethylaminopurine dissolved in
TCM199 containing 10% FCS, 0.2 mg/ml PVA, 3 mg/ml
BSA plus 0.0 and 1.0 mM SAH. Activated reconstituted
oocytes were cultured in groups of ten in wells (36, 37)
drained in 10 µl droplets of mSOF embryo culture medium
at the same conditions described for ICSI embryos.
On days three and seven after fusion, the reconstructed
embryos were checked for cleavage and blastocyst rates,
respectively. In this study, SCNT with non-transfected
somatic cells was considered as control.
Polymerase chain reaction screening for site specific
integration in transgenic embryos
For screening of site specific recombination junctions
in transgenic embryos, EGFP positive embryos (selected
using fluorescent microscope) at day 8 of embryo
development were pooled and lysed by freezing/thawing.
Nested PCR was performed for detection of seven site
specific recombination junction.
RNA extraction and reverse transcription
Total mRNA was extracted from blastocysts at day 8 of
embryo culture using the RNeasy Micro Kit (QiagenTM,
Germany) and subsequently, cDNAwas synthesized by the
RevertAidTM First Strand cDNA Synthesis Kit (Thermo,
USA) according to their manufacturer’s recommendation.
Real-time polymerase chain reaction
To detect the presence of EGFP mRNA in SMGT
derived embryos, real-time polymerase chain reaction
(RT-PCR) was conducted using
rEGFP-R: 5´GTGCTCAGGTAGTGGTTGTC-3´ primers.
In this regards, cDNA from SMGT derived embryos were
subjected to RT-PCR with following programs: 94°C
for 5 minutes as an initial denaturation step, followed
by 35 repetitive cycles at 94°C for 30 seconds, 60°C for
30 seconds, and 72°C for 20 seconds. Final extension of
72°C for 5 minutes was performed at last stage of PCR.
Identification of phiC31-mediated recombinant sites
in bovine genome
Bovine fibroblasts after successful co-transfection with
pCMVInt and pDB2, EGFP-EF1 alpha showed constant
EGFP expression throughout 11 passages (Fig .2,). IPCR
detected three new pseudo attP sitesin bovine genome
which were named BF5, BUN1 and BUN2 with 32, 16
and 48% of identity with wild type of attP sequence. To
screen the recombinant sites which were mediated by
phiC31 integrase, we designed nested primers for BF5,
BUN1 and BUN2 as well as four other recombinant
sites which previously reported in bovine genome
(Table 1,) (28, 29). Nested PCR amplified whole
expected recombinant sites with exception of BpsM1
site in co-transfected bovine fibroblasts by pCMVInt
and pDB2 vectors (Fig .3,). During colony selection
procedure, only two individual cell clones with EGFP
integrated into the BF4 and BF10 sites were selected
for each co-transfection strategy (CMVInt-pDB2 and
CMVInt-EGFP EF1 alpha) (Fig .2,).
- Microscopic observation in bovine fibroblast cells and by somatic cell nuclear transfer (SCNT) derived embryos. A. EGFP expression under two
different promoters regulation in transgenic bovine fibroblast cells and SCNT derived embryos which obtained by phiC31 integrase systems in BF4 and
BF10 pseudo sites, I. From left to right, column 1; Nested PCR product for detection of the BF4 and BF10 sites, column 2; Targeted stable transgenic bovine
in BF4 and BF10 sites which obtained by co-transfection of pCMVInt and pDB2, column 3 and 4; Phase contrast and fluorescence microscopic observation
from SCNT embryos obtained by BF4 and BF10 targeted bovine fibroblast cells, respectively, II. Nested PCR product for detection of the BF4 and BF10
sites, column 2; Targeted stable transgenic bovine in BF4 and BF10 sites which obtained by co-transfection of pCMVInt and EGFP-Ef1 alpha, column 3 and
4; Phase contrast and fluorescence microscopic observation from SCNT embryos obtained by BF4 and BF10 targeted bovine fibroblast cells, respectively,
B. EGFP expression under OCT4 promoters in transgenic bovine fibroblast cells and its SCNT derived embryos according to Jafarpour et al. (35) column 1
and 2; Phase contrast and fluorescence microscopic observation of EGFP-OCT4 cell line, respectively and column 3 and 4; Phase contrast and fluorescence
microscopic observation from SCNT embryos obtained by EGFP-OCT4 cell line, and C. Genomic PCR for amplification of complete EGFP Open Reading
Frame in EGFP positive bovine cells and embryos (scale bars: 100 µm).
- Identification of recombinant sites created by phiC31 integrase in bovine genome.
T; Transfected bovine fibroblast and UT; Untransfected bovine fibroblast.
