From the Departamento de Inmunología y
Oncología and the ¶ Departamento de Biotecnología
Microbiana, Centro Nacional de Biotecnología, Consejo Superior
de Investigaciones Científicas, Campus de la Universidad
Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain
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ABSTRACT |
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The development of new strategies for the
in vivo modification of eukaryotic genomes has become an
important objective of current research. Site-specific recombination
has proven useful, as it allows controlled manipulation of murine,
plant, and yeast genomes. Here we provide the first evidence that the
prokaryotic site-specific recombinase ( Several methods have been developed allowing the manipulation of
mammalian genomes in order to elucidate the relevance and function of
particular genes of interest. Among them, the development of transgenic
mouse strains and gene targeting technologies has been particularly
useful (1, 2). These techniques have experienced a new advance with the
characterization and application of site-specific recombinases (3).
Site-specific recombinases can be clustered into two major families.
The Int family comprises those enzymes that catalyze recombination
between sites located either in the same DNA molecule (resolution and
inversion) or in separate DNA molecules (integration) (4-7). The
latter property has been exploited to allow targeted insertion of
specific sequences at precise locations (8, 9). The recombinases
currently used to manipulate mammalian genomes are mainly the Cre and
Flp proteins, both members of the Int family (3). The target sequences
for these enzymes, loxP sites for the Cre enzyme and FRT for
Flp, consist of a short inverted repeat to which the protein binds. The
recombination process is operative through long distances (up to 70 kilobases) in the genome. Using these enzymes, several authors have
reported site- and tissue-specific DNA recombination in murine models
(10-13), chromosomal translocations in plants and animals (14-16),
and targeted induction of specific genes (17). For instance, expression
of Cre from the lck proximal promoter leads to specific
recombination in thymus (10). The gene encoding DNA polymerase The second family of recombinases includes those enzymes that catalyze
recombination only when the sites are located in the same DNA molecule
(resolution and/or inversion); they are collectively termed
resolvases/invertases (18). In this study, we have explored the use of the prokaryotic
site-specific Plasmids and Cloning--
Plasmids pBT338 and pCB8, carrying
either one or two directly oriented six sites (19), and
pLXSN, which carries the resistance marker for neomycin (G418) (24),
have been previously described. A eukaryotic expression vector with the
SV40 early promoter, pSV2 (25), was kindly provided by Dr. J. Ortín (Centro Nacional de Biotecnología). The
expression plasmid pSV Culture and Cell Lines--
Transient expression assays were
performed in the simian COS-1 cell line, kindly provided by Dr. J. Ortín. Stable clones with the DNA substrate for Transfection Conditions and Plasmid DNA Extraction--
The
transient expression experiments were performed in COS-1 cells by
DEAE-dextran transfection as described (26). Cells were harvested
48 h after transfection, and the extrachromosomal DNA was
extracted using the method described by Hirt (27). In brief, cell
pellets were lysed with SDS (Merck) and treated with proteinase K
(Boehringer, Mannheim, Germany) at 37 °C. The genomic DNA was
precipitated with 1 M NaCl (Merck). Upon centrifugation, the supernatant was phenol-extracted, and plasmid DNA was precipitated with ethanol and resuspended in water for further experiments.
Stable cell clones with pCB8 DNA randomly inserted at different genome
sites were obtained by electroporation, in a Bio-Rad Gene Pulser, of
2 × 106 NIH/3T3 cells at 220 V and 960 microfarads
with 20 µg of pCB8 DNA and pLXSN DNA at a 10:1 ratio. Transfected
cells were selected with 1 mg/ml G418 (Sigma) for ~2 weeks. The
stable clones obtained were analyzed in Southern experiments (26) or by
immunofluorescence as described below.
