Efficient Flp-Int HK022 dual RMCE in
Eugenia Voziyanova1, Natalia Malchin2, Rachelle P. Anderson1, Ezra Yagil2,
Mikhail Kolot2,* and Yuri Voziyanov1,*
1School of Biosciences, Louisiana Tech University, 1 Adams Boulveard, Ruston, LA 71272, USA and
2Department of Biochemistry and Molecular Biology, Tel-Aviv University, Tel-Aviv 69978, Israel
Received November 8, 2012; Revised April 7, 2013; Accepted April 10, 2013
RMCE, is a clean approach of gene delivery into a
desired chromosomal location, as it is able to insert
only the required sequences, leaving behind the
unwanted ones. RMCE can be mediated by a
single site-specific DNA recombinase or by two re-
combinases with different target specificities (dual
RMCE). Recently, using the Flp–Cre recombinase
pair, dual RMCE proved to be efficient, provided
therelativeratio of the
reaction is optimal. In the present report, we
analyzed how the efficiency
mediated by the Flp–Int (HK022) pair depends on
amount of the recombinase expression vectors
added at transfection—and on the order of the
addition of these vectors: sequential or simultan-
eous. We found that both in the sequential and the
simultaneous modes, the efficiency of dual RMCE
was critically dependent on the absolute and the
relative concentrations of the Flp and Int expression
vectors. Under optimal conditions, the efficiency of
‘simultaneous’ dual RMCE reached ?12% of the
transfected cells. Our results underline the import-
ance of fine-tuning the reaction conditions for
achieving the highest levels of dual RMCE.
Random DNA insertions in the eukaryotic genome may
be undesirable owing to their mutagenic and position
effects. Moreover, if the activities of different alleles of
a certain gene are to be compared, random integration
of each allele may distort the results. Site-specific
recombinases that have become efficient tools for site-
specific gene manipulations can overcome these problems,
as they can promote gene insertions, deletions or inversion
at predefined loci of the eukaryotic genome. Among the
most popular site-specific recombination systems currently
used are the bacteriophage P1 Cre-lox and yeast’s Flp-
FRT that belong to the tyrosine family recombinases, as
well as the Int-att system of phage fC31 that belong to
the serine family recombinases. Each family is named after
the conserved nucleophylic residue in the active site of the
recombinase (1–4). In these systems, the site-specific inte-
grations of desired genes are catalyzed by a recombination
reaction between two specific short DNA target sites
(?30–40bp long), one located in a chromosome and the
other one, identical or similar, located on a plasmid that
carries the gene(s) to be inserted (Figure 1A). Such inte-
gration reaction, however, leads to the insertion of the
entire vector plasmid that may include undesired resist-
ance markers and/or replication origins. Moreover, in
the case of tyrosine-family recombinases mentioned
above, the insertion results in two identical tandem
target sites that flank the inserted DNA and are substrates
for the more efficient reverse excision reaction (stronger
arrow in Figure 1A).
A more elegant and ‘clean’ approach of gene insertions
is the recombinase-mediated cassette exchange (RMCE)
reaction (5) that is able to replace a plasmid borne gene
of interest with a genomic target leaving out undesired
plasmid sequences provided both DNA fragments are
flanked by a set of incompatible recombination targets
(Figure 1B). Moreover, the reversibility of this cassette
exchange reaction is low because the compatible recom-
bination sites are located on different DNA molecules.
The RMCE reaction can be catalyzed by one recombin-
ase. In this, original type of RMCE (6,7), the two incom-
patible target sites that flank the DNA fragments to be
exchanged are usually the respective cognate wild-type re-
combination target and its incompatible mutated version,
*To whom correspondence should be addressed. Tel: +1 318 257 2694; Fax: +1 318 257 4574; Email: email@example.com
Correspondence may also be addressed to Mikhail Kolot. Tel: +972 3 6409823; Fax: +972 3 6406834; Email: firstname.lastname@example.org
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
Published online 29 April 2013Nucleic Acids Research, 2013, Vol. 41, No. 12e125
? The Author(s) 2013. Published by Oxford University Press.
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both targets being substrates for the same recombinase:
homotypic targets. Alternatively, RMCE can be per-
formed by two recombinases. In this type of the replace-
ment reaction, dual RMCE, each target site in the pair of
the incompatible recombination targets that flank the re-
placement cassettes is recognized by the respective recom-
binase: heterotypic targets (8).