Vector assay integration
To evaluate the phiC31 integrase functionality, we used
pBCPB+ as an intra-molecular assay vector. This vector
carries phiC31 attP and attB sites in direct orientation
flanking with a LacZ gene. SincephiC31 integrase has
a precise target recombinant activity; it can distinguish
two att sites on pBCPB+ and delete the LacZ sequence.
The recombinant site could be identified by a PCR
reaction with specific primers that can amplify a 401 bp
product as a detector for phiC31 site specific activity.
The positive recombination control was performed by
in vitro incubating pBCPB+ vector with a crude protein
extract from integrase-expressing E.coli (20). In vitro
produced phiC31 protein had precise activity and PCR
reaction amplified an expected 401 bp product when
reaction buffer was used as a template. In parallel, PCR
on co-injected oocytes by phiC31 mRNA and pBCPB+
also amplified an expected 401 bp product, indicating that
recombination has occurred for integrase-mediated site-
specific recombination between attP and attB. Therefore,
in vitro produced phiC31mRNA had accurate activity in
bovine oocyte cytoplasm.
Exogenous DNA uptake by heparin-glutathione
After incubation with exogenous DNA, sperm cells
were treated with heparin-GSH which resulted in three
types of decondensation pattern according to Sekhavati
et al. (9). Assessment of these decondensed sperm cells
under fluorescent microscope indicated that spontaneous
uptake of labeled pDB2 plasmid was mostly confined to
the head of spermatozoa. The pattern of DNA uptake was
observed either in acrosomal ridge or in the post-acrosomal
region (Figes.1A,, 3,). Interestingly, DNA uptake was not
influenced by the degree of sperm head decondensation
(data not shown).
Sperm mediated gene transfer and enhanced green
fluorescent protein expression assessment
EGFP expression was not detected after fluorescent
assessment of SMGT embryos obtained by injection of
sperm cells exposed to 200 or with 1000 ng of exogenous
pDB2 DNA. Microscopic observations showed that
morphological aspects of the SMGT-derived embryos
were not influenced by exogenous DNA concentration
(Fig .1B,). However, EGFP expression was detected at
mRNA level by real-time polymerase chain reaction (RTPCR)
in SMGT-derived embryos of both groups (Fig .1C,).
Targeted sperm-mediated gene transfer
PhiC31 mRNA may be transcribed in the cytoplasm
using cytoplasm transcription machinery and subsequently
active integrase could import into the nucleus where it
would possibly catalyze the target gene in the bovine
pseudo attP sites. Because the recombinant sites attR and
attL are not substrate for the phiC31 integrase, the reaction
is unidirectional. TSMGT combined with microinjection
of phiC31 mRNAresulted in eight EGFP positive embryos
out of 310 oocytes injected (approximately: 2.5%). The
majority of TSMGT-derived embryos had low quality but
the intensity of their fluorescent EGFP was considerable
(Fig .4A,). The results of PCR screening for seven possible
recombinant junctions, which could be generated by
phiC31 integrase system in EGFP positive embryos,
showed that just BF10 pseudo attP site was amplified
in the expected size (Fig .4B,). Subsequently, sequencing
the amplified fragment confirmed the presence of BF10
pseudo site in pooled EGFP positive derived embryos by
Targeted somatic cell nuclear transfer-mediated gene
EGFP signal was clearly observed in fibroblasts
transfected with either CMV-EGFP or EF1-EGFP
vectors (Fig .2,).Targeted transfection had no apparent
effect on the competence of the reconstituted oocytes
to cleave and to further develop to the blastocyst stage
compared to control (Table 2,). EGFP signal i. Was not
detected in any stage of SCNT embryos reconstructed
with CMV-EGFP transgenic fibroblasts, ii. Was clearly
observed throughout in vitro development of EF1EGFP
reconstructs, and iii. Was observed only after
8-16 cell stage in SCNT embryos reconstructed with
OCT4-EGFP transgenic fibroblasts.
- Targeted sperm mediated gene transfer using phiC31 system and PCR screening for detection of recombinant junction. A. Green positive bovine
TSMGT derived embryos in deferent stage of pre-implantation development and B. Nested PCR screening for identifying the likely recombinant junction
which could be created by phiC31 integrase system.
A; Green positive bovine TSMGT derived embryos, B; SMGT derived embryos, and C; Negative control without DNA in PCR reaction.
The rate of cleavage and blastocyst formation in SCNT derived embryos using transfected and non-transfected cells
SCNT control (non-transfected)
91 (84.26 ± 4.5)
30 (32.97 ± 5.3)
SCNT transfected (BF10 )
122 (90.4 ± 6.1)
34 (27.87 ± 3.9)
SCNT transfected (BF5)
107 (85.5 ± 4.7)
32 (29.99 ± 3.9)
SCNT; Somatic cell nuclear transfer and BF10; Bovine fibroblast chromosome10.