Immunoblotting and Immunofluorescence--
Rabbit polyclonal
antibodies against the purified
Transfected cells were cultured on coverslips. After 48 h, cells
were fixed in methanol/acetone (1:1) at
For immunoblotting analysis, transiently transfected cells were
harvested 48 h after transfection and lysed in radioimmune precipitation assay buffer (137 mM NaCl, 20 mM
Tris-HCl (pH 8), 1 mM MgCl2, 1 mM
CaCl2, 10% glycerol, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS; Merck). The lysed fraction was separated on SDS-polyacrylamide gel; blotted onto nitrocellulose membrane (Bio-Rad); and incubated with polyclonal anti-
Subcellular fractionation was performed by detergent lysis of
transiently transfected cells essentially as follows. 48 h after transfection, cells were trypsinized, washed twice with
phosphate-buffered saline, and harvested by centrifugation. Each cell
pellet was resuspended in TM-2 buffer (10 mM Tris-HCl (pH
7.4), 2 mM MgCl2, and 0.5 mM
phenylmethylsulfonyl fluoride) and incubated on ice for 5 min. Then,
Triton X-100 was added to each pellet to a final concentration of 0.5%
and incubated on ice for 5 min. Cells were sheared by gentle pipetting,
monitoring the appearance of free nuclei in a phase-contrast
microscope. When essentially all nuclei were free of cytoplasmic tags,
they were collected by centrifugation. The proteins of the cytoplasmic
fraction were stored frozen for further Western analysis. The nuclei
were washed twice with TM-2 buffer, and the proteins were extracted as
described before. Analysis of Recombination Products--
PCR was performed with
the GeneAmp PCR System 2400 from Perkin-Elmer equipped with a heating
cover. Each reaction was carried out using 0.5 µg of genomic DNA or
one-tenth of the Hirt preparation according to the supplier's
instructions. Taq polymerase (2.5 units; Perkin-Elmer) was
added with Perfect Match PCR Enhancer (Stratagene, La Jolla, CA) after
an initial denaturation (94 °C, 10 min). The procedure (Touch-Down)
was thereafter performed as follows: 80 °C for 2 min, five cycles of
denaturation (94 °C, 1 min) and annealing/extension (72 °C, 2 min), and five cycles of 1 min at 94 °C and 2 min at 70 °C. This
was coupled to 25 cycles of denaturation (94 °C, 1 min), annealing
(68 °C, 30 s), and extension (72 °C, 2 min) and one
additional extension step at 72 °C for 5 min. For the PCR analysis
of the Hirt preparations, we used the 16-mer reverse sequencing primer
(No. 1201) and the 17-mer universal sequencing primer (No. 1211) from
New England Biolabs Inc. (Beverly, MA). These primers are hereafter
referred to as a and b, respectively.
The primers used for PCR amplification of the Hirt preparations were
unsuitable for the analysis of genomic DNA preparations (low
Tm). A new pair of primers was thus designed:
pBT338UP158, 5'-CCGGCTCGTATGTTGTGTGGAAT-3'; and pBT338DO802,
5'-TGGCGAAAGGGGGATGTGCTG-3'. These primers are hereafter referred to as
a' and b', respectively.
Southern analysis of the PCR products was performed by blotting the DNA
separated on agarose gels onto nylon membranes (Amersham Pharmacia
Biotech). Filters were hybridized at 42 °C in 250 mM phosphate buffer (pH 7.2), 50% formamide, 250 mM NaCl, 1 mM EDTA, and 7% SDS and washed in 1× SSC and 0.1% SDS at
room temperature for 30 min, at least twice. The washing temperature
was increased when needed. The radioactive labeling of probes was
performed with the Prime-It random primer labeling kit (Stratagene).
Nucleotide sequences from the PCR bands of interest were determined by
automated fluorescent sequencing and were analyzed using Seq-Ed 1.0.3 software (Applied Biosystems Inc.).
Recombination-activated Gene Expression--
To obtain further
evidence of recombination due to Expression of the Prokaryotic
Transiently transfected cells were stained with rabbit polyclonal
anti-
These results indicate that
To determine whether eukaryotic cells could provide this host factor,
recombination activity due to
To provide further experimental evidence of the
Upon transfection of these plasmids in a stable
However, the
The fidelity of the recombination mechanism was also confirmed by DNA
sequencing of the amplified bands in the case of clones 1 and 2 (data
not shown). The regenerated six site (see Fig.