The introduction of new site-specific recombination
systems for eukaryotic gene manipulations is important,
in particular when these manipulations become more
complex, i.e. in cases in which a particular recombinase
becomes useless if its target site(s) has remained in the
genome owing to a previous manipulation. We have re-
cruited the site-specific recombination system of coliphage
HK022 to be active in human cells (9). This system, which
is similar to that of the better known coliphage ?, has two
different but compatible target sites: the 21bp attB site
that originates from the host (Escherichia coli) chromo-
some and the longer 230bp attP site that comes from
the phage chromosome. The phage-encoded integrase
(Int) catalyzes the integrative attP?attB site-specific re-
combination reaction that leads to the formation of the
recombinant attL and attR sites, which serve as substrates
for the Int-catalyzed excisive reaction. In the natural
E. coli host, as well as in in vitro reactions, both integra-
tion and excision reaction require accessory proteins
(10,11), which are dispensable when the system is
introduced into human and plant cells (12), though their
presence alleviates the reactions (13).
In preliminary dual RMCE reactions that were per-
formed in E. coli cells using HK022’s Int and yeast’s Flp
systems, we have shown that the most efficient RMCE
reaction occurs when the recombinases are supplied se-
quentially rather than simultaneously. In this mode, one
recombinase was supplied first to catalyze recombination
between one pair of target sites that led to the integration
of the entire incoming plasmid, followed by the supply of
the second recombinase that subsequently completes the
RMCE reaction (14).
In this article, we report results of experiments that test
the ability of Flp and HK022 Int to mediate a dual RMCE
reaction in mammalian cells in the sequential and simul-
taneous modes. To this end, we created a model Chinese
Hamster ovary (CHO) cell line that bears an integrated
together with the incoming reporter plasmid, makes up a
dual RMCE reporter system designed to detect separate
fluorescent signals from the Flp and Int recombination
events that allows monitoring of the activity of each re-
combinase in both the sequential and in the simultaneous
dual RMCE modes.
Using the CHO model system, we show that Flp and Int
can mediate dual RMCE in both sequential and simultan-
eous modes. We also show that the replacement reaction
performed in the simpler faster simultaneous mode can be
more efficient than the one performed in the sequential
mode. We found that the sequential ‘Flp-then-Int’ mode,
when Flp catalyzed the integration reaction while Int
catalyzed the deletion reaction, was less efficient than
the ‘Int-then-Flp’ mode, when Int catalyzed the integra-
tion reaction while Flp catalyzed the deletion reaction. We
also found that the efficiency of the dual RMCE reaction
in both sequential and simultaneous modes was critically
dependent on the absolute and the relative concentrations
of the Int and Flp expression vectors underlining the im-
portance of optimizing the parameters of the reaction for
reaching the highest levels of dual RMCE.
MATERIALS AND METHODS
Cell line and transfection
Derivatives of CHO cells, CHO TD-In cells (15), were
used as model mammalian cells. The cells were propagated
in F12-K medium. The medium for the intact CHO TD-In
cells was supplemented with zeocin (100mg/l). On integra-
tion of the platform reporter plasmid pNA1372plat, the
resultant CHO TD-1372 cells were grown in the medium
supplemented with hygromycin (550mg/l). Cell transfec-
tions were performed using Polyfect (Qiagen).
The expression vector for Flp recombinase, pOG-Flp, is a
derivative of pOG44 (Flp-In system, Invitrogen), in which
Flp(F70L) is replaced with Flpe. The construction of
pOG-Flp is described in Anderson et al. (15).
HK022 oInt, IHF and Xis were pNA979oInt, pIHF2cP
and pNA998, respectively (13).
Both the platform and the incoming reporter vectors are
based on the vector pcTD (15), which is a derivative of the
pcDNA5/FRT vector of the Flp-In system (Invitrogen).