This study introduced that the two new bovine pseudo
attP sites are in favor of site directed transgene expression.
It was also demonstrated that phiC31 integrase can be
used for production of transgenic bovine embryos by
SCNT and SMGT techniques. Connected to two most
routine techniques of animal transgenesis site-specific
transgenesis system for efficient generation of bovine
embryos carrying targeted reporter gene. Therefore,
these results in agreement with other studies in bovine
(28-31) show that attP inclusion into a selection cassette
can be used as a powerful tool to conduct site directed
transgenesis in mammalian cells and embryos.
In our study, completely decondensed sperm still had
the ability to store the exogenous DNA. By incubation of
sperm cells decondensed with an egg extract with linear
DNA, Ishibashi et al. (38) successfully produced transgenic
Xenopus. Even though, our results revealed that sperm
chromatin decondensation with heparin-GSH improved
development of ICSI-SMGT embryos (data not shown),
EGFP expression was detected only at mRNA level but
there was no any detectable EFGP protein in blastocyte.
This is consistent with the previous reports that SMGT-
derived bovine embryos are not able to express EGFP
protein and only EGFP mRNA is detectable (38, 39).
When cytoplasmic injection of phiC31 mRNA was
carried out with the aim of gene targeting before
TSMGT,only 8 green positive embryos out of 310
oocytes injected. This low efficiency of is compatible
with the report of Hoelker et al. (40) who detected only
3.6% transgenic bovine embryos following conventional
SMGT. Screening for recombinant junction by nested PCR
amplified BF10 junction in TSMGT-derived embryos.
This pseudo attP site was previously reported by Qu et al.
(29) as a preferred site recognized by phiC31 integrase in
bovine genome. Sequencing also confirmed the presence
of BF10 junction sequence in TSMGT derived embryos.
It has been shown that the half-life of phiC31 integrase in
liver cells is about 6 hours and a small fraction of active
integrase may gain access to the nucleus, whereas the
bulk being cytoplasmic (40, 41). So, it is likely that the
injected phiC31 mRNA could be translated into protein
and reached to the nucleus for site specific recombination
of donor plasmid harboring attB into bovine pseudo attP
sites. Indeed, it seems that phiC31 integrase system has a
proper potential for gene targeting in the SMGT protocol.
To investigate whether the EGFP expression is resulted
from stable gene integration or extra chromosomal
expression, we carried out SCNT experiments with stable
targeted (for BF10 and BF5 sites) transgenic (for CMVEGP
and EF1-EGFP) fibroblasts. Interestingly, none of
the cloned embryos showed EGFP expression under CMV
promoter regulation but EGFP was expressed successfully
and efficiently under EF1 promoter regulation. Control
SCNT embryos carrying EGFP-OCT4 showed EGFP
signals only at the morula and blastocyst stages. This
results may lend support for the notion that de novo
methylation of pre-implantation embryo development can
silence integrated viral DNA (32, 41). It seems that green-
positive embryos derived by TSMGT have expressed
EGFP as an extra-chromosomal gene which remained
unaccessible to the de novo methylation machinery of
embryo while remains non-integrated (32). Accordingly,
detection of EGFP signal in almost all cloned embryos
carrying EF1-EGFP and OCT4-EGFP could be due to the
lack of any viral DNA in their plasmid structure.
PhiC31 has been successfully used for site-directed
transgenesis in a variety of tissues and organs under in
vivo and ex vivo conditions in several species. This study
introduced that the two new bovine pseudo attP sites
are in favor of site directed transgene expression. It was
also demonstrated that phiC31 integrase can be used for
production of transgenic bovine embryos by SCNT and
SMGT techniques. Therefore, these results in agreement
with other studies in bovine show that attP inclusion into
a selection cassette can be used as a powerful tool to
conduct site directed transgenesis in mammalian cells and
Authors would like to thanks HR Ghanaie for help
in figure preparation. The present study was funded by
Royan Institute. The authors declare that there is no
conflict of interest.
M.H.S.; Conception and design, collection and/
or assembly of data, data analysis and interpretation,
manuscript writing. S.M.H.; Conception and design, data
analysis and interpretation. M.T.; Conception and design,
data analysis and interpretation, manuscript writing,
final approval of manuscript. K.G., F.J.; Conception and
design, data analysis and interpretation. M.H., K.D.; Data
analysis and interpretation. M.H.N-E; Conception and
design, data analysis and interpretation. All authors read
and approved the final manuscript.
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