1A) obtained after recombination was unaltered. These data
indicate that The common genome manipulation techniques, including transgenesis
and gene targeting, have opened a new path for the understanding of a
wide variety of mechanisms involving diverse genetic functions. The
utility of these systems becomes limited, however, when the overexpression or inactivation of a given gene has fatal effects on
embryo development (as an example, see Refs. 11 and 30) or when the
lack of gene function can be bypassed or compensated by redundant
mechanisms (31, 32). Moreover, the effects of gene inactivation outside
the tissue or cell lineage of interest are usually unknown and
uncontrollable (33).
These problems have been overcome to some extent by the development and
application of the site-specific recombination techniques (reviewed in
Ref. 7) that allow spatiotemporal control of the targeting event. This
is the case of the Cre-loxP and Flp-FRT systems (reviewed in
Refs. 3 and 4).
We show that the prokaryotic Transient Since in vitro recombination requires a chromatin-associated
protein (28), we assume that this factor is provided by the host.
Indeed, it is known that the mammalian HMG1 chromatin-associated protein can efficiently stimulate in vitro We have also studied the ability of We provide the first evidence, in eukaryotic cells, for the activity of
a DNA recombinase belonging to the prokaryotic resolvase/invertase family. Enzymes of this family promote DNA recombination through a
mechanism different from that of DNA integrases. Integrases such as Cre
or Flp promote intramolecular as well as intermolecular recombination,
whereas recombinases of the resolvase/invertase family are highly
specialized in intramolecular recombination. If confirmed in animal
models, the availability of a tool such as -recombinase), which
catalyzes only intramolecular recombination, is active in eukaryotic
environments.
-Recombinase, encoded by the
gene of the
Gram-positive broad host range plasmid pSM19035, has been functionally
expressed in eukaryotic cell lines, demonstrating high avidity for the
nuclear compartment and forming a clear speckled pattern when assayed by indirect immunofluorescence. In simian COS-1 cells, transient
-recombinase expression promoted deletion of a DNA fragment lying between two directly oriented specific recognition/crossing over sequences (six sites) located as an extrachromosomal DNA
substrate. The same result was obtained in a
recombination-dependent lacZ activation system
tested in a cell line that stably expresses the
-recombinase
protein. In stable NIH/3T3 clones bearing different number of copies of
the target sequences integrated at distinct chromosomal locations,
transient
-recombinase expression also promoted deletion of the
intervening DNA, independently of the insertion position of the target
sequences. The utility of this new recombination tool for the
manipulation of eukaryotic genomes, used either alone or in combination
with the other recombination systems currently in use, is discussed.
INTRODUCTION
Top
Abstract
Introduction
References
has
been tissue-specifically deleted using the same strategy (11). In a
different approach, the SV40 tumor antigens have been specifically
activated in the lenses of mice, resulting in tumors at that location
and not in the rest of the animal (17). The Cre-loxP
strategy has also been used in combination with inducible promoters, as
in the case of an interferon-responsive promoter that was used to
provoke gene ablation in liver with high efficiency and, to a lesser
extent, in other tissues (12).
-Recombinase, which belongs to this
family, catalyzes exclusively intramolecular deletions and inversions
of DNA sequences located between two target sites for the recombinase,
called six sites (19, 20). Each six site comprises 90 bp1 (see Fig. 1)
and is composed of two subsites, termed I and II, to which the
recombinase binds (19, 21).
-Recombinase is encoded by the
gene
of the Gram-positive broad host range plasmid pSM19035 (22, 23).
-recombinase for the manipulation of mammalian
genomes. We describe the cloning and expression in eukaryotic cells of the gene coding for
-recombinase and show its ability to catalyze site-specific resolution (deletion) of DNA sequences when the target
sequences are either in a plasmid (extrachromosomal target) introduced
into the cell by transfection or integrated in the genome as
chromatin-associated structures at several locations. The possible
applications and potential advantages of this new system, specifically
in combination with those already in use, are discussed.
EXPERIMENTAL PROCEDURES
2 was constructed by PCR amplification of the
coding sequence for the
gene from plasmid pBT233 (22). The primers
used for PCR were as follows: betaUP,
5'-GAGAGAAAGCTTGGTTGGTTGAAAATGGCT-3'; and betaDO,
5'-GAGAGATGATCAGTACTCATTAACTATCCC-3'. These oligonucleotides contain
restriction sites for HindIII and BclI,
respectively, which were used to clone the amplified gene in the pSV2
vector following standard methods (26). Since BclI is
sensitive to methylation, the pSV2 plasmid was isolated from the BZ101
(dam
) bacterial strain. The relevant structures are
depicted in Fig. 1.
-recombinase
integrated at different chromatin sites were established in the murine
cell line NIH/3T3. Both cell lines were cultured in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) supplemented with
10% fetal calf serum (Cultek, Madrid, Spain), 2 mM
L-glutamine (Merck, Darmstadt, Germany), and the
antibiotics streptomycin (0.1 mg/ml; Sigma) and penicillin (100 units/ml; Sigma).
-recombinase were obtained by
conventional techniques (26).
-Recombinase was detected by indirect
immunofluorescence or by SDS-polyacrylamide gel electrophoresis
followed by immunoblotting.
20 °C for 5 min, air-dried, and rehydrated with phosphate-buffered saline. Cells were
then incubated with rabbit polyclonal anti-
-recombinase antibodies
(1:5000 dilution) at room temperature for 30 min, washed three times
for 5 min with phosphate-buffered saline, and reincubated with a
fluorescein-conjugated anti-rabbit IgM antibody (Dako, Glostrup,
Denmark) for 1 h at 37 °C in phosphate-buffered saline. The
cells were mounted on microscope slides and photographed in a
fluorescence microscope.
-recombinase
antibodies, previously blocked with COS-1 total cell lysate (1:500
dilution). Peroxidase-conjugated anti-IgM antibody (Dako) was used as
secondary antibody. Membranes were processed using the ECL
chemiluminescence detection kit (Amersham Pharmacia Biotech, Aylesbury,
United Kingdom).
-Recombinase detection was performed on Western
blots as described. A monoclonal anti-
-actin antibody (Sigma) was
used as a marker for cytoplasmic fraction detection. Alternatively, the
presence of nuclear fraction proteins was monitored with a monoclonal
anti-histone antibody (Chemicon International, Inc., Temecula, CA).
Peroxidase-conjugated anti-mouse IgM antibody (Dako) was used as
secondary antibody for both purposes.
-recombinase, a
recombination-dependent gene expression system was
constructed, as depicted in Fig. 5, for the reporter gene
lacZ. For analysis of
-galactosidase expression, plasmids
were transiently transfected in a cell line constitutively expressing
-recombinase activity,2
and 48 h after transfection, the proteins were extracted according to the protocol from Luminescent
-galactosidase detection kit II
(CLONTECH). lacZ gene expression was
measured in a scintillation counter for each condition.
RESULTS
-Recombinase in Mammalian
Cells--
The coding sequence for
-recombinase was cloned in the
pSV2 vector under the control of the SV40 early promoter. A control plasmid that does not contain the
-gene was also generated during this process (pSVc). The resulting constructs, pSV
2 (Fig.
1B) and pSVc, respectively,
were transiently transfected in COS-1 cells, which express SV40
T-antigen. Under these conditions, the expression from plasmids that
contain the SV40 early promoter (included in pSV
2) is amplified.
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Fig. 1.
Schematic representation of the site-specific
recombination mechanism mediated by
-recombinase and the plasmids used in this
study. A,
-recombinase (shown as a dimer; open
circles) interacts, in the presence of a host factor (HMG1, shown
as a monomer; open polygons), with two identical copies of
the six site (shaded and open arrows)
to form a synaptic complex. If the reaction occurs within the host
genome, this complex is resolved, giving rise to two recombination
products. One of those, a circular intermediate harboring the
intervening sequences, would be lost. The other, containing one full
six site, would remain intact in the host cell.
B, the essential features of plasmid pSV
2 and pCB8 and
pBT338 DNAs are indicated. The six site is shown
schematically, and sites I and II are highlighted. Orientation of the
six sites is denoted by the direction of the shaded
arrows. The coding region of the xylE gene, initially
designed for expression in bacteria, is indicated. Also indicated are
the hybridization sites of the specific primer pairs a/b and a'/b' used
for PCR amplification to detect the recombination products and of the
probe used for specific hybridization controls.
-recombinase antibodies. Fluorescence microscopy of the
pSV
2-transfected cells showed a strong speckled signal located specifically in the cell nucleus (Fig. 2,
D and E). However, very faint staining was
detected in the mock and control transfections (Fig. 2, A
and B, respectively) as well as in pSV
2-transfected cells
incubated first with preimmune rabbit serum instead of the polyclonal
anti-
-recombinase antibodies (Fig. 2C). Similar results were obtained when expression was tested by immunoblotting (Fig. 2F). A specific 25-kDa band, with a mobility corresponding
to that of purified
-protein (Fig. 2F, c
lane), was developed by the anti-
-recombinase antibodies when
COS-1 cells were transfected with the pSV
2 plasmid (+ lane), but not in the mock-transfected cells (
lane). Definitive evidence for the preferential nuclear location of
-recombinase was provided by subcellular fractionation experiments. As shown in Fig. 3, the
specific band corresponding to
-recombinase appeared only on the
nuclear enriched fraction of the pSV
2-transfected cells.
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Fig. 2.
-Recombinase expression in
mammalian cells detected by indirect immunofluorescence and Western
blotting. COS-1 cells, transfected with the indicated plasmids,
were cultured on glass coverslips. After 48 h, cells were fixed
and stained as indicated. A and B correspond to
mock- and pSVc-transfected cells, respectively, incubated with
polyclonal anti-
-recombinase antibodies and developed with
fluorescein-conjugated anti-rabbit IgG. C-E show
pSV
2-transfected cells. In C, the pSV
2-transfected
cells were incubated first with preimmune rabbit serum and then
developed as described above. In D and E, showing
two different fields of the pSV
2-transfected cells, they were
incubated with polyclonal anti-
-recombinase antibodies and developed
as described above. In F, COS-1 cells, transfected as
indicated above, were harvested after 48 h and lysed as described
under "Experimental Procedures." The whole cell extract proteins
were resolved by SDS-polyacrylamide gel electrophoresis, blotted onto
nitrocellulose, and analyzed by Western blotting. Shown is the
autoradiograph of a Western blot incubated first with polyclonal
anti-
-recombinase antibodies and then developed with
peroxidase-conjugated anti-IgM.
lane, mock-transfected
COS-1 cells; + lane, pSV
2-transfected COS-1 cells;
c lane, puriied
-recombinase (10 ng). The molecular mass
of the band of interest is indicated.
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Fig. 3.
Nuclear localization of
-recombinase detected by subcellular fractionation
of nuclei and cytoplasm. COS-1 cells were transfected with the
indicated constructs. After 48 h, cells were harvested and lysed
as described under "Experimental Procedures" to prepare the nuclear
(N) and cytoplasmic (C) fractions. The proteins
from each fraction/condition were resolved by 10% SDS-polyacrylamide
gel electrophoresis (15 µg/lane) and blotted. Shown are three
autoradiographs of the Western blots corresponding to
anti-
-recombinase, anti-
-actin, and anti-histones primary
antibodies, incubated separately using the same membrane (stripped
after each blotting) and developed with the appropriate
peroxidase-conjugated anti-IgM antibodies. Lane 1, no DNA;
lane 2, transfection with the pSVc plasmid; lane
3, transfection with pSV
2; + lane, purified
-recombinase protein (10 ng). The sizes of the bands of interest are
indicated.
-recombinase can be expressed in
eukaryotic environments, showing strong avidity for the nuclear compartment. Additional experiments with stable
-recombinase-expressing clones showed the same cellular
distribution, without affecting cellular
viability.3
-Recombinase Catalyzes Site-specific Recombination in
Transiently Transfected Mammalian Cells--
Unlike integrases with
simple recombination sites, such as Cre and Flp, which catalyze inter-
and intramolecular recombination and do not require additional protein
factors (4, 5, 7),
-recombinase catalyzes intramolecular
recombination and has a strict requirement for a chromatin-associated
protein to mediate DNA recombination (19, 20).
-Recombinase binds to
the six sites and, with the help of a chromatin-associated
protein, promotes strand exchange (Fig. 1A). The accessory
factor is a chromatin-associated protein such as prokaryotic HU or
eukaryotic HMG1 protein (20, 28, 29).
-recombinase was first tested by
transient cotransfections in COS-1 cells with plasmids pSV
2 (bearing
the
-recombinase gene) and pCB8 (the substrate DNA containing two
target sites for
-recombinase in direct orientation flanking the
xylE gene; see Fig. 1B). Upon recombination, two derivatives of pCB8, with a single six site each, should be
obtained. The presence of one of these recombination products can be
easily monitored by PCR amplification of Hirt extracts using primers complementary to the sequences located upstream of one of the six sites (primer a in Fig. 1B and under
"Experimental Procedures") and downstream of the second
six site (primer b in Fig. 1B and under
"Experimental Procedures"). In pCB8, these two primers hybridize to
sequences located >2.7 kilobases apart. Under our PCR conditions, this
fragment was not efficiently amplified; nevertheless, a 555-bp DNA
segment should be amplified from the recombination product. A band of
similar length should be obtained when using the same primers and
plasmid pBT338 as template, which contains a single six site
and was used as positive control (Fig. 1B). After
transfection of the COS-1 cells (48 h), the extrachromosomal fraction
(Hirt extraction) of the cells was therefore purified, and the presence of recombination products was analyzed by PCR. An amplified band of the
expected length (555 bp) was observed only when both pCB8 and pSV
2
plasmids were cotransfected (Fig. 4);
this band was absent when the two DNAs were transfected separately or
when pCB8 was cotransfected with pSVc, the negative control plasmid.
The specificity of the amplified band was further confirmed by Southern hybridization (Fig. 4, lower panel) with a probe specific
for the six site (see Fig. 1A). A positive signal
of the correct size was detected only in the positive control lane
(Fig. 4, pBT338 (+))and in the pCB8/pSV
2 cotransfection
sample. In lanes corresponding to transfections containing the pSV
2
plasmid, the additional band of smaller size detected on the agarose
gel was demonstrated to be nonspecific, as it did not hybridize to the
probe containing the six site sequence. These results
indicate that
-recombinase is active in a eukaryotic environment,
using the machinery/factors provided by the host cell.
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Fig. 4.
Functional expression of
-recombinase assessed in transient transfection
experiments. COS-1 cells were transfected with the indicated
combinations of plasmids: pSVc, pSV
2, pCB8, pSVc + pCB8, and pSV
2 + pCB8. Hirt extracts were obtained 48 h after transfection. The
upper panel shows the result of PCR amplification from the
Hirt extracts using the a/b primer pair indicated in Fig.
1B. The mock lane shows the negative controls of
the PCR amplification, and the pBT338 (+) lane shows the
positive control (100 pg of plasmid). The size marker lane (kilobases)
corresponds to BstEII-digested
DNA (500 ng). The
lower panel shows the Southern blot analysis of the agarose
gel presented in the upper panel using a probe specific for
the six site (see Fig. 1B). The position of the
band of interest is highlighted. kb, kilobases.
-recombinase-mediated process in eukaryotic cells, a new set of
vectors was constructed for recombination-activated gene expression
(Fig. 5A). The assay vector
consisted of the lacZ gene separated from the SV40 early
promoter by the pac gene (which confers resistance to
puromycin in bacteria and eukaryotic cells) flanked by two six sites in direct orientation. Upon recombination, the
pac gene should be excised from the plasmid, leaving the
lacZ gene under the control of the SV40 promoter, thus
rendering expression of
-galactosidase activity. This reporter gene
can easily be monitored and quantified in cell extracts. The negative
control (plasmid pPursixgal) lacks the first six site and is
not a suitable substrate for recombination. A positive control (plasmid
pgal) was obtained by in vitro recombination (19) of the
Recombiner plasmid using purified
-recombinase and further isolation
and characterization.
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Fig. 5.
Recombination-activated gene expression
mediated by -recombinase activity.
A shows the schematic structure of the plasmids used to
transfect a cell line constitutively expressing
-recombinase. The
six sites (triangles) and the genes
pac and lacZ (rectangles) are
indicated. Each transfection was performed in triplicate. After 48 h, the proteins were extracted, and
-galactosidase activity was
measured as described under "Experimental Procedures." B
shows the representation of the mean cpm ± S.D. from each
triplicate condition.
-recombinase-expressing cell line, the whole protein fraction was
extracted from each condition and assayed for
-galactosidase
activity. As shown in Fig. 5B, transfection of the
Recombiner construct promoted
-galactosidase expression several
orders of magnitude higher than the mock and pPursixgal transfections,
indicating that recombination had occurred on that substrate.
Equivalent transfection experiments on the parental cell line not
expressing
-recombinase rendered no detectable
-galactosidase
activity, demonstrating
-recombinase dependence of the measured activity.
-galactosidase activity induced by transfection of the
Recombiner construct was not in the same range as the one obtained with
the positive control (pgal transfection). One plausible reason for this
result could be that recombination occurs in a time period close to
that used in the experimental conditions. Since pgal is already
recombined, expression of
-galactosidase from this plasmid occurs
early after transfection. This is not the case of Recombiner, which has
to become recombined prior to lacZ gene expression. As a
result, the number of cells with recombined plasmid is less in
Recombiner transfection 48 h later than in pgal transfection, and
therefore,
-galactosidase accumulation is reduced.
-Recombinase Promotes Recombination in Structured
Chromatin--
The need of supercoiled DNA has been described as a
critical condition for
-recombinase-mediated deletions between two
directly oriented six sites (20). To explore whether
-recombinase can promote DNA rearrangement when two six
sites form part of the chromatin structure, we established NIH/3T3 cell
clones in which the pCB8 construct was integrated at different
locations within the mammalian genome. Several stable clones were
analyzed by Southern hybridization. Five of them, each carrying a
different copy number of the substrate plasmid (5-75) (data not
shown), were chosen for transient transfection with the
-recombinase
expression plasmid pSV
2. The presence of recombination products was
determined by PCR of genomic DNA preparations using two primers
(pBT338UP158 and pBT338LO802 (see "Experimental Procedures"),
termed primers a' and b', respectively, in Fig. 1B), which
should generate a 668-bp amplified DNA fragment. Amplified DNA
fragments in high copy number clones could be seen directly on agarose
gels (data not shown). In Southern blot assays performed with a probe
specific for the six site, however, a band (~660 bp) was
detected in all cases in the pSV
2-transfected samples (Fig.
6, + lanes). This DNA fragment
did not appear when plasmid pSV
2 was not included in the
transfection (mock transfection;
lanes). Signal strength appeared to correlate with the copy number of the target construction integrated in the chromosome, suggesting that recombination had occurred at many of the integrated target sequences and regardless of
the integration site. Control PCR experiments in mock-transfected NIH/3T3 cells or NIH/3T3 cells transfected with the pSV
2 plasmid were carried out routinely, and no amplified band of 660 bp was detected (Fig. 6, lanes c and d).
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Fig. 6.
-Recombinase-mediated
site-specific recombination in chromatin-associated targets. Five
NIH/3T3 clones (lanes 1-5) harboring several copies (~75,
72, 12, 18, and 5, respectively) of pCB8 DNA integrated in different
chromosomal locations were isolated and characterized. These clones
were transiently transfected, either with (+ lanes) or
without (
lanes) plasmid pSV
2. Genomic DNA was purified
48 h after transfection, and 500 ng of each sample were analyzed
by PCR under the conditions described using the a'/b' primer pair (see
Fig. 1B). One-tenth of each sample was electrophoresed on
0.8% agarose gel, blotted onto nylon membrane, and hybridized with an
appropriate probe. C +,positive controls pBT338 (100 pg)
(lane a) and pBT338 (100 pg) + 500 ng of NIH/3T3 genomic DNA
(lane b); C
, negative controls of NIH/3T3
cells without integrated pCB8 DNA transfected with no DNA (lane
c) and pSV
2 (lane d). The size (0.66 kilobases) and
position of the band of interest are indicated.
-recombinase can catalyze site-specific recombination
in mammalian genomes. It therefore seems that the chromatin structure provides superhelical torsion suitable for
-recombinase-mediated recombination.
DISCUSSION
-recombinase, which belongs to the
resolvase/invertase family of enzymes, can be functionally expressed in
eukaryotic cells and can promote the deletion of DNA sequences located
between directly oriented target sites in mammalian cells.
-Recombinase appears to have high avidity for the nuclear
compartment since, following transfection, it was detected mainly in
the nuclear region, forming a very condensed and speckled pattern on
indirect immunofluorescence. This point was reassessed in subcellular
fractionation experiments (see "Results" and Fig. 3). This behavior
is similar to that observed for the Cre enzyme (13). Cre and
-recombinase do not present a canonical or bipartite nuclear
localization motif in their primary sequence (34, 35). Since they have
access to the nuclear compartment, it is assumed that this localization
occurs by diffusion through the nuclear membrane or following the
transient disorganization of this membrane during mitosis.
-recombinase expression by plasmid pSV
2 promoted
site-specific recombination between the two directly oriented six sites in the substrate plasmid pCB8 when both plasmids
were cotransfected in mammalian cells. As a result, the sequences
between the two target sites were deleted from the DNA substrate. The site-specific recombination product was detected by PCR amplification of the Hirt extracts and reassessed by Southern hybridization of the
amplified products. The presence of this recombination product was
strictly dependent on the cotransfection of plasmids pSV
2 and pCB8;
no recombination products were observed when plasmids pSV
2 and pCB8
were transfected separately. It therefore seems that
-recombinase
can promote strand exchange of an extrachromosomal DNA (pCB8 DNA) in
the mammalian environment, with no detectable spontaneous
recombination. Similar results were obtained in recombination-activated
-galactosidase expression experiments. This reporter gene was designed to be expressed only upon recombination due to
-recombinase (plasmid Recombiner; see Fig. 5). As expected, high expression of
-galactosidase was obtained compared with the negative controls. This experiment not only provides additional evidence of recombination due to
-recombinase in mammalian cells independent of PCR detection, but also confirms the possibility of designing experiments to activate
the expression of genes of interest with an analogue approach.
-mediated
recombination (20, 28). It has recently been observed that
chromatin-associated proteins from plants can also assist
-recombinase in mediating DNA recombination (29), suggesting that
-recombinase might be also suitable for manipulation of plant genomes.
-recombinase to act on
chromatin-integrated target substrates. Several stable NIH/3T3 clones
were established bearing different copy numbers (5-75) of the
substrate plasmid pCB8 randomly integrated in the host chromatin.
Transient
-recombinase expression led to the excision of the
sequences between the two directly oriented six sites; the
recombination product was detected by PCR amplification from purified
genomic DNA and Southern hybridization, and its identity was confirmed
by direct DNA sequencing of the amplified product (data not shown).
-recombinase will expand
the possibilities for the programmed modification of eukaryotic genomes
currently under use.
-Recombinase, used alone or in combination with
the already existing recombination systems, will allow a more specific
spatiotemporal control of the recombination events. Researchers would
have the opportunity to design several independently controlled
recombination events in the same animal or cell, thus providing new,
more flexible solutions to general research. In this respect, different
approaches to assess whether all these recombination systems can work
simultaneously will be of great interest for further investigations.
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ACKNOWLEDGEMENTS |
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We thank Drs. Juan Ortín and Inés Canosa for providing strains and plasmids and Drs. Juan Ortín and Miguel Torres for critical reading of this manuscript. Automated fluorescent sequencing was done by A. Varas and M. A. Gallardo. We also thank Coral Bastos and Catherine Mark for secretarial and editorial support, respectively.
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FOOTNOTES |
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* This work was supported in part by Grant 08.6/0021/1997 from the Comunidad Autónoma de Madrid, Grant SAF95-1548-CO2-02 from the Comisión Interministerial de Ciencia y Tecnología, and European Project RE:BIO4-CT95-0284 (to A. B.) and by Grant PB96-0817 from the Comisión Interministerial de Ciencia y Tecnología (to J. C. A.). The Department of Immunology and Oncology was founded and is supported by the Consejo Superior de Investigaciones Científicas and Pharmacia & Upjohn.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a fellowship from the Ministerio de Educación y Ciencia.
To whom correspondence should be addressed. Tel.:
34-91-585-4562; Fax: 34-91-372-0493; E-mail: abernad{at}cnb.uam.es.
2 V. Díaz, F. Rojo, C. Martínez-A., J. C. Alonso, and A. Bernad, manuscript in preparation.
3 V. Díaz, F. Rojo, C. Martínez-A., J. C. Alonso, and A. Bernad, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: bp, base pair(s); PCR, polymerase chain reaction.
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REFERENCES |
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