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INTRODUCTION |
Site-specific recombinases have become a routine tool for
conditional gene modifications, as an alternative to classical gene targeting technologies (1-3). These systems, which allow programmed intermolecular recombination, overcome some of the limitations of the
classical knock-out systems, such as embryonic lethality or generation
of compensation mechanisms. Cre-loxP is currently the system
of choice, because of its ability to induce targeted changes in model
animals (1, 4). Using this system, tissue-specific, conditional, and
inducible gene targeting events in a wide variety of tissues and organs
have been reported (5-8). Although the utility of these systems is
unquestionable, to date only the Cre-loxP, and to a lesser
extent, the Flp/FRT systems are currently under use. Only very
recently, the Int/attP site-specific recombination system from
phage has proven to work successfully in plants (9). Moreover, there
are relatively few reports on the combined use of these systems (10).
It thus becomes important to study and characterize other site-specific
recombination systems (SSR)1
that could be used as an alternative to or in combination with existing ones.
-Recombinase from the Gram-positive plasmid pSM19035 induces
specific intramolecular recombination in mammalian cells, both in
episomal plasmids and in chromatin-associated substrate structures (11). Plasmid pSM19035 has an unusual structure, because almost 80% of
the molecule consists of a repeated sequence and two replication origins (12). Because replication of this plasmid follows the classical
theta model, a mechanism must exist to ensure its complete replication
(inversion) and maximization of plasmid segregation (resolution). The
development of an in vitro recombination system based on the
use of purified
-recombinase demonstrated both inversion and
deletion activities associated to the protein (13), delimited the
sequences required for directing the SSR reactions (13, 14), and
characterized the requirements for both reactions (13). Unlike Cre and
Flp, which belong to the Int recombinase family,
-recombinase is
included in the family of resolvases/invertases and has the interesting
property of catalyzing exclusively intramolecular recombination events
(13, 15). At difference from Cre and Flp site-specific recombinases,
which do not require additional factors (reviewed in Refs. 16 and 17),
-recombinase requires for deletions a supercoiled substrate and a
chromatin-associated protein (e.g. bacterial Hbsu or
eukaryotic HMG1 proteins) (14, 15, 18). We showed previously that the
mammalian cell environment can provide such a host factor and that
nuclear genomic DNA supercoiling is suitable for
-recombinase
function (11).
To obtain additional insights regarding
-recombinase system
application for the manipulation of eukaryotic genomes, we have developed a recombination-activated gene expression (RAGE) system dependent on
-recombinase activity that consists of the
lacZ reporter gene separated from the promoter by the
pac gene flanked (sixed) by two minimal
six sites (19) in direct orientation, equivalent to those
described for other models (1-3, 20). In addition, we have also
obtained and characterized a novel hybrid protein, composed of
-recombinase fused through its C-terminal end to enhanced green
fluorescent protein (EGFP) from Aequorea victoria. As for
the Cre-loxP system (21), combination of these two
approaches results in a simple, rapid way to enrich cell clones that
have undergone the desired recombination event after
-EGFP transient
expression, using fluorescence-activated cell sorting (FACS). This
approach may be suitable for increasing the efficiency of
locus-specific recombination events during the generation of mouse
conditional knock-out approaches or for the deletion of the positive
selection cassette in mouse conventional knock-out models, which might
alter or influence the final phenotype (10, 22).
Using these novel approaches we have studied the host protein factor
requirement for
-recombinase protein in mammalian cells. In previous
works, several proteins were demonstrated to replace in
vitro the chromatin-associated factor required by
-recombinase reaction (15, 18). More precisely, histone-like proteins Hbsu and HU
can efficiently contribute to the recombination process. The identity
of this host factor in eukaryotic environments is still unknown,
although different HMG1-like proteins of mammalian and plant origin
have the ability to facilitate the formation of nucleoprotein
structures to different extents, because they can efficiently replace a
bacterial chromatin-associated protein required for the site-specific
-mediated recombination (15, 18, 23, 24). HMG1/2, which are abundant
components of the eukaryotic nucleus, bind any linear DNA with moderate
affinity and no sequence specificity (25) but bind with high affinity to DNA that is already sharply bent (26-28); thus they are considered structural chromatin proteins expressed in all eukaryotic cell types.
However, recent evidences have demonstrated that HMG1/2 are also
recruited to DNA by interactions with proteins required for basal and
regulated transcription (29-34) and for SSR machinery, as the
RAG/V(D)J recombination system (reviewed in Ref. 35), the major
determinant for diversity generation of antibody and T-cell receptor.
These novel data strongly suggest an additional role as expression
regulator of specific gene families or physiological molecular
events mediated by the recombination machinery (36). Therefore, HMG1
and other closely related mammalian proteins (i.e. HMG2 and
HMG4) are potential candidates to act as host factors in
-recombinase reactions in mammalian cells, although HMG2 is expressed at a much lower level than HMG1 (37), and HMG4 is almost
exclusively expressed during embryonic stages of development (38).
Here we show that overexpression of HMG1 does not induce a significant
enhancement on
-mediated recombination both on episomal plasmid-based targets or chromatin-integrated constructs. Furthermore,
-mediated recombination is efficiently performed in a HMG1 knock-out cell line. These data clearly suggest that in eukaryotic environments there are other chromatin-associated proteins that could act as helpers
in
-mediated reactions, and therefore it is expected that those
reactions could occur in most cell types and tissues. In conclusion,
the results presented highly support the future usefulness of the
/six system as a new tool for conditional modification of eukaryotic genomes.
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EXPERIMENTAL PROCEDURES |
Plasmids--
Plasmids pBT233 and pCB103 have been
previously described (12, 19). Plasmids pEGFP-N1, p
gal-Basic and
pPur (CLONTECH, Palo Alto, CA), and pGEM-T
(Promega, Madison, WI) were from commercial sources. Plasmid pHMG1 (36)
was kindly provided by Dr. M. Bianchi (Milan, Italy).
To fuse the
-recombinase gene to EGFP in the pEGFP-N1 vector, we
introduced appropriate 5' HindIII and 3' EcoRI
sites on the
gene from pBT233. Using primers
Hind5
(5'-GAGAGAAAGCTTGGTTGAAAATGGCT-3') and
Eco3C
(5'-ACTATCCCTCTTTCCC-3'), we performed PCR amplification (30 cycles at
94 °C 1 min, 48 °C 1 min, 72 °C 1 min, followed by one cycle
of elongation at 72 °C 5 min). The 635-bp amplification product was
joined to HindIII/EcoRI-digested pEGFP-N1
following standard cloning procedures. The resulting plasmid,
p
-EGFP, was purified for transfection experiments with Qiagen columns.
For recombination-activated gene expression, we designed a set of
three vectors that were obtained as follows. A 93-bp six site was obtained by PCR amplification from pCB103, for which we used
the pUC reverse primer and a primer containing a novel SmaI/XmaI site needed for further cloning which
we called 103M (5'-CAGTGCCAAGCTTCCCGGGCTGCAGG-3'). The amplified
product was purified and cloned using the pGEM-T Easy system, giving
rise to the pGEM-T/103M plasmid. This was digested with KpnI
and HindIII, and the resulting fragment containing the
six site was cloned in the same restriction sites of the
p
gal-Basic vector, resulting in the psixgal vector containing the
lacZ gene downstream of a six site. The
BamHI fragment containing the six-lacZ cassette from this vector was cloned in pPur vector
(CLONTECH), resulting in the pPursixgal plasmid,
the negative control vector used in all experiments (see Fig.
2A). The last six site was obtained from psixgal
by digestion with XmaI and cloned in the AgeI
site from pPursixgal to obtain Recombiner, the substrate for
recombination used in this study (see Fig. 2A). The pgal
vector (positive control) was obtained by in vitro
recombination essentially as described (12, 13), using 1 µg of
Recombiner as substrate, 320 nM purified
-recombinase, and 100 nM Hbsu. The resulting reaction
was digested with SacII, which only cuts in
Recombiner molecules but not in pgal. Transformation of this
restriction reaction rendered pgal containing bacterial colonies for
further purification of this plasmid.
For retroviral transduction of the HMG1-defective cell line, the
-EGFP open reading frame from p
-EGFP plasmid was cloned in
pLZR-CMV-GFP retroviral vector (39) upon digestion with
BamHI and NotI restriction enzymes and subsequent
ligation. The resulting plasmid, pLZR-
-EGFP, was further packaged in
retroviral particles as described below.
Cell Lines and Cultures--
NIH/3T3 cell line was obtained from
the American Type Culture Collection (CRL-1658). 293T cells were
originally called 293tsA1609ne (40) and derived from 293 cells, which
are a human embryonic kidney cell line. HMG1 KO and wt counterpart cell
lines were a gift from Dr. M. Bianchi (36). All cell lines were
cultured in Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) supplemented with 10% fetal calf serum (Cultek, Madrid, Spain) and 2 mM L-glutamine (Merck).
Cell Transfection and Retroviral Transduction--
To obtain
stable transformants of the
-EGFP fusion or the recombination
activable Recombiner construct, plasmid DNA (20 µg) was
introduced in NIH/3T3 cells by electroporation of 2 × 106 cells at a concentration of 107 cells/ml in
supplemented Dulbecco's modified Eagle's medium, pulsed at 220 V, 950 microfarads (Bio-Rad Gene Pulser). Cells were replated, and after
48 h of further culture, selection antibiotics were added to the
medium. For selection of stable clones bearing the
-EGFP fusion
construct we used 1 mg/ml G418, and for the Recombiner
construct, 2 µg/ml puromycin. Clones were collected and analyzed
10-15 days after electroporation. Transient transfection was done in
all cases using Superfect reagent (Qiagen), according to
manufacturer's instructions. FACS analysis and
-Gal detection were
performed 48 h after transfection.
Packaging of pLZR-
-EGFP retroviral vector was performed transiently
in 293T as described (39). Viral supernatants were titrated on NIH-3T3
cells on the basis of GFP fluorescence. 35-40 TU/cell were used to
transduce both HMG1-defective cells and the corresponding +/+ control
cell line. Cells expressing
-EGFP protein were purified by sorting
and recultured for transfection with Recombiner construct.
Plasmidic DNA fraction was purified for PCR analysis 48 h after
transfection using Qiagen miniprep purification kit.
Fluorescence Microscopy and FACS--
In vivo
expression of
-EGFP fusion was easily detected by direct observation
of cultures in a phase-contrast microscope equipped with a fluorescent
lamp and blue filter. Indirect immunostaining was performed after
fixing cells with 3% formaldehyde, 5% Triton X-100 for 20 min at room
temperature. Blocking was performed with 100 mM
NH4Cl in phosphate-buffered saline for 10 min.
Anti-
-recombinase rabbit antibody (11) was used at a 1:500 dilution.
Secondary Cy3-labeled goat anti-rabbit IgG (H+L) antibody was purchased from Amersham Pharmacia Biotech and used at 1:400 dilution. For Hoechst
33258 staining, cells were incubated with 0.8 µg/ml Hoechst 33258 (Molecular Probes, Eugene, OR) for 5 min, immediately after second
antibody incubation. Samples were mounted and analyzed using a Leitz
DMINB epifluorescence microscope and a Leica TCSNT confocal scanning
microscope. Images were noise-filtered, corrected for background, and
processed using Adobe Photoshop.
FACS analysis using an EPICS Elite sorter was performed on transfected
cells that were trypsin-treated, centrifuged, and resuspended in 1 ml
of phosphate-buffered saline. Once isolated, each population was
recultured in 100-mm dishes with antibiotics, which were eliminated after 2 days. Isolated clones normally appeared 7-10 days
post-sorting.
Analysis of Recombination Products--
Isolated clones were
harvested from 24-well plates when they reached confluence, resuspended
in 1× PCR buffer with MgCl2 containing 0.5% Tween-20 and
100 µg/ml proteinase K and incubated 45 min at 56 °C. Proteinase K
was heat inactivated (10 min, 95 °C) and 1/10 of the resulting
reaction was used for PCR detection in a Perkin Elmer thermocycler
under the following conditions: 94 °C, 5 min; 30 cycles of 94 °C
for 30 s, 60 °C for 30 s, 72 °C for 1 min, and final
elongation at 72 °C for 5 min. Primers used for PCR detection were
sixUP (5'-GCCCAGTTCCGCCCATTCTC-3') and sixDO (5'-CCAGACCGGCAACGAAAATC-3'). Although in the unrecombined substrate a
1.5-kb product could be obtained, our PCR protocol was optimized for
the exclusive amplification of the recombination-diagnostic product
corresponding to a 0.4-kb band, which must result specifically from the
presence of recombined molecules.
For normalization of PCR reactions, an internal (351 bp) fragment of
the LacZ gene was amplified, using the following primers: lacZ-1
(5'-GTGGTGGTTATGCCGATCG-3') and lacZ-2 (5'-TACCACAGCGGATGGTTCGG-3'). The reaction conditions were the same as described for sixUP and sixDO primers.
Detection of THS
gene as internal template loading control was
performed using primers THS
UP (5'-TCCTCAAAGATGCTCATTAG-3') and
THS
LO (5'-GTAACTCACTCATGCAAAGT-3') under the following conditions: 94 °C for 2 min and 30 cycles of 94 °C for 30 s, 60 °C
for 30 s, 72 °C for 30 s, and final elongation at 72 °C
for 5 min. Quantitative PCR was performed on a
LightCyclerTM system (Roche Molecular Biochemicals).
Samples were amplified during 40 cycles in the same conditions
described above, adding SYBR green dye to the reaction. The amount of
recombination product (template for sixUP/sixDO primers) was calculated
using a duplicate standard curve of 10-fold dilutions of the pgal
plasmid, and the results were normalized after subtracting the
nontemplate control, by the amount of total lacZ template
(see above). Three independent transfection experiments were analyzed,
and for each one, at least four amplification reactions were performed.
Southern analysis of PCR products was performed by blotting DNA
separated on agarose gels onto nylon membranes (Hybond-N+;
Amersham Pharmacia Biotech). Filters were hybridized at 65 °C in
Rapid-hyb buffer (Amersham Pharmacia Biotech), using supplier's recommendations. Southern analysis of genomic DNA was performed after
overnight digestion with EcoRV and StuI (Roche)
of 10 µg of genomic DNA obtained following standard procedures (41)
and subsequent agarose gel separation and blotting.
The specific probe for hybridization was obtained by digestion of
Recombiner with ClaI and EcoRV and
purification of the resulting 289-bp DNA fragment. Probes were
radiolabeled using the Rediprime kit (Amersham Pharmacia Biotech).
RT-PCR--
Total RNA from transfected cells was extracted using
Tri-Reagent (Sigma) using conditions recommended by supplier. 1 µg
from each condition was treated with RNase-free DNase (Promega) to avoid contamination from genomic and/or plasmid DNA from the
transfection. RT reaction was performed using Superscript II RT kit
(Life Technologies, Inc.) according to the supplier conditions. HMG1
cDNA detection was detected by PCR using the specific primers
HMG1RTUP (5'-CCTCGCGGAAAATCAAC-3') and HMG1RTLO
(5'-CAAAAACCAAACTACTGTCC-3'). PCR conditions were as follows:
94 °C for 4 min and 25 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min, and final elongation at 72 °C for 5 min.
Loading controls were performed upon detection of GAPDH gene under the
same conditions, using the primers GAPDH5' (5'-CATCACCATCTTCCAGGAGC-3') and GAPDH3' (5'-CATGAGTCCTTCCACGATACC-3').
Northern Blotting--
Total RNA from transiently
transfected cells was extracted using Tri-Reagent (Sigma), according
manufacturer's instructions, and 40 µg from each condition were
loaded on agarose gels (1× MOPS, 37% paraformaldehyde). Once
separated electrophoretically, the RNAs were transferred to nylon
membranes (Hybond-N+, Amersham). Blotting and labeling of
the specific probe was performed as for Southern hybridizations.
-Gal Detection--
Cells transfected in 35-mm dishes were
harvested after 48 h. Proteins were extracted using the
luminescent
-Gal detection kit from CLONTECH.
Total protein was quantified, and equal amounts of each extract were
analyzed. Luminescence was measured in a scintillation counter
(Wallac, EG&G Instruments, Madrid, Spain).
 |
RESULTS |
-EGFP Fusion Behaves as a Nuclear Protein That Can Be
Constitutively Expressed in Mammalian Cells--
The
-recombinase
open reading frame was cloned into the pEGFP-N1 vector to fuse this
gene with the 5' end of the EGFP gene. The resulting p
-EGFP plasmid
was transiently transfected in NIH/3T3 cells. In contrast to
transfections with pEGFP-N1, fluorescence microscopy showed a
nucleus-restricted pattern in the transfected cells (Fig.
1A). This nuclear signal was
observed more clearly using confocal microscopy (Fig. 1B).
The nuclear pattern is not homogeneous, because
-EGFP recombinase
localizes in heterochromatin regions. This compartmentalization was
confirmed by counterstaining other gene products that localize in these
regions (not shown). Furthermore, living mitotic cells expressing the
fusion protein show brighter signals in the centromeric areas of the
chromosomes (Fig. 1B), which have been assigned to
heterochromatin (42). The
-EGFP signal was confirmed by
immunostaining with anti-
-recombinase specific antibodies (not
shown). These results confirm and extend our previous observation on
the spontaneous subcellular localization of
-recombinase in
eukaryotic cells (11).

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Fig. 1.
Intracellular distribution of the
-EGFP protein. NIH 3T3 cells were transfected
with the pEGFP-N1 vector or -EGFP expression plasmid (p -EGFP) and
analyzed 48 h later, as indicated. A, fluorescence
microscopy of cells transfected with plasmid pEGFP-N1 (left
panel) or with p -EGFP (right panel). B,
confocal microscopy images showing -EGFP fluorescence of transfected
living cells at different cycle stages. C, differential
expression of -EGFP protein in stable clones transfected with
p -EGFP in NIH 3T3 cells line. Lane 1, phase
contrast microscopy of living cells. Lane 2, in
vivo fluorescence of fields shown in lane 1. Lane
3, fluorescence distribution of each clone analyzed by flow
cytometry.
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To assess the feasibility of stable
-EGFP fusion protein expression
in living cells, plasmid p
-EGFP was transfected in NIH/3T3 cells,
and stable clones were obtained. Different levels of expression of
-EGFP were observed when analyzed by fluorescence microscopy (Fig.
1C, clones B3, D3, and D4) and by cytometry (Fig.
1C, lane 3). We were unable to obtain clones with
very high expression levels, suggesting a deleterious effect of either
high doses of
-recombinase or EGFP expression, although results in
other systems argue for a EGFP-mediated toxic effect (43). Selected
-EGFP-expressing clones developed normally (Fig. 1C,
lane 1), and growth rate parameters were equivalent to those
of wt cells (not shown), demonstrating that sustained
-EGFP
expression is compatible with normal cell processes. The NIH/3T3 clone
/GFP.D4 was selected for further experiments because of its high,
stable, and uniform
-EGFP expression pattern.
The
-EGFP Protein Catalizes SSR--
To elucidate whether
fusion to EGFP would alter the enzymatic activity of
-recombinase,
/GFP.D4 cell clone was transiently transfected with the
Recombiner construct (11) containing the minimal directly
oriented six sequences (93 bp), which direct
-recombinase-mediated deletions (19). The Recombiner
reporter system was designed for in vivo expression of
-Gal activity after
-recombinase-mediated elimination of the
sixed pur open reading frame (Fig.
2A). The structure of the
Recombiner reporter system was tested in vitro as
a suitable substrate for
-recombinase activity (Fig. 2B),
and the control reporter plasmid pgal was isolated from the in
vitro reaction (Fig. 2B, lane C2). The
insertion of six sites upstream of or flanking the reporter
genes used in this RAGE system does not appear to interfere with normal
gene expression (Fig. 2C). This is important in genomic
tissue-specific modification experiments, in which it is essential to
maintain the original expression levels of the gene to be deleted
before conditional manipulation, and in tissues other than that
designed to express
-recombinase. Conversely, constitutive
expression through the recombination substrate does not appear to
affect
-recombinase function (Fig. 2D). After
transfection with Recombiner or with control plasmids,
pPursixgal and pgal, cell protein extracts were obtained for
quantitative analysis of
-Gal expression.
-Gal activity was
detected only in transfections with Recombiner and pgal
constructs (Fig. 2D), but not in the negative control and pPursixgal, which is not a suitable
-recombinase substrate because it lacks one of the two directly oriented six sites required
for the recombination process. Taken together, these results
demonstrate that
-EGFP protein maintains the recombinase activity
and suggest that fusion of other protein domains at the
-recombinase
C terminus would not affect its activity.

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Fig. 2.
Generation of a RAGE system for
-recombinase. A, schematic
representation of the plasmids used. Open arrows indicate
the SV40 early promoter. Triangles correspond to minimal
six sites (93 bp). The relative positions of primers sixUP
and sixDO used in PCR assays are indicated. B, in
vitro recombination assay using purified -recombinase, Hbsu
protein as accessory factor, and plasmid Recombiner as
substrate. The reactions were performed as described under
"Experimental Procedures," digested with EcoRV and
SacII, and resolved in agarose gels. Restriction fragments
from the unrecombined plasmid (NR, 2.9 and 6.2 kb) and of
the recombined plasmid (R, 7.9 kb) are indicated. The
fragment of 1.2 kb runs off the gel under these conditions and is not
shown in the figure. Lanes C1 (purified
Recombiner) and C2 (purified pgal) show the
restriction patterns of the constructs not subjected to in
vitro recombination. C, Northern blot detection (40 µg of total mRNA) of pac gene mRNA in NIH 3T3
cells transfected with the indicated plasmids. Lanes A and
B correspond to different plasmid preparations of
Recombiner. D, /GFP.D4 cells transfected with
the indicated plasmids were evaluated for -Gal activity. The graph
shows the mean relative units (RU) ± S.D.
corresponding to -Gal activity from each triplicate condition
48 h after transfection. One unit corresponds to the cpm value in
pUC18 transfection.
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Combined Use of RAGE and
-EGFP Fusion for the Improved Selection
of Site-specific Genome-modified Cellular Clones--
The use of
-EGFP fusion in combination with the developed RAGE system has been
also useful for isolation of cellular clones harboring site-specific
recombined chromatin-associated targets. The Recombiner
plasmid was transfected in the NIH 3T3 cell line, and several resistant
clones were obtained after puromycin selection. Clone 3T3/D6.3 was
selected for further study, because it demonstrated the highest
-Gal
levels after
-EGFP expression (not shown). This clone harbored
several copies of the target sequence (not shown). In a first approach,
we generated resistant clones after transfection of the 3T3/D6.3 line
with p
-EGFP or pEGFP-N1 plasmids, followed by G418 selection. Clones
from the p
-EGFP transfection (Fig.
3A, lanes 1-6) and
from pEGFP-N1 (lane C) were recultured and analyzed. Both
the Southern analysis and
-Gal detection showed heterogeneous
recombination levels in the different clones when compared with the
untransfected clone (Fig. 3A, lane C). These results indicate the feasibility of the approach and suggest a distinct
accessibility of
-recombinase to the copies of Recombiner structure within the chromatin of clone 3T3/D6.3, probably dependent on
the distinct level of the
-recombinase induced in the cell clones.

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Fig. 3.
A, -EGFP-mediated recombination on
chromatin associated targets. Clone 3T3/D6.3 was transfected with
plasmid p -EGFP, and several double-stable clones were analyzed.
Protein extracts of these clones were prepared, and -Gal activity
was measured as described under "Experimental Procedures." The
upper panel graphs the -Gal activity for each clone; the
lower panel shows a Southern analysis of genomic DNA from
these clones digested with EcoRV and SacII and
hybridized with a lacZ gene-specific probe. The positions of
bands of interest are indicated. Lane C, untransfected
clone. Lanes 1-6, different stably transfected clones.
Intensity ratios (1.9-kb band/1.4-kb band) for each lane: 1.8, 2.0, 1.0, 0.46, 1.74, and 1.58. B, recombination-activated gene
expression of cells expressing -EGFP fusion protein after cell
sorting. Genomic DNA of several individual clones was prepared and
analyzed by PCR using sixUP and sixDO primers to detect recombination
events. The resulting products were separated on agarose gels
amplification and blotted. The upper panel shows the result
of hybridization with a lacZ-specific probe. The positive
control of this PCR reaction (+) was prepared using 100 pg of plasmid
pgal as template. To ensure homogeneous loading of genomic DNA in each
sample, a control PCR reaction was performed to detect the TSH gene
(0.4-kb fragment, lower panel). In this case, the positive
control consisted of 500 ng of mouse genomic DNA. In both PCR
reactions, the negative control ( ) corresponds to a mock reaction
amplification without template DNA.
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In an experimental design similar to that previously described with the
Cre-loxP system (21), clone 3T3/D6.3 was transiently transfected with
the p
-EGFP plasmid; 48 h later, cells were trypsinized and
analyzed by flow cytometry. The typical fluorescence distribution in
these cells after transfection with p
-EGFP or pEGFP-N1, rendered ~12-20% positive cells (not shown). We sorted populations of
moderate-high expression (+, levels similar to those obtained in stable
clones; see Fig. 1B), very high expression (++), and no or
very poor expression (
). Sorted cells (85-95% enriched) were
further cultured in growth medium without antibiotic selection until
isolated colonies had formed, and each one was processed separately for
genomic DNA isolation and PCR analysis (see Fig. 2A for
scheme). The PCR amplified fragments were separated on agarose gels,
blotted, and hybridized with a probe for the lacZ gene to
ensure detection and specificity of the amplified diagnostic product
(0.4 kb). Almost 80% of the + group (intermediate-high expression)
showed signal amplification (Fig. 3B), whereas in the ++
group (very high expression), only 50% were recombination-positive
clones. This result again suggests some type of deleterious effect in
the ++ group, concurring with the expression levels observed in stable
plasmid p
-EGFP clones.
HMG1 Is Not an Essential Requirement for the
-Recombinase-mediated Reactions in Mammalian Environments--
The
need for a host factor to ensure in vitro recombination in
the
/six system has been previously reported (13, 15). The host
factor is also provided in eukaryotic environments, but its identity
remains unknown. In vitro reactions with HMG1 proteins of
both plant and animal origins have proven to work as efficient accessory factors for
-mediated recombination (18, 23). HMG1 and
other members of the same family are thus good candidates for host
factors in
-mediated recombination reactions in eukaryotic cells. We
tested the influence of ectopic HMG1 expression in in vivo
reactions carried out with Recombiner as substrate.
Transient cotransfections with a HMG1 expression plasmid (36) and
Recombiner in
/GFP.D4 cell line, resulted in a positive
influence on the
-mediated recombination on the episomal substrate
when detected by RAGE (Fig.
4A). Overexpression of HMG1 in
the transfection experiments was confirmed by semiquantitative PCR
(Fig. 4C) using specific primers for the corresponding
mRNA (see "Experimental Procedures"). It has been previously
described that ectopic HMG1 expression could promote an enhancer effect
on expression levels through different promoters (34). To evaluate
whether this effect could be mediating in the increment observed in
-Gal expression, we performed transient pHMG1 and/or pgal
transfections in the
/GFP.D4 line (not shown), demonstrating such an
effect (approximately 2-fold) on SV40-directed
-Gal expression. This
increment seems to account for most of the total effect obtained in the
RAGE recombination analysis (Fig. 4A). This conclusion has
been confirmed with independent criteria, making use of quantitative
PCR for the monitoring of the recombination events (Fig.
4D). In this set of experiments we were not able to
demonstrate any reproducible enhancement of the
-mediated
recombination of the Recombiner substrate when HMG1 was also
cotransfected in the cells. We therefore conclude that the enhancement
of RAGE
-Gal expression observed (Fig. 4A) is the
consequence mainly of the increment in SV40 promoter activity and not
of a significant improvement in the recombination rate.

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Fig. 4.
Influence of HMG1 expression in
-mediated recombination in the
/GFP.D4 clone (A) and in the
3T3/D6.3 clone (B) of the indicated plasmids. At
48 h after transfection, proteins were extracted, and -Gal
activity was measured. Each transfection was performed in triplicate;
the representations correspond to the mean value obtained from each
triplicate condition ± S.D. expressed in relative units
(RU). 1 unit is arbitrarily assigned to -Gal activity
detected in pUC18 transfection (background levels). C,
upper panel, detection of the expression of HMG1 mRNA
upon transfection with the indicated plasmids. Total RNA from
transfected cells was extracted 48 h after transfection, and the
cDNAs corresponding to mRNAs was obtained using RT. HMG1
cDNA levels were estimated using semi-quantitative PCR conditions.
The number of cycles was adjusted to nonsaturation conditions. The
lower panel corresponds to the detection of the housekeeping
gene GAPDH. HMG1 KO cell line was used as negative control for this
detection. D, quantification of recombination levels upon
Recombiner cotranfection with (+ pHMG1 lane) or
without pHMG1 plasmid ( pHMG1 lane) in /GFP.D4 cell
line using quantitative PCR (see "Experimental Procedures").
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The influence of HMG1 in chromatin-associated targets was also
evaluated. Fig. 4B shows the result of the transfection of p
-EGFP alone or combined with pHMG1 in the 3T3/D6.3 line. In this
case, overexpression of HMG1, also monitored by semiquantitative PCR
(not shown) did not show a remarkable effect. In addition, we also
confirmed that overexpression of HMG1 by cotransfection with the
p
-EGFP plasmid does not appear to influence the efficiency of the
-mediated recombination nor the distribution of
-recombined clones in 3T3/D6.3 line, after sorting/cloning experiments equivalent to those previously described (Fig. 3B). Again, the
recombined clones are detected exclusively within the intermediate
positive clones with a mean efficiency of the 70%, remarking some type of toxic or deleterious effect because of high
-EGFP expression levels.
Finally, to address whether HMG1 is a critical factor for the
-mediated recombination processes, we analyzed the efficiency of the
specific
/six system recombination in a HGM1-defective skin
fibroblast murine cell line, derived from the knock-out model (36). For
that purpose we developed a retroviral vector for the stable
transduction and expression of the
-EGFP protein in this
HMG1-knock-out cell line in comparison with a control one, derived from
wt littermates. Transduced and GFP-sorted HMG1 knock-out and wt cell
populations were further transfected with the reporter Recombiner plasmid. Analysis by semiquantitative PCR of the
recombination products generated during the first 24 h after the
transfection of Recombiner in
-EGFP-transduced
HMG1-defective cells showed that
-mediated recombination occurs, in
absence of HMG1, at a very comparable level with respect to the HMG1 wt
control cell line (Fig. 5), indicating
the existence of other eukaryotic cellular factors that can act as
helpers of the recombination process. These results were further
confirmed by quantitative PCR analysis of independent experiments (not
shown).

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Fig. 5.
-Mediated SSR in HMG1-deficient
cells. Upper rows, detection of recombination events by
PCR using specific primers (see "Experimental Procedures") in HMG1
wt and HMG1 KO cell lines. lane, Recombiner
plasmid. + lane, pgal plasmid. HMG1 KO cell line and the
HMG1 wt control cell line were transduced with the pLZR- -EGFP
retroviral vector for constitutive and stable expression of -GFP
fusion protein. After sorting and reculture of the GFP positive cells,
these were transfected with the indicated plasmids and the levels of
recombination were evaluated by semi-quantitative PCR in the indicated
conditions (see "Experimental Procedures"). -GFP transduced
lanes correspond to transfections in HMG1 wt or KO cells retrovirally
transduced with pLZR- -EGFP retroviral vector (see "Experimental
Procedures"). The lower rows show PCR detection of
LacZ gene, which was used as an internal loading control for
the reaction.
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DISCUSSION |
SSR technologies have widely expanded the possibilities of genomic
manipulation in living organisms. With these systems, strict spatio-temporal control of the induced manipulation can be achieved, eliminating the consequences of undesired lethal effects because of
systemic lack of an essential gene (see Refs. 44 and 45) or bypass
effects because of uncontrollable redundant mechanisms that result in
the absence of detectable phenotype changes (46, 47).
In contrast to Cre-loxP and Flp/FRT systems,
-six model
requires a host protein factor and a supercoiled substrate to catalyze resolution reactions (18). In a previous report, we showed that
-recombinase is active in eukaryotic cells (11), implying the existence of at least one eukaryotic host factor that can contribute to
the recombination reaction and that the eukaryotic chromatin organization can provide the supercoiling needed for
-mediated recombination.
To evaluate in more depth
-recombinase activity in the eukaryotic
environments, we developed an easily detectable fusion protein that
maintains both the recombinase activity and the subcellular localization properties described for this protein (Ref. 11 and Figs. 1
and 2). The data presented here clearly demonstrate that the
-EGFP
fusion presents no major modifications in its recombination activity.
It is therefore possible to assume that the C-terminal region of
-recombinase is flexible and that fusion of other protein regulatory
domains would behave similarly. This result opens the future
possibility of introducing regulatory domains for temporal-inducible
control of
-recombinase activity, as the fusion of the ligand
binding domain from receptors corresponding to several hormone systems,
as shown for the Cre-loxP model (48-50).
The use of
-EGFP protein also revealed a stronger preference of
-recombinase for the heterochromatin regions within the nucleus. The
preferential nuclear localization can be explained by the bipartite
nuclear localization signal (51-53) present in the C-terminal portion
(amino acids 184-200) of
-recombinase, potentially implicated in
active transport to the cell nucleus via specific mechanisms
in eukaryotic cells. We also show that sustained
-recombinase
activity is compatible with normal cell growth (Fig. 1). Stable
-EGFP expressing clones were developed after p
-EGFP transfection
of NIH/3T3 cells, and they show homogeneous expression of the chimeric
protein, which is easily detected both by in vivo microscopy
and by flow cytometry.
The existence of recombination in a transcriptionally active structure
(Recombiner) ensures that the transcription machinery does
not interfere with the recombination process. Conversely, the insertion
of six sites between the promoter regions and the gene to be
expressed has no significant effect on the transcription level (data
not shown). This is an important feature for a system intended for use
in conditional gene manipulation, because the goal of such an approach
is to maintain unmodified levels of the gene product of interest
(designed as a "floxed/sixed" construct) before induction of the
targeted deletion, or outside the tissue/organ in which recombination
is expected to occur. The development of the described RAGE system
coupled to the use of the
-EGFP version of the
-recombinase has
allowed the efficient enrichment of populations expressing
-recombinase activity and the evaluation of several mechanistic and
practical aspects of the
/six system applied to the targeted
modification of the mammalian genome.
The role of HMG1 as a putative critical host factor in
-mediated
recombination was first evaluated through the analysis of the
consequences of HMG1 overexpression on the
-mediated recombination efficiency. Cotransfection of a plasmid encoding HMG1 in experiments parallel to those described above seemed to show a clear enhancing effect on recombination efficiency (Fig. 4A). When
Recombiner is introduced as an episomal substrate, HMG1
overexpression promotes a 3-fold increment in
-Gal expression
compared with transfections with Recombiner alone. However,
previous reports showed that HMG1 expression enhances the expression of
promoters such as SV40 early promoter or cytomegalovirus (34).
By analyzing the effect of HMG-1 overexpression on the levels of
-galactosidase expression in the recombined product and measuring
recombination efficiency by using quantitative PCR, we determined that
the observed effect was mainly due to this recombination-independent
effect (Fig. 4D), although we could not rule out completely
a slight enhancement on the recombination reaction. In this regard, the
only mammalian SSR system, the RAG/V(D)J mechanism, critically involved
in generation of antibody and T cell receptor diversity, is highly
stimulated by HMG1 and 2 in vitro (reviewed in Ref. 35).
HMG1/2 proteins are proposed to stabilize the DNA bending because of
the binding of RAG1 protein to its target sequences, behaving like a
clamp to ensure a stable and favorable recombination structure.
HMGs may stabilize the recombination complex by providing minor groove DNA contacts in the vicinity of the specific recombination sequences in
this system (35). However, this mechanistic implication was not
formerly substained from the results obtained recently on the HMG1
knock-out model (36), where no great defect was noted in the immune repertoire.
In the
-mediated six-specific recombination system,
the exogenous introduction of HMG1 expression constructs does not exert a remarkable effect, neither on episomic target plamids nor in chromatin-associated target structures, suggesting that HMG1 is not a
critical factor for the
-mediated recombination in eukaryotic environments. To confirm this suggestion, we attempted to define whether HMG1 was the principal contributor to
-recombinase-mediated reaction in eukaryotic cells. We performed transfections of
Recombiner in a HMG1-defective cell line expressing
-EGFP
fusion and searched for recombination products. PCR detection showed
the existence of efficient recombination caused by
-recombinase in
this HMG1-defective environment (Fig. 5). These data indicate that
there are other cellular factors that, at least, can substitute for
HMG-1 in the recombination reaction. Moreover, episomal targets could
adopt in the mammalian nucleus a recombination-efficient structure
caused by organization of a chromatin-like structure in such
environment, regardless of specific factors. This would minimize
possible problems derived from a theoretical unavailability of factor
in experimental applications of the
-recombinase system.