To construct the platform plasmid pNA1372plat, the
CMV promoter of pcTD was replaced by an MluI-KpnI
fragment carrying the EF-1a promoter, following by
cloning of a polymerase chain reaction (PCR)-generated
Figure 1. Comparison between integration and cassette exchange reac-
tions to deliver a gene of interest into a desired genome location. (A) A
single site-specific recombination reaction integrates a gene of interest
along with entire vector sequences. (B) RMCE integrates just a gene of
interest. GOI, gene of interest; gt, genomic target; AbR, antibiotic re-
sistance gene; RT, recombination target site; RTa and RTb, incompat-
ible recombination target sites that can be recognized either by a single
recombinase or by two different recombinases.
e125Nucleic Acids Research, 2013,Vol.41, No. 12PAGE 2 OF 8
KpnI-EcoRV FRT-neoRfragment and an EcoRV-NotI
To construct the incoming pNA1345inc plasmid, the
NheI-MluI CMV fragment was deleted from the pcTD
vector, followed by cloning of a PCR-generated FRT-
EGFP fragment between the NheI and XhoI sites and a
CMV-attR fragment between the RsrII and SacII sites.
Plasmid CMV-LEF (15) that expresses EGFP under the
CMV promoter was used to estimate the rate of transfec-
tion in the RMCE experiments. CMV-LEF has the same
backbone and similar size as the incoming reporter vector
Construction of CHO TD-1372 cell line
To construct CHO TD-1372 cell line, CHO TD-In cells
pNA1372plat and pOG-TD1-40 (15), which expresses
the TD1-40 variant of the TD recombinase (16). The
transfection was done in six-well plates. The amount of
the reporter and the expression plasmids added at trans-
fection was 0.2 and 2mg, respectively. Forty-eight hours
after transfection, one-sixth of the cells were transferred
into a 100-mm plate into the medium supplemented with
hygromycin. After ?10 days, several hygromycin resistant
colonies were transferred into 96-well plate and their sen-
sitivity to zeocin and neomycin was tested. The colonies
that were sensitive to zeocin and resistant to neomycin
were used in the RMCE experiments.
Sequential dual RMCE
Sequential dual RMCE experiments were performed
either in the ‘Flp-then-Int’ mode or in the ‘Int-then-Flp’
mode. In both cases, the first integrative step of the se-
quential RMCE was initiated by co-transfecting cells that
carried the integrated platform reporter (CHO TD-1372)
with the respective expression vector—either pOG-Flp or
pNA979oInt—and with the incoming reporter vector
The experiments were performed in six-well plates. The
amount of the incoming reporter pNA1345inc at transfec-
tion was kept at 0.5mg. The amount of the expression
vectors pOG-Flp and pNA979oInt at transfection is
indicated where the respective experiments are described.
The number of the transfected cells in the RMCE experi-
ments was estimated by calculating the transfection effi-
ciency in the control experiments, which were always
performed in parallel with each experimental transfection.
The control transfections were done by transfecting
the platform reporter cells with the EGFP-expressing
plasmid CMV-LEF. The amount of CMV-LEF added at
transfection was 0.5mg; the ratio of green cells to the total
number of the cells was calculated 24hr after transfection.
The range of the efficiency of the control transfections was
between 20 and 25%.
Forty-eight hours after transfection, one-sixth of the
cells from each experimental well were transferred into
100-mm plates, the cells were allowed to become confluent
and the number of the green (in the ‘Flp-then-Int’ mode)
or red (in the ‘Int-then-Flp’ mode) colonies was counted.
Several green or red colonies from these plates were
expanded and analyzed. For the second excisive step of
RMCE, the green cells were transfected with various
amounts of pNA979oInt, and the red cells were trans-
fected with various amounts of pOG-Flp. The transfec-
tions were done in six-well plates. The number of the
transfected cells was estimated as above. Forty-eight
hours after transfection, one-sixth of the cells were
transferred into 100-mm plates, the cells were allowed to
become confluent, and the number of the red (in the ‘Flp-
then-Int’ mode) or green (in the ‘Int-then-Flp’ mode)
colonies was counted. Several green or red colonies from
these plates were expanded and analyzed.
Simultaneous dual RMCE
Simultaneous dual RMCE experiments were performed
essentially as described above except that the CHO TD-
1372 cells that harbor the platform reporter were co-trans-
fected with the incoming reporter pNA1345inc and with
both expression vectors: pOG-Flp and pNA979oInt. The
replacement reactions were done in six-well plates.
The number of the transfected cells was estimated as in
the sequential RMCE experiments. The amount of the
incoming reporter pNA1345inc at transfection was kept
at 0.5mg. The various concentrations of the expression
vectors pOG-Flp and pNAo979Int added at transfection
are indicated in the respective parts of the ‘Results’
section. Forty-eight hours after transfection, one-sixth of
the cells were transferred into 100-mm plates, the cells
were allowed to become confluent and the number of
the green, red and green-red colonies was counted.
Several colonies that were both green and red were
expanded and analyzed.
In all RMCE experiments with Int, 0.6mg of the IHF
expression plasmid and 0.6mg of the Xis expression
plasmid were also included.
Plasmid DNA was isolated using GeneJET Plasmid
Miniprep Kit (Thermo). Amplification of the DNA frag-
ments used for cloning was performed using Pfu-Ultra
polymerase (Agilent Technologies). PCR analysis of the
mammalian genomic DNA was performed using Taq
polymerase (New England Biolabs). Genomic DNA
from cultured mammalian cells was isolated using
GeneJET Genomic DNA Purification Kit (Thermo).
General genetic engineering experiments were performed
as described in Sambrook and Russell (17).
Reporter vectors for Flp-Int dual RMCE
To monitor the activity of each recombinase during dual
RMCE, we have constructed a set of two reporter
plasmids that, via activating the expression of two differ-
ent fluorescent markers, can assess the efficiency of a re-
placement reaction catalyzed by Flp and Int in the absence
of a selection force (Figure 2). Moreover, with this set of
reporters one can compare the efficiency of the dual
RMCE reaction when the recombinases are supplied
PAGE 3 OF 8 Nucleic Acids Research, 2013,Vol.41, No. 12e125
sequentially or simultaneously. One of the two reporter
plasmids serves as a platform that is integrated into an
actively transcribed locus of the CHO genome, and the
other is an incoming reporter (Figure 2A).
pNA1372plat contains the NeoRgene under the control
of the EF1a promoter. The NeoRgene is followed by the
transcription terminator STOP (18) and the promoterless
DsRed gene (Figure 2A). The Flp cognate sequence FRT
is located between the EF1a promoter and the NeoRgene;
the Int cognate sequence attL is located between
STOP and the DsRed gene. The platform reporter
pNA1372plat, which is a derivative of the pcTD plasmid
of the TD-In system (15), was integrated into the TDRT
site located in the genome of the CHO TD-In cells using
the TD-40 variant of TD recombinase to obtain the CHO
TD-1372 cell line. The incoming plasmid pNA1345inc
carries a reporter cassette composed of the promoterless
EGFP gene followed by the CMV promoter. FRT and
attR that can recombine with their counterparts in the
plasmid pNA1372plat, flank the EGFP-CMV reporter
cassette (Figure 2A).
The attL and attR sites in the platform and incoming
reporters were chosen as substrates for Int because this
pair of recombination targets supports the most efficient
recombination in human cells (13,14).
Flp-catalyzed recombination between the FRT sites
located on the platform and the incoming reporters leads
to the swap between the NeoRand the EGFP genes
and therefore activates the expression of the EGFP gene
(Figure 2B–D). Int-catalyzed recombination between the
attL and attR sites leads to the swap between the transcrip-
tion terminator STOP and the CMV promoter thus
activating the expression of the DsRed gene (Figure 2B–D).
Figure 2. Schematics of the sequential and simultaneous RMCE reactions. (A) The incoming and the chromosomally integrated platform reporters
used in this study. Only essential elements of the reporters are shown. The reporters are designed to activate the expression of the EGFP gene from
the EF1a promoter on recombination between the FRT sites, and to activate the expression of the DsRed gene from the CMV promoter on
recombination between the attL and attR sites. NeoR, neomycin resistance gene; STOP, transcription terminator. (B and C) Sequential RMCE.
(B) In the ‘Flp-then-Int’ mode of the sequential RMCE reaction, the first integration reaction is mediated by Flp. The successful integration can be
detected by the appearance of the green cells. The second deletion reaction is mediated by Int. The successful deletion can be detected by the
appearance of the red cells that also maintain the expression of the EGFP gene. (C) ‘Int-then-Flp’ mode of the sequential RMCE reaction. In this
mode of the replacement reaction, the integration reaction is mediated by Int. The successful integration can be detected by the appearance of the red
cells. The deletion reaction is mediated by Flp. The successful deletion can be detected by the appearance of the green cells that also maintain the
expression of the DsRed gene. (D) Simultaneous RMCE. The replacement of the NeoR-STOP cassette with the EGFP-Pcmvcassette proceeds via a
seemingly one-step reaction. The successful replacement can be detected by the appearance of the cells that are both green and red.
e125Nucleic Acids Research, 2013,Vol.41, No. 12PAGE 4 OF 8
In a sequential mode of dual RMCE, the first integrative
step is catalyzed by supplying either Flp or Int (Figure 2B
and C). A successful integration reaction is signaled by the
appearance of the green or red cell, respectively. The second
step of the sequential dual RMCE—deletion—is catalyzed
by supplying the other recombinase: either Int or Flp, re-
spectively (Figure 2B and C). A successful deletion is
indicated by the activation of the expression of the
second fluorescent gene, thus allowing the expression of
both the EGFP and DsRed genes.
A ‘simultaneous’ dual RMCE reaction between the
reporter cassettes located in the incoming and the
platform plasmids is catalyzed by a simultaneous supply
of both Flp and Int recombinases (Figure 2D). Successful
‘simultaneous’ dual RMCE is expected to replace the
NeoR-STOP cassette in the integrated platform reporter
with the EGFP-CMV cassette in the incoming plasmid. As
a result, the expression of both EGFP and DsRed genes is
activated, which can be detected by the appearance of cells
that are both green and red (Figure 2D). The ‘simultan-
eous’ dual RMCE proceeds as a seemingly one-step
reaction although it is proposed to progress in a sequential
integration-then-excision mode (6). In this work, we refer
to the ‘simultaneous’ dual RMCE only in a context of the
simultaneous supply of Flp and Int.
Sequential dual RMCE reactions catalyzed by Flp and Int
Sequential dual RMCE is performed as follows: one of the
two recombinases is used to integrate the entire incoming
plasmid into the respective recombination site of the
chromosomally located platform plasmid and the cells,
in which the successful integration occurs, are identified
and expanded (Figure 2B and C). After that, the second
recombinase is used to perform the excision recombin-
ation between its cognate sites, and the cells with correctly
replaced reporters are identified and expanded. To
compare the efficiency of the sequential dual RMCE
initiated by Flp or Int, we performed sequential dual
RMCE in two modes: ‘Flp-then-Int’ and ‘Int-then-Flp’
(Figures 3 and 4).
To perform the reaction in the Flp-then-Int mode
(Figure 3), the Flp-expressing plasmid and the incoming
reporter pNA1345inc were co-transfected into the CHO
TD-1372 cells. As in our recent Flp-Cre dual RMCE ex-
periments (15), we considered only those green cells as
positive integrants that were able to form groups after
the post-transfection expansion (Figure 3A). When the
amount of the Flp-expressing plasmid added at transfec-
tion was 2mg, a fraction of the groups of the green cells
constituted ?0.1% of the transfected cells. When the
amount of the Flp-expressing plasmid added at transfec-
tion was lowered, the fraction of the groups of the green
cells was lower (data not shown). Several groups of the
green cells were expanded and analyzed by PCR and
sequencing confirming that the pNA1345inc reporter
plasmid was integrated into the CHO TD-1372 cells cor-
rectly (Figure 3B). At the next step, the expanded green
cells were transfected with the Int-expressing plasmid to
complete the RMCE reaction by an excision of the DNA
fragment between the attL and attR sites. The transfected
cells were expanded and groups of the red cells that
formed were counted. In these experiments, we noticed
that the fraction of the transfected cells that were able to
form groups of the red cells was inversely dependent on
the amount of the Int-expressing plasmid added at trans-
fection: 1mg of the Int-expressing plasmid was able to
generate groups of the red cells in ?0.5% of the trans-
fected cells, while 0.01mg of the Int-expressing plasmid
generated groups of red cells in ?10% of the transfected
cells (Figure 3C).
The sequential Int-then-Flp dual RMCE experiments
(Figure 4) were performed methodologically similar to
the Flp-then-Int one described above. The CHO TD-
1372 cells were co-transfected with the Int-expressing
plasmid and the pNA1345inc reporter, and the groups
Figure 3. Sequential dual RMCE reaction in the ‘Flp-then-Int’ mode.
(A) Schematics of the reaction. The color scheme of the functional
elements shown is the same as in Figure 2. Typical group of green
cells formed as a result of the first integration reaction mediated by
Flp, are shown to the right of the respective integration product. The
expanded green/red cells formed as a result of the second deletion
reaction mediated by Int, are shown to the right of the respective
deletion product. (B) The PCR analyses of typical expanded green
and green/red colonies. The horizontal green and red bars in panels
(A) and (B) schematically represent the control PCR products that can
be generated if the respective recombination reactions are successful.
The sequencing of the PCR products obtained confirmed their identity.
G, PCR analysis of the expanded green cells obtained as a result of the
Flp recombination. C1, control PCR analysis of the cells with the
integrated platform reporter using the same primers as in lane ‘G’.
R, PCR analysis of the expanded green/red cells obtained as a result
of the Int recombination. C2, control PCR analysis of the green cells
obtained as a result of the Flp recombination using the same primers as
in lane ‘R’. M, 2-log DNA ladder (New England Biolabs). (C) The
efficiency of the Int-mediated deletion reaction inversely depends on
the amount of the Int expression vector added at transfection. The
amount of the vector added is indicated. The efficiency of the
deletion reaction is represented by green bars. The green bars show
the mean value of three experiments; the error bars indicate standard
PAGE 5 OF 8Nucleic Acids Research, 2013,Vol.41, No. 12e125
of the red cells that formed after the expansion of the
transfected cells were counted (Figure 4A). When the
amount of the Int-expressing plasmid at transfection was
2 or 1mg, no groups of the red cells were observed. Rare
groups of the red cells started to form only when the
amount of the Int-expressing plasmid at transfection was
lowered. The fraction of the red cell groups reached
?0.8% of the transfected cells when the amount of the
plasmid was lowered to 0.01mg (Figure 4C). Several
groups of the red cells were expanded and analyzed by
PCR and sequencing confirming that Int integrated the
pNA1345inc reporter plasmid into the CHO TD-1372
cells correctly (Figure 4B). The expanded red cells were
then transfected with the Flp-expressing plasmid to delete
the DNA fragment between the FRT sites in the
pNA1372plat-pNA1345inc co-integrant thus activating
the expression of the EGFP gene (Figure 4A). When the
‘standard’ amount of the Flp-expressing plasmid (2mg)
was added at transfection, the fraction of the green cells
that were able to form groups after expansion was ?50%.
Lower amounts of the Flp-expressing plasmid added at
transfection generated fewer groups of the green cells.
Successful simultaneous dual RMCE requires a balanced
supply of Int and Flp
Simultaneous dual RMCE is performed by co-transfecting
the platform reporter cells CHO TD-1372 with both re-
combinase-expressing plasmids and the incoming reporter.
The expected positive outcome of this type of RMCE is
the appearance of cells that are both green and red
(Figures 2D and 5). The experiments on sequential dual
RMCE described above suggested that high amounts of
the Int-expressing plasmid added at transfection may not
be optimal neither for the integration reaction nor for the
deletion reaction. If the same holds true for the simultan-
eous dual RMCE, then there will be few, if any, positive
cells when the amounts of the Flp- and Int-expressing
plasmids added at transfection are ?2mg. Indeed, when
Figure 4. Sequential dual RMCE reaction in the ‘Int-then-Flp’ mode.
(A) Schematics of the reaction. Typical group of red cells formed as a
result of the integration reaction mediated by Int, are shown to the
right of the respective integration product. The expanded red/green
cells formed as a result of the deletion reaction mediated by Flp, are
shown to the right of the respective deletion construct. (B) The PCR
analyses of typical expanded red and red/green colonies. The horizontal
green and red bars in panels (A) and (B) schematically represent the
PCR products that are expected if the respective recombination reac-
tions are successful. The sequencing of the PCR products obtained
confirmed their identity. R, PCR analysis of the expanded red cells
obtained as a result of the Int recombination. C1, control PCR
analysis of the cells with the integrated platform reporter using the
same primers as in lane ‘R’. G, PCR analysis of the expanded red/
green cells obtained as a result of the Flp recombination. C2, control
PCR analysis of the red cells obtained as a result of the Int recombin-
ation using the same primers as in lane ‘G’. M, 2-log DNA ladder
(New England Biolabs). (C) The efficiency of the Int-mediated integra-
tion reaction inversely depends on the amount of the Int expression
vector added at transfection. The amount of the vector added is
indicated. The efficiency of the integration reaction is represented by
green bars. The green bars show the mean value of three experiments;
the error bars indicate standard deviation.
Figure 5. Simultaneous dual RMCE. (A) Schematics of the reaction.
Typical group of green/red cells formed when the expression vectors
that code for Flp and Int are simultaneously added to the transfection
mixture, are shown to the right of the replacement product. (B) The
PCR analyses of a typical expanded green/red colony. The horizontal
green and red bars in panels (A) and (B) represent the expected PCR
products characteristic of a successful simultaneous dual RMCE
reaction. The sequencing of the PCR products obtained confirmed
their identity. G and R, PCR analysis of the expanded green/red cells
with the primers that anneal on the EF1a promoter and the EGFP
gene and on the CMV promoter and the DsRed gene, respectively.
C1 and C2, control PCR analysis of the cells with the integrated
platform reporter using the same set of primers as in lanes ‘G’ and
‘R’. M, 2-log DNA ladder (New England Biolabs). (C) The efficiency
of the simultaneous dual RMCE reaction depends on the amount of
the Flp and Int expression vectors added at transfection. The amounts
of the vectors added are indicated. The efficiency of the replacement
reaction is represented by green bars. The green bars show the mean
value of three experiments; the error bars indicate standard deviation.
e125Nucleic Acids Research, 2013,Vol.41, No. 12PAGE 6 OF 8
the initial experiments on the simultaneous dual RMCE
were performed by adding 2mg and 1mg of the Flp- and
Int-expressing plasmids, respectively, no red/green or just
red cells were observed. Only when the amount of the Int-
expressing plasmid was significantly lowered (to ?0.1mg),
we started to see red/green cells (Figure 5C). When the
amount of the Int-expressing plasmid was lowered to
0.01mg, a fraction of the colony-forming red/green cells
grew to ?5% of the transfected cells. This number became
even larger—?12% of the transfected cells—when the
amount of the Flp-expressing plasmid added at transfec-
tion was decreased to 0.3mg (Figure 5C). Under these
optimized conditions, just green and just red groups of
cells, which reflect the integration of the incoming
reporter plasmid into FRT and attL, respectively,
constituted ?1.5% of the transfected cells each. Several
groups of the red/green cells were expanded and
analyzed by PCR and sequencing confirming their
nature as those that underwent dual RMCE (Figure 5B).
In this report, we presented an analysis of the dual RMCE
reaction catalyzed by Flp and HK022 Int in a model
setting of CHO cells. The analysis was aimed to identify
the conditions that permit and maximize the efficiency of
the replacement reaction performed in a sequential (Flp-
(Flp+Int) mode. We demonstrate that HK022 Int
paired with Flp can mediate efficient replacement
reaction that does not require a selection force to
identify the desired DNA rearrangements. We found
that low amounts of the Int-expressing plasmid added at
transfection is a key for the efficient RMCE in both the
sequential and the simultaneous dual RMCE modes, while
lowering the amount of the Flp-expressing plasmid helps
improve the efficiency of dual RMCE in the simultaneous
To simplify the analysis of the dual RMCE reaction, we
developed a reporter system that detects the recombin-
ation events mediated by each recombinase (Figure 2).
The detection of the recombination events is accomplished
by the independent activation of the expression of the
EGFP and DsRed genes on recombination at FRT and
att sites, respectively. Our dual RMCE reporter system is
functionally complete: in a population of cells transfected
with the incoming reporter and the Flp and Int expression
plasmids, the system can differentiate between the cells
that underwent a complete dual RMCE reaction and
those in which an incomplete RMCE reaction, that is,
just integration of the incoming reporter into either FRT
or att sites, has occurred. Our reporter system can be
modified to analyze the dual RMCE reaction mediated
by other recombinases by replacing FRT and att sites
with the respective recombination targets.
It was rather unexpected that the efficient simultaneous
dual RMCE reaction required a low amount of the Int-
expressing plasmid. This amount was significantly lower
than the amount of the Flp-expressing plasmid: 0.01mg
versus 0.33mg, respectively, for the reaction performed
in six-well plates (Figure 5). This low amount of the Int-
expressing plasmid mirrors the conditions that are optimal
for the sequential dual RMCE reaction (Figures 3 and 4)
where Int acts alone and needs to perform either deletion
(Flp-then-Int mode) or integration (Int-then-Flp mode).
Overall, these results suggest that the inability of Int to
support simultaneous dual RMCE when the amount of
the Int-expressing plasmid added at transfection is high
may be mainly due to its inability to perform any type
of recombination reaction under these conditions. This
contrasts sharply with the properties of Flp that
mediates the highest level of recombination when the
amount of the Flp expression plasmid is high.
One explanation for the difference in the functional
properties of the recombinases is based on the difference
in the complexity of the synaptic complexes they form. Flp
is a simple recombinase that assembles a simple synaptic
complex (19), while Int is a complex recombinase that
requires additional factors to assemble its synaptic
complex. It is possible that the results on the Int activity
described here reflect the fact that Int can assemble a pro-
ductive synaptic complex only when the concentration of
Int in a cell is below certain threshold. If the concentration
of Int is above that threshold, a synaptic complex formed
becomes unproductive, which might result from Int aggre-
gation. Indeed, in a bacterial assay, using a regulatable
promoter, we observed that insolubility of Int increases
as the level of its expression increases (unpublished
data). This property of Int suggests an additional or alter-
native explanation for the inverse correlation between the
amount of the Int expression plasmid added at transfec-
tion and the activity of Int: Int aggregates can sequester
Int molecules, thus limiting its ability to form functional
In the model setting tested here, Flp, when it acts alone,
exhibits the highest activity when the amount of the Flp-
expressing plasmid is the highest tested (2mg per six-well
plate). It is interesting to note that this is not the case in
the simultaneous dual RMCE reaction, when both recom-
binases are present (Figure 5). We found that the effi-
ciency of the replacement reaction is the highest at a
lower amount of the Flp-expressing plasmid added at
transfection (0.33mg per six-well plate). One possible ex-
planation of this phenomenon is that at higher concentra-
tions Flp and Int mediated reactions or their synaptic
complexes may interfere with each other.
Our earlier work on dual RMCE, aimed to identify the
conditions that maximize the efficiency of the reaction
performed by simple recombinases Flp and Cre (15),
determined that the low input of Cre helps improve
the replacement reaction. It was suggested that such
input increases the integration activity of Cre (without
significantly decreasing its deletion activity), which, in
turn, improves the overall efficiency of RMCE. In the
present study, we observed that lowering the input of Int
improves the efficiency of the Flp-Int dual RMCE.
Despite the apparent similarity between the conditions
that increase the efficiency of the replacement reaction
in the Flp-Cre and Flp-Int systems, it is unlikely that
the mechanisms behind the phenomena are the same.
In contrast to Cre, both the integration and the
PAGE 7 OF 8 Nucleic Acids Research, 2013,Vol.41, No. 12e125
deletion activities of Int are equally dependent on the
input of its expression plasmid (Figures 3–5), which, we
believe, reflects the assembly of the productive synaptic
complex discussed above.
In our previous work on dual Flp-Int RMCE reaction
performed in E. coli, we have shown that the replacement
reaction performed in the sequential modes was by far
more efficient than the one performed in the simultaneous
mode (14). As the results of the present work show, the
reason might be because in the bacterial experiments we
were unable to control the levels of Flp and Int expression.
In the sequential dual RMCE modes, the integrative
trans reactions mediated by Flp and Int was somewhat
inefficient (?0.1% and ?1%, respectively) owing to the
known high reversibility rate of the integration reaction
[(3) and Figure 1A]. In contrast, the subsequent cis
excision reactions (mediated by Int and Flp, respectively)
led to the final RMCE product and were efficient (?10%
and ?50%, respectively). Optimized conditions of the sim-
ultaneous dual RMCE mode led to the complete RMCE
products in respectful 12% of the transfected cells. The
progenitors of RMCE (6) envisioned the reaction as a
fast succession of integration and excision. If that is
correct, the relatively high yield of the dual RMCE
products seen in the simultaneous mode, despite the con-
siderably inefficient integrative reaction, is probably due
to the presence of both recombinases that thermodynam-
ically enable the high potential of the efficient excision
reaction to overcome the instability of the integrative
intermediate. The successive integration-excision model
is also supported by the detection of small fraction of
each intermediate incoming plasmid integrant in the sim-
ultaneous RMCE mode.
In summary, in mammalian cells, the simpler simultan-
eous supply of both Flp and Int in a dual RMCE reaction
is preferable over the sequential protocol. Our work also
shows that Int-HK022, as an efficient recombinase, can be
added to the repertoire of recombinases used in mamma-
lian genome engineering.
National Institutes of Health (NIH) [R01GM085848
to Y.V.]; German Israeli Foundation for Scientific
Research and Development (G.I.F) [1062-62.3/2008];
Israel Science Foundation [702/11 to E.Y. and M.K.].
Funding for open access charge: NIH.
Conflict of interest statement. None declared.
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