# Development of CRTEIL and CETRIZ, Cre-loxP-Based  Systems, Which Allow Change of Expression of Red to Green or  Green to Red Fluorescence  upon Transfection with a

Authors: Masato Ohtsuka; Takayuki Warita; Takayuki Sakurai; Satoshi Watanabe; Hidetoshi Inoko; Masahiro Sato
Journal: Journal of Biomedicine and Biotechnology
 (2009)

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## Abstract

We developed Cre-loxP-based systems, termed CRTEIL and CETRIZ, which allow gene switching in a noninvasive manner. Single transfection with pCRTEIL resulted in predominant expression of red fluorescence. Cotransfection with pCRTEIL and Cre-expression plasmid (pCAG/NCre) caused switching from red to green fluorescence. Similarly, cotransfection with pCETRIZ and pCAG/NCre resulted in change of green to red fluorescence. These noninvasive systems will be useful in cell lineage analysis, since descendants of cells exhibiting newly activated gene expression can be continuously monitored in noninvasive fashion.


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## Body

## 1. Introduction

The
bacteriophage  P1 Cre recombinase, a 38-kDa protein, recognizes  specific 34-bp sequences calledloxP sites and catalyzes site-specific recombination between two loxP sites [1, 2]. The Cre-loxP system has been considered an
important tool for manipulation of genomic sequences and gene expression
including tissue-specific activation or inactivation of genes as well as cell-type-specific
gene targeting [3]. Araki et al. [4] first utilized this system for
manipulation of mouse preimplantation embryos. They constructed a CAG-CAT-Z
transgene, in which expression of the floxed chloramphenicol acetyltransferase
(CAT) gene occurs under control of the ubiquitously active cytomegalovirus
enhancer/chicken β-actin
promoter (CAG) [5]. Expression of the lacZ gene coding for bacterial β-galactosidase protein,
which is located downstream of the floxed CAT sequence in the CAG-CAT-Z
transgene, does not occur in the absence of Cre recombinase protein. When Cre-expression vector DNA
was injected into the pronuclei of fertilized eggs carrying the CAG-CAT-Z
transgene, expression of lacZ protein was induced, indicating that expression
of the lacZ gene is induced after removal of the floxed CAT sequence by Cre
recombinase. Once such recombination occurs, expression of the lacZ gene in
descendant cells will continue throughout embryogenesis.Using a strategy similar to that of Araki
et al. [4], we previously produced a double-reporter mouse line carrying a
pCETZ-17 transgene, which consists of CAG, floxed DNA sequence (containing
enhanced green fluorescent protein (EGFP) cDNA), and the lacZ gene [6]. This
Cre-loxP-based system was useful for
cell lineage analysis in mice. For example, when one blastomere of the 2-cell
embryos derived from CETZ-17 transgenic mice was microinjected with Cre-expression vector
(pCAG/NCre-5) [7], its descendant exhibited Cre-mediated recombination in the
integrated transgenes and also exhibited lacZ activity. However, the offspring
of the other blastomere, into which no injection was performed, remained silent
for lacZ activity. In this case, histochemical detection of lacZ activity is
always associated with fixation of tissues and subsequent staining with X-Gal,
a substrate for lacZ, or by immunodetection using anti-β-galactosidase antibodies. This procedure
often hampers serial microscopic observation of cells expressing newly
activated gene products, and some tissues (i.e., adult ovaries, oviducts, and
epididymides) are rich in endogenous activity for lacZ (unpublished data).In this study, we constructed two new
plasmids, pCRTEIL and pCETRIZ, as double-reporter transgenes. pCRTEIL contains
CAG, floxed sequence (HcRed1 cDNA and CAT gene), EGFP cDNA, internal ribosomal
entry site (IRES) from the encephalomyocarditis virus, and firefly luciferase
(luc) cDNA. pCETRIZ contains CAG, floxed EGFP cDNA and CAT gene, HcRed1 cDNA,
IRES, and lacZ gene. For example, cells carrying pCRTEIL exhibit HcRed1 (red
fluorescence) but neither EGFP nor luc. However, upon transfection with a
Cre-expression vector, they will exhibit EGFP (green fluorescence), because theloxP-flanked sequence in the
integrated pCRTEIL transgenes is removed by transiently-expressed Cre protein.
Thus, the behavior of descendants of the cells exhibiting Cre-mediated gene
activation, which will exhibit EGFP continuously, can be monitored in real-time
in vivo and in vitro.


## 2. Materials and Methods

### 2.1. Plasmid Construction

The
reporter plasmid pCRTEIL (Figure1(a)) was constructed through several cloning
steps. First, pCRT-17, an intermediate product for pCRTEIL, was constructed by
inserting an 8.8-kb fragment containing HcRed1 cDNA + poly(A) sites of the SV40
gene isolated from pHcRed1-N1 (Clontech Laboratories, Inc., Palo Alto, Calif,
USA) in front of the 5′  end of CAT in the pCAG-CAT-lacZ [4] from which the lacZ
gene had already been removed. A DNA fragment containing EGFP cDNA, IRES, and
luc cDNA was then placed immediately downstream of the loxP site located at the 3′ end of the CAT gene in pCRT-17 to
obtain pCRTEIL-6. The resulting pCRTEIL has a backbone of pBluescript SK(-)
(Stratagene, La Jola, Calif, USA). The reporter plasmid pCETRIZ (Figure 1(a))
was constructed in a multistep process. First, pACS, an intermediate product
carrying “I-Sce I-Sfi I-Xba I-Spe I-Sty I-Nhe I-Sfi I-I-Sce I” cassette, was generated by
introducing linker oligo into a pBluescript II-based vector. A fragment
containing IRES, lacZ gene, and poly(A) sites was introduced into a Sty I site (which had been blunt) of
pACS to generate pACSIZA. Next, pCETIZ was generated by ligation of a 4.5-kb Spe I fragment containing CAG promoter, floxed EGFP cDNA and CAT
gene, and poly(A) sites into an Xba I
site of pACSIZA. Finally, pCETRIZ was constructed by introducing PCR-amplified
HcRed1 cDNA into a Spe I site of pCETIZ.(a) Scheme for Cre-loxP-mediated recombination using pCRTEIL and pCETRIZ as reporter
transgenes. Before recombination, the floxed HcRed1/CAT hybrid sequence is
expressed under control of the CAG promoter in cells carrying pCRTEIL, while
the EGFP and luc cDNAs are silent. Similarly, the floxed EGFP/CAT hybrid
sequence is expressed in cells carrying pCETRIZ, but the HcRed1 cDNA and lacZ
genes are silent. Cre-mediated recombination results in deletion of the floxed
sequence, and expression of the EGFP and luc cDNAs in pCRTEIL-carrying cells or
expression of HcRed1 and lacZ in cells carrying pCETRIZ. (b) Plasmids (pCE-29, pCL,
and pCAG/NCre) are used for expression of EGFP, luc, and NCre, respectively.
All plasmids have a pBluescript SK(-) backbone. Abbreviations are CAG:
cytomegalovirus enhancer + chicken β-actin promoter; CAP site: transcription start site; CAT: chloramphenicol
acetyltransferase gene; EGFP: enhanced green fluorescent protein cDNA; IRES:
internal ribosomal entry site; Luc: firefly luciferase cDNA; NCre: nuclear
location signal + Cre gene; pA: poly(A) sites.
(a)(b)For expression of Cre, we used pCAG/NCre
plasmid ([7]; see Figure 1(b)). pCE-29 plasmid [8] was also used as a positive
control vector for expression of EGFP in murine cells (Figure 1(b)). pCL was
constructed by inserting luc cDNA downstream of the CAG promoter (Figure 1(b))
and used as a positive control vector for expression of luc.


### 2.2. Cell Line and Transfection

NIH3T3
cells were first seeded onto gelatin-coated 6-well dishes (number 4810-020;
Iwaki Glass Co., Tokyo, Japan) at a density of
106 cells/well one day before transfection and
grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Co., Carlsbad, Calif,
USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen Co.) at 37°C in
an atmosphere of 5% CO2 in air at 37°C. For transfection of a single
plasmid, four μg of
plasmid DNA was mixed with 8 μl
of LF2000 (number 11668-027; Invitrogen Co.) in Dulbecco’s modified
phosphate-buffered saline without Ca2+ and  Mg2+, pH 7.4 (PBS(-)), and a total of 100 μl solution was prepared
according to the manufacturer’s protocol. For cotransfection, two plasmids (3 μg for each) were mixed
with 12 μl of
LF2000 in PBS(-). These DNA/liposome complexes were added to the cell culture
and incubated for 1 day at 37°C. After transfection, cells were observed for
fluorescence, as described in what follows.


### 2.3. Histological Analysis

Cells and tissue sections were observed using an Olympus
BX60 fluorescence microscope (Olympus, Tokyo, Japan) with DM505 filters
(BP460-490 and BA510IF; Olympus) and DM600 filters (BP545-580 and BA6101F; Olympus),
which were used for EGFP and HcRed1 monitoring, respectively. For detection of
fluorescence in dissected oviducts, an Olympus BX40 dissecting microscope was
used. Microphotographs were taken using a digital camera (FUJIX HC-300/OL; Fujifilm,
Tokyo, Japan) attached to the fluorescence microscope and printed out using a Mitsubishi
digital color printer (CP700DSA; Mitsubishi, Tokyo, Japan).


### 2.4. In Vivo Gene Transfer

Intraoviductal injection was performed as described
previously [9]. One μl
of solution containing plasmid DNA and trypan blue (TB; 0.05% final
concentration) was slowly injected with a glass micropipette, which had been
attached to a mouthpiece,
into the ampulla of an oviduct of B6C3F1 females (4- to 6-week-old; purchased
from CLEA Japan, Inc., Tokyo, Japan). The DNA introduced per oviduct was pCE-29
(0.2 μg), pCRTEIL
(0.2 μg), pCAG/NCre
(0.2 μg), pCRTEIL
(0.2 μg) +
pCAG/NCre (0.2 μg),
or pCL (0.2 μg). In
each transfection group, 0.02 μg of phRL-SV (Promega Co., Madison, Wis, USA) was included as a
control for normalization of luc activity as noted in what follows. After
finishing the injection, the micropipette was rapidly removed. The oviductal
regions were then subjected to in vivo
EP. Eight square-wave pulses with pulse duration of 50 milliseconds and
electric field intensity of 50 V were administered from a square-wave pulse
generator (T820; BTX Genetronics, Inc., San Diego, Calif, USA). One day after in vivo EP, the oviducts were
subjected to observation for fluorescence and then to lysis for  measurement of
luc activity.Oviducts from the mice receiving plasmid DNA
were fixed in 4% paraformaldehyde (PFA) in PBS(-) for 2 days at 4°C, then immersed in 30% sucrose in PBS(-) for
more than 2 days at 4°C, embedded in Tissue-Tek OCT compound (Sakura Finetek Co., Tokyo,
Japan), and finally frozen before serial cryostat sectioning (10–30 μm in thickness).
Sections were mounted in VectraShield mounting medium (Vector Laboratories,
Burlingame, Calif, USA) and observed for fluorescence.To deliver DNA-coated gold particles to the
abdominal skin of adult ICR females in
vivo, we used the Helios gene gun (Bio-Rad Laboratories, Hercules, Calif,
USA). Before the introduction of DNA, ether-anaesthetized mice were shaved in
the abdominal area. Transfection particles were made per Helios gene gun
protocol giving a microcarrier loading quantity of 0.5 mg per shot and a DNA
loading ratio of 2 or 4μg
DNA/mg gold particles (1.0 μm).
For single transfection with pCETRIZ (control), 1 μg of DNA was used. For cotransfection using
pCETRIZ plus pCAG/NCre (experiment), 1 μg of each DNA was used. Mouse abdominal
skin was bombarded with the DNA-coated gold particles using the gene gun
pressurized by a helium tank at 300 psi. One bombardment was carried out per
mouse using 6 mice. In total, 3 transfections were performed for each group.
Animals were killed at 48 hours after gene gun bombardment.Mouse skin was excised and immediately
subjected to observation for fluorescence under UV illumination. The samples were
then fixed with 4% PFA in PBS(-) for 1 day at 4°C. Subsequently, the skin was washed in
PBS(-) twice and stained with X-Gal staining solution (X-gal Staining Assay Kit, Genlantis, Calif,
USA) at 30°C for
24 hours. The stained samples were then washed twice in PBS(-) and observed for
lacZ activity under light.


### 2.5. Assay for Luciferase Activity

Luc
assay was performed using a kit (Dual-Luciferase Reporter Assay System (number
E1910); Promega Co.). The oviducts isolated 1 day after in vivo EP were homogenized in 1 mL of 1 X reporter lysis buffer
(Promega Co.). After centrifugation at
15 000 rpm for 10 minutes at 4°C, the supernatant (200 μl) was transferred to a
fresh Eppendorf tube. Cells 1 day after
transfection were collected with a cell scraper and precipitated after brief
centrifugation. Cell pellets
were then lysed with 1 mL of 1 X reporter lysis buffer. Relative light units
(RLUs) obtained with luc were measured for 5 seconds following a 2-second delay
after the addition of the lysate (10 μl) to 50 μl
of luc assay substrate (Promega Co.) using a luminometer (number TD-20/20;
Turner Designs Instrument, Sunnyvale, Calif, USA). RLUs were normalized to micrograms of
tissue protein added in the luc assay. Tissue protein determinations were
performed using Bradford reagent (Bio-Rad Laboratories, Inc., Hercules, Calif,
USA). At least four oviducts were tested in each transfection group, and values
were presented as means ±
standard  deviation (SD).


### 2.6. Statistical Analysis

Results are presented as means ± SD of
separate experiments. The RLU obtained from transfection into oviductal
epithelium  was analyzed using the SPSS 9.0/PC statistics package (SPSS Inc.,
Chicago, Ill, USA). Results were compared using one-way analysis of variance
(ANOVA) following arcsin transformation of the proportions. The Duncan new multiple
range test or Tukey test was performed if ANOVA revealed significant differences
among compared values.


## 2.1. Plasmid Construction

The
reporter plasmid pCRTEIL (Figure1(a)) was constructed through several cloning
steps. First, pCRT-17, an intermediate product for pCRTEIL, was constructed by
inserting an 8.8-kb fragment containing HcRed1 cDNA + poly(A) sites of the SV40
gene isolated from pHcRed1-N1 (Clontech Laboratories, Inc., Palo Alto, Calif,
USA) in front of the 5′  end of CAT in the pCAG-CAT-lacZ [4] from which the lacZ
gene had already been removed. A DNA fragment containing EGFP cDNA, IRES, and
luc cDNA was then placed immediately downstream of the loxP site located at the 3′ end of the CAT gene in pCRT-17 to
obtain pCRTEIL-6. The resulting pCRTEIL has a backbone of pBluescript SK(-)
(Stratagene, La Jola, Calif, USA). The reporter plasmid pCETRIZ (Figure 1(a))
was constructed in a multistep process. First, pACS, an intermediate product
carrying “I-Sce I-Sfi I-Xba I-Spe I-Sty I-Nhe I-Sfi I-I-Sce I” cassette, was generated by
introducing linker oligo into a pBluescript II-based vector. A fragment
containing IRES, lacZ gene, and poly(A) sites was introduced into a Sty I site (which had been blunt) of
pACS to generate pACSIZA. Next, pCETIZ was generated by ligation of a 4.5-kb Spe I fragment containing CAG promoter, floxed EGFP cDNA and CAT
gene, and poly(A) sites into an Xba I
site of pACSIZA. Finally, pCETRIZ was constructed by introducing PCR-amplified
HcRed1 cDNA into a Spe I site of pCETIZ.(a) Scheme for Cre-loxP-mediated recombination using pCRTEIL and pCETRIZ as reporter
transgenes. Before recombination, the floxed HcRed1/CAT hybrid sequence is
expressed under control of the CAG promoter in cells carrying pCRTEIL, while
the EGFP and luc cDNAs are silent. Similarly, the floxed EGFP/CAT hybrid
sequence is expressed in cells carrying pCETRIZ, but the HcRed1 cDNA and lacZ
genes are silent. Cre-mediated recombination results in deletion of the floxed
sequence, and expression of the EGFP and luc cDNAs in pCRTEIL-carrying cells or
expression of HcRed1 and lacZ in cells carrying pCETRIZ. (b) Plasmids (pCE-29, pCL,
and pCAG/NCre) are used for expression of EGFP, luc, and NCre, respectively.
All plasmids have a pBluescript SK(-) backbone. Abbreviations are CAG:
cytomegalovirus enhancer + chicken β-actin promoter; CAP site: transcription start site; CAT: chloramphenicol
acetyltransferase gene; EGFP: enhanced green fluorescent protein cDNA; IRES:
internal ribosomal entry site; Luc: firefly luciferase cDNA; NCre: nuclear
location signal + Cre gene; pA: poly(A) sites.
(a)(b)For expression of Cre, we used pCAG/NCre
plasmid ([7]; see Figure 1(b)). pCE-29 plasmid [8] was also used as a positive
control vector for expression of EGFP in murine cells (Figure 1(b)). pCL was
constructed by inserting luc cDNA downstream of the CAG promoter (Figure 1(b))
and used as a positive control vector for expression of luc.


## 2.2. Cell Line and Transfection

NIH3T3
cells were first seeded onto gelatin-coated 6-well dishes (number 4810-020;
Iwaki Glass Co., Tokyo, Japan) at a density of
106 cells/well one day before transfection and
grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Co., Carlsbad, Calif,
USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen Co.) at 37°C in
an atmosphere of 5% CO2 in air at 37°C. For transfection of a single
plasmid, four μg of
plasmid DNA was mixed with 8 μl
of LF2000 (number 11668-027; Invitrogen Co.) in Dulbecco’s modified
phosphate-buffered saline without Ca2+ and  Mg2+, pH 7.4 (PBS(-)), and a total of 100 μl solution was prepared
according to the manufacturer’s protocol. For cotransfection, two plasmids (3 μg for each) were mixed
with 12 μl of
LF2000 in PBS(-). These DNA/liposome complexes were added to the cell culture
and incubated for 1 day at 37°C. After transfection, cells were observed for
fluorescence, as described in what follows.


## 2.3. Histological Analysis

Cells and tissue sections were observed using an Olympus
BX60 fluorescence microscope (Olympus, Tokyo, Japan) with DM505 filters
(BP460-490 and BA510IF; Olympus) and DM600 filters (BP545-580 and BA6101F; Olympus),
which were used for EGFP and HcRed1 monitoring, respectively. For detection of
fluorescence in dissected oviducts, an Olympus BX40 dissecting microscope was
used. Microphotographs were taken using a digital camera (FUJIX HC-300/OL; Fujifilm,
Tokyo, Japan) attached to the fluorescence microscope and printed out using a Mitsubishi
digital color printer (CP700DSA; Mitsubishi, Tokyo, Japan).


## 2.4. In Vivo Gene Transfer

Intraoviductal injection was performed as described
previously [9]. One μl
of solution containing plasmid DNA and trypan blue (TB; 0.05% final
concentration) was slowly injected with a glass micropipette, which had been
attached to a mouthpiece,
into the ampulla of an oviduct of B6C3F1 females (4- to 6-week-old; purchased
from CLEA Japan, Inc., Tokyo, Japan). The DNA introduced per oviduct was pCE-29
(0.2 μg), pCRTEIL
(0.2 μg), pCAG/NCre
(0.2 μg), pCRTEIL
(0.2 μg) +
pCAG/NCre (0.2 μg),
or pCL (0.2 μg). In
each transfection group, 0.02 μg of phRL-SV (Promega Co., Madison, Wis, USA) was included as a
control for normalization of luc activity as noted in what follows. After
finishing the injection, the micropipette was rapidly removed. The oviductal
regions were then subjected to in vivo
EP. Eight square-wave pulses with pulse duration of 50 milliseconds and
electric field intensity of 50 V were administered from a square-wave pulse
generator (T820; BTX Genetronics, Inc., San Diego, Calif, USA). One day after in vivo EP, the oviducts were
subjected to observation for fluorescence and then to lysis for  measurement of
luc activity.Oviducts from the mice receiving plasmid DNA
were fixed in 4% paraformaldehyde (PFA) in PBS(-) for 2 days at 4°C, then immersed in 30% sucrose in PBS(-) for
more than 2 days at 4°C, embedded in Tissue-Tek OCT compound (Sakura Finetek Co., Tokyo,
Japan), and finally frozen before serial cryostat sectioning (10–30 μm in thickness).
Sections were mounted in VectraShield mounting medium (Vector Laboratories,
Burlingame, Calif, USA) and observed for fluorescence.To deliver DNA-coated gold particles to the
abdominal skin of adult ICR females in
vivo, we used the Helios gene gun (Bio-Rad Laboratories, Hercules, Calif,
USA). Before the introduction of DNA, ether-anaesthetized mice were shaved in
the abdominal area. Transfection particles were made per Helios gene gun
protocol giving a microcarrier loading quantity of 0.5 mg per shot and a DNA
loading ratio of 2 or 4μg
DNA/mg gold particles (1.0 μm).
For single transfection with pCETRIZ (control), 1 μg of DNA was used. For cotransfection using
pCETRIZ plus pCAG/NCre (experiment), 1 μg of each DNA was used. Mouse abdominal
skin was bombarded with the DNA-coated gold particles using the gene gun
pressurized by a helium tank at 300 psi. One bombardment was carried out per
mouse using 6 mice. In total, 3 transfections were performed for each group.
Animals were killed at 48 hours after gene gun bombardment.Mouse skin was excised and immediately
subjected to observation for fluorescence under UV illumination. The samples were
then fixed with 4% PFA in PBS(-) for 1 day at 4°C. Subsequently, the skin was washed in
PBS(-) twice and stained with X-Gal staining solution (X-gal Staining Assay Kit, Genlantis, Calif,
USA) at 30°C for
24 hours. The stained samples were then washed twice in PBS(-) and observed for
lacZ activity under light.


## 2.5. Assay for Luciferase Activity

Luc
assay was performed using a kit (Dual-Luciferase Reporter Assay System (number
E1910); Promega Co.). The oviducts isolated 1 day after in vivo EP were homogenized in 1 mL of 1 X reporter lysis buffer
(Promega Co.). After centrifugation at
15 000 rpm for 10 minutes at 4°C, the supernatant (200 μl) was transferred to a
fresh Eppendorf tube. Cells 1 day after
transfection were collected with a cell scraper and precipitated after brief
centrifugation. Cell pellets
were then lysed with 1 mL of 1 X reporter lysis buffer. Relative light units
(RLUs) obtained with luc were measured for 5 seconds following a 2-second delay
after the addition of the lysate (10 μl) to 50 μl
of luc assay substrate (Promega Co.) using a luminometer (number TD-20/20;
Turner Designs Instrument, Sunnyvale, Calif, USA). RLUs were normalized to micrograms of
tissue protein added in the luc assay. Tissue protein determinations were
performed using Bradford reagent (Bio-Rad Laboratories, Inc., Hercules, Calif,
USA). At least four oviducts were tested in each transfection group, and values
were presented as means ±
standard  deviation (SD).


## 2.6. Statistical Analysis

Results are presented as means ± SD of
separate experiments. The RLU obtained from transfection into oviductal
epithelium  was analyzed using the SPSS 9.0/PC statistics package (SPSS Inc.,
Chicago, Ill, USA). Results were compared using one-way analysis of variance
(ANOVA) following arcsin transformation of the proportions. The Duncan new multiple
range test or Tukey test was performed if ANOVA revealed significant differences
among compared values.


## 3. Results

Successful in Vitro Cre-Mediated Recombination Results in Switching of Gene Expression
In
a preliminary test, we first examined what percentage of single cells can
incorporate two constructs simultaneously; NIH3T3 cells were cotransfected with
pCRTEIL and pCE-29 by the lipoplex method. One day after transfection,
fluorescent cells were inspected under a fluorescence microscope. Eight percent
(12/142) and 10% (14/142) of cells exhibited EGFP-derived green and
HcRed1-derived red fluorescence, respectively, (data not shown). Twenty-two percent
(31/142) of cells expressed both types of fluorescence (data not shown). It was
thus estimated that in approximately 20% of single cells, incorporation of two
constructs into a single cell occurs at the same time. To test whether
Cre-mediated excision of the floxed HcRed1/CAT sequence in the pCRTEIL
construct results in generation of EGFP fluorescence (Figure1(a)), NIH3T3
cells were cotransfected with pCRTEIL and pCAG/NCre by the lipoplex method. One
day after transfection, some (9% (10/112)) of these cells exhibited both red
and green fluorescence (indicated by arrowheads in Figures 2(d)–2(f)), suggesting
incomplete Cre-mediated recombination in the introduced pCRTEIL construct
(Table 1). There were a few cells (4% [4/112]) preferentially expressing green
fluorescence (indicated by arrows in Figures 2(d)–2(f)), suggesting
the occurrence of complete Cre-mediated recombination in the transfected cells
(Table 1). Twenty-four percent (27/112) of cells (Table 1) expressed
only red fluorescence, suggesting incorporation of two constructs at the same
time in a single cell (though gene switching failed) or incorporation of
pCRTEIL alone. Considering that 20% of cells can incorporate two constructs
simultaneously, the rate of Cre-mediated recombination in this system appears
to be greater than 50% at a minimum. In the control experiment in which cells
were transfected with pCRTEIL alone, some cells (35% (36/102); Table 1)
exhibited HcRed1-derived red fluorescence but not EGFP-derived green
fluorescence (arrows in Figures 2(b) versus
2(c)).
Similar results were also obtained when
pCETRIZ was transfected singly or cotransfected with pCAG/NCre (Figures2(g)–2(n)). In this
system, expression of lacZ protein arises from the recombined form of pCETRIZ
together with HcRed1 after Cre-mediated recombination. In fact, cells
expressing HcRed1 exhibited lacZ, which could be visualized after fixation of
cells and subsequent staining with X-Gal (arrows in Figures 2(k)–2(n)). Transfection
of cells with a single pCETRIZ resulted in expression of EGFP but not of both
HcRed1 and lacZ (arrows in Figures 2(g)–2(j)). These
findings indicate that pCRTEIL and pCETRIZ as reporter plasmids work well with
cultured murine cells.Table 1
Summary of efficiency of Cre-mediated recombination in NIH3T3 cells after cotransfection  with tester and Cre-expression constructs.
No gene switching1Complete gene switching1Incomplete gene switching1Plasmid construct(s) usedTotal number of cells examinedNo. of cells expressing
only HcRed1-derived
fluorescence (%)No. of cells expressing
only EGFP-derived
fluorescence (%)No. of cells expressing
both types of fluorescence (%)pCRTEIL10236 (35)0 (0)0 (0)pCRTEIL
+
pCAG/NCre11227 (24)4 (4)10 (9)1Cells one day after transfection were observed for fluorescence under a fluorescence microscope, and regions containing fluorescent and neighboring nonfluorescent cells were photographed. The fluorescent cells were classified into groups with no gene switching (cells exhibiting only red fluorescence; as exemplified by the cells (arrowed) in Figure 2(b)), complete gene switching (cells exhibiting only green fluorescence; as exemplified by the cell (indicated by arrowheads) in Figures 2(e), 2(f)), or incomplete gene switching (cells exhibiting both fluorescence; as exemplified by the cell (arrowed) in Figures 2(e), 2(f)). Regions comprising 30–40 cells were photographed, and fluorescent cells were scored. The numbers of cells in a total of 3 regions are listed in the table.Figure 2
Cre-mediated excision in vitro. (a)–(c) NIH3T3 cells
transfected with pCRTEIL alone. When cells were inspected for fluorescence 1
day after transfection, HcRed1-derived red fluorescence (but not EGFP-derived
green fluorescence) was predominant (indicated by arrows in (b)). (a)–(c) Microphotographs
indicate the same cells. (d)–(f) NIH3T3 cells
cotransfected with pCRTEIL and pCAG/NCre. Some cells predominantly exhibited
green fluorescence (indicated by arrows in (e) and (f)), while other cells
exhibited both types of fluorescence (indicated by arrowheads in (e) and (f)). (d)–(f) Microphotographs
indicate the same cells. (g)–(j) NIH3T3 cells
transfected with pCETRIZ alone. When cells were inspected for fluorescence 1
day after transfection, EGFP-derived green fluorescence (but not HcRed1-derived
red fluorescence and lacZ expression) was predominant (indicated by arrows in (h)).
(g)–(j) Microphotographs
indicate the same cells. (k)–(n) NIH3T3 cells
cotransfected with pCETRIZ and pCAG/NCre. Cells exhibited both green and red
fluorescence as well as lacZ expression (indicated by arrows in (l), (m), and
(n)). (k)–(n) Microphotographs
indicate the same cells. (a), (d), (g), and (k) Photographs taken under light
(Light); (b), (e), (i), and (m) photographs taken under UV illumination using
filters for detection of red fluorescence (HcRed1); (c), (f), (h), and (l)
photographs taken under UV illumination using filters for detection of green
fluorescence (EGFP); (j) and (n) photographs taken under light after staining
with X-Gal (lacZ). Bar: 10μm.Successful in Vivo Cre-Mediated Recombination in pCRTEIL System
We
next examined whether this Cre-loxP system using pCRTEIL as a reporter plasmid can also be used in vivo. For this purpose, gene
delivery into oviductal epithelium was performed by DNA injection into the
lumen of oviducts and subsequent EP [9]. Instillation of pCE-29 plasmid yielded
bright green fluorescence throughout the ampulla ((b) in Figure 3(A))
but not red fluorescence ((c) in Figure 3(A)).
When pCRTEIL plasmid DNA was
singly introduced, no fluorescence for EGFP was observed ((e) in Figure 3(A)). Instead,
red fluorescence was observed in some oviductal epithelial cells ((f) in Figure
3(A)). Coinjection with pCRTEIL and pCAG/NCre plasmids resulted in
generation of both red and green fluorescence, although green fluorescence
appeared to be weaker than red fluorescence (indicated by arrows in (h) and (i)
in Figure 3(A)).
To examine the localized expression of EGFP and HcRed1 in
the oviducts after cotransfection, cryostat sections were prepared and
inspected for EGFP and red fluorescence. Strong, but patchy, EGFP fluorescence
was observed in some oviductal epithelial cells (indicated by arrows in (k) in
Figure3(A)), together
with weak red fluorescence throughout the EGFP-positive area (data not shown).
These findings indicate that the Cre-loxP system using pCRTEIL works well even in
vivo, and as expected HcRed1 exhibits a nonoverlapping spectral profile
from EGFP.
The IRES sequence is
present between EGFP and luc cDNAs in the pCRTEIL plasmid (Figure1(a)). Therefore, the two proteins (EGFP and
luc) should be produced at the same time in a cell into which pCRTEIL and
pCAG/NCre have been cotransfected, since an IRES sequence allows
cap-independent translation initiation [10, 11] from the dicistronic mRNA
generated. In fact, we observed increase in luc activity repeatedly when
oviductal epithelial cells were cotransfected with pCRTEIL and pCAG/NCre
(Figure 3(B)). This finding also
suggests successful in vivo
Cre-mediated recombination.Cre-mediated excision in vivo.
(A): (a)–(c) Oviducts
transfected with pCE-29 alone. EGFP-derived green fluorescence (but not
HcRed1-derived red fluorescence) was observed. (d)–(f) Oviducts
transfected with pCRTEIL alone. HcRed1-derived red fluorescence (but not
EGFP-derived green fluorescence) was observed. (g)–(i) Oviducts
cotransfected with pCRTEIL and pCAG/NCre. Both types of fluorescence (indicated
by arrows in (h) and (i)) were seen, although green fluorescence appeared to be
weaker than red fluorescence. (j) and (k) Cryostat sections of oviducts sampled
1 day after introduction of pCRTEIL and pCAG/NCre DNA (0.2μg each) and subsequent in vivo electroporation. Note that
strong, but patchy, EGFP fluