BENCHMARKS
460 BioTechniques Vol. 40, No. 4 (2006)
We are interested in identifying and
characterizing recombinases that can
achieve efficient site-specific insertion
or cassette exchange in mammalian
cells. The combination of gene targeting
techniques and site-specific recombi-
nation systems (Cre/loxP and Flp/FRT)
have been widely exploited in genetic
analysis and in engineering complex
chromosomal alterations in higher
eukaryotes (1). The Cre/loxP system
has been the preferred tool for genome
engineering in murine embryonic stem
cells (ESCs) and in mice. Cre and Flp
are best suited for generating site-
specific deletions (knockouts), because
the 34-bp loxP and FRT recombination
target sites, respectively, are recreated
during recombination, and excision is
preferred over integration. Mutant loxP
sites have been developed to stabilize
the integration event, but the efficiency
of integration is still low (2). Attempts
to exploit integrases from λ phage and
closely related HK022 phage have
resulted in limited success because
of cofactor requirement and poor
efficiency of intermolecular integration
and intrachromosomal deletion (3,4).
Recent work on integrases from ΦC31,
R4, and TP901-1 phages demonstrated
that these enzymes catalyze site-
specific recombination between attP
(phage attachment) and attB (bacterial
attachment) sites in mammalian
cells (5–8). These integrases belong
to the resolvase/invertase or serine
recombinase family that utilizes an
N-terminal catalytic serine to mediate
recombination and are structurally
different from Cre, Flp, and λ int of
the λ integrase or tyrosine recom-
binase family that utilizes a C-terminal
catalytic tyrosine (9).
Recently it was shown that phage
Bxb1 integrates into Mycobacterium
smegmatis groEL1 gene, and Bxb1
integrase can catalyze recombination
between the attP and attB sites in vitro
in the absence of supercoiled DNA,
cofactors, and divalent cations (10–13).
Here we report that Bxb1 integrase,
a serine recombinase, is functional
in mammalian cells and catalyzes
highly efficient unidirectional recom-
bination between short heterologous
attP and attB target sites resulting in
the integration or deletion of DNA
depending on the orientation and
location of attP and attB sites.
To determine if the Bxb1 integrase
functions in mammalian cells,
we created a recombination assay
plasmid pCMV-attP/attB containing
a transcription termination or stop
sequence, flanked by 52-bp attP and
46-bp attB sites (10), placed between
the cytomegalovirus (CMV) promoter
and the luciferase reporter gene (Figure
1A). Recombination between the
attP and attB sites catalyzed by Bxb1
integrase would result in deletion of
the stop sequence and activation of
luciferase gene. Since the integrase
gene is from bacteriophage, we totally
synthesized the gene using codons
optimized for high-level human and
mouse expression without changing
the encoded amino acid sequence
(GenBank® accession no. NP_075302)
and cloned it into a CMV promoter-
driven mammalian expression plasmid
to obtain pCMV-Bxb1. To test the
recombination in various cell types,
we obtained and cultured HEK 293
(ATCC, Manassas, VA, USA), NIH
3T3 (ATCC), mouse 129/S6 ESCs
(Primogenix, St. Louis, MO, USA),
mouse C17.2 neural stem cells (NSCs;
Evan Snyder, The Burnham Institute,
La Jolla, CA, USA), and rat bone
marrow stromal cells (BMSCs; Osiris
Therapeutics, Baltimore, MD, USA)
following the recommended protocols.
One day before transfection, cells were
plated in a 96-well (HEK 293, NIH
3T3) or 48-well plate (BMSC, ESC,
NSC) at different densities depending
on the cell type (HEK 293, 20,000 cells;
NIH 3T3, 5000 cells; ESC, 10,000
cells, NSC, 120,000 cells; and BMSC,
3200 cells). The cells were then trans-
fected with 25 ng recombination assay
plasmid alone or along with varying
amounts of pCMV-Bxb1 DNA (0, 25,
50, or 100 ng) using Lipofectamine™
2000 (Invitrogen, Carlsbad, CA, USA)
according to the manufacturer’s instruc-
tions. Constitutively expressed Renilla
luciferase reporter plasmid (pRL-
CMV; Promega, Madison, WI, USA)
was cotransfected (2 ng/well) and used
as an internal control to normalize the
transfection efficiency. Twenty-four
hours (HEK 293, NIH 3T3, ESC) or
48 h (BMSC, NSC) after transfection,
the media was discarded, and the cells
were lysed with 50 μL passive lysis
buffer (Promega) and 25-μL extracts
were then assayed using the Dual
Luciferase® Assay kit (Promega) on
a plate reader equipped with injectors
(Dynex Technologies, Chantilly, VA,
USA). Introduction of pCMV-attP/attB
and pCMV-Bxb1 plasmid DNAs into
human HEK 293 cells, mouse NIH
3T3, NSCs, and ESCs, and rat BMSCs
resulted in 215- to 2886-fold induction
of luciferase activity depending on the
cell type (Figure 1B, only the values
obtained for 100 ng pCMV-Bxb1 are
shown). Thus, the results clearly show
that the Bxb1 integrase can catalyze
the site-specific recombination reaction
in mammalian cells, and its activity
in cells from three different species
suggests that the integrase may function
autonomously of cellular factors.
The ability of the Bxb1 integrase
to catalyze recombination between
chromosomally placed attB and attP
sites was assessed by stably integrating
a single copy of CMVp-attP-STOP-
attB-luciferase sequence (Figure 1A)
at the FRT locus in Flp-In™-293 cells
(Invitrogen) following the manufac-
turer’s instructions and transiently
introducing pCMV-Bxb1. To place
a single copy of CMVp-attP-STOP-
attB-luciferase sequence in Flp-In-293
cells, we first cloned the sequence into
the pcDNA5/FRT plasmid (Invitrogen)
to create pFRT-attP/attB and then
Phage Bxb1 integrase mediates highly efficient
site-specific recombination in mammalian cells
John P. Russell, David W. Chang, Anna Tretiakova, and Malla Padidam
RheoGene, Inc., Norristown, PA, USA
BioTechniques 40:460-464 (April 2006)
doi 10.2144/000112150

BENCHMARKS
462 BioTechniques Vol. 40, No. 4 (2006)
cotransfected Flp-In-293 cells with
pFRT-attP/attB DNA and Flp recom-
binase expression plasmid pOG44
DNA. We then selected several stable
clones that were resistant to hygro-
mycin and sensitive to zeocin and
that lost β-galactosidase. A represen-
tation of the FRT locus is shown in
Supplementary Figure S1B available
online at www.BioTechniques.com.
Cells derived from a single stable
clone (15,000 cells/well, 96-well plate)
were then transfected with 0, 25, 50,
100, or 200 ng pCMV-Bxb1, and the
luciferase activity was measured after
48 h of incubation as described above.
As shown in Figure 1C, increasing
amounts of the pCMV-Bxb1 DNA
resulted in a dose-dependent increase
in luciferase expression. The Bxb1
integrase, therefore, functioned within
the environment of the mammalian
cell nucleus and deleted chromosomal
DNA flanked by attP and attB sites.
In order to test the ability of the
integrase to insert a circular plasmid
DNA containing an attP site into an
attB site present on the chromosome
(or an attB site into an attP site), we
made Flp-In-293 stable cell lines
containing a single copy of the CMV
promoter and an attB site (or the CMV
promoter and an attP site) at the FRT
locus. First, we inserted the 46-bp attB
or 52-bp attP oligonucleotide after the
CMV promoter in pcDNA5/FRT to
make plasmids pFRT-attB or pFRT-
attP. Next, a single copy of either pFRT-
attB or pFRT-attP was introduced at
the FRT locus of Flp-In-293 cells as
described above to create two sets of
stable clones. Two integration assay
plasmids, pPURO-attP and pPURO-
attB, were then constructed in which
an attP or attB site was placed before
a promoter-less puromycin resistance
gene. Site-specific recombination
between pPURO-attP and the chromo-
somal attB site (or pPURO-attB and
the chromosomal attP site) would place
the promoter-less puromycin gene next
to the CMV promoter and confer a
drug resistance phenotype to the cells.
Random integration of pPURO-attP
or pPURO-attB is not expected to
yield any drug-resistant clones, unless
the integration is next to a promoter.
A representation of the FRT locus
before and after the integration of
puromycin resistance gene is shown in
Supplementary Figure S1C.
Cells derived from a single stable
clone containing either pFRT-attB or
pFRT-attP (500,000 cells/well, 6-well
plate) were then transfected with either
1 μg pPURO-attP or pPURO-attB alone
Figure 1. Site-specific recombination activity of Bxb1 phage integrase in mammalian cells. (A) Representation of pCMV-attP/attB construct and activation
of luciferase gene expression after deletion of stop sequence. (B) Bxb1 integrase activity in human HEK 293 (n = 6), mouse NIH 3T3 (n = 5), mouse embry-
onic stem cells (ESCs; n = 3), mouse neural stem cells (NSCs; n = 4), and rat bone marrow stromal cells (BMSCs; n = 8). Cells were cotransfected with 2 ng
Renilla luciferase (RLuc) plasmid pRL-CMV and 25 ng pCMV-attP/attB plasmid in the absence (-) or presence (+) of 100 ng pCMV-Bxb1 plasmid. Luciferase
(Luc) and RLuc activities were assayed after 48 h incubation using the Dual Luciferase Assay kit reagents and a luminometer equipped with injectors. Values
shown are the mean ± sd of ratios of Luc and RLuc activities. Fold-inductions (ratio of activity in the presence of pCMV-Bxb1 to the activity in the absence
of pCMV-Bxb1) are shown above the bars. (C) Deletion of chromosomal DNA flanked by attP and attB sites. Luciferase activity in Flp-In-293 cells stably
transfected with pCMV-attP/attB and transiently cotransfected with the indicated amounts of pCMV-Bxb1 plasmid along with 2 ng pRL-CMV plasmid. Values
were measured 48 h posttransfection and are the mean ± sd (n = 8) of ratios of Luc and RLuc activities. (D) Integration-specific PCR analysis. Confirmation of
attL and attR sites in clones after the integration of pPURO-attB into attP cells (attB > attP, lanes 4, 8, and 12) and integration of pPURO-attP into attB cells
(attP > attB, lanes 3, 7, and 11), and attB and attP sites present in the cells before the integration (attB, lanes 1, 5, and 9; attP, lanes 2, 6, and 10) is achieved by
primer combinations that amplify attL (lanes 1–4), attR (lanes 5–8), and attB and attP (lanes 9–12) sites. CMVp, SV40t, and Luc are cytomegalovirus (CMV)
promoter, simian virus 40 (SV40) terminator, and luciferase gene, respectively.
A B
C D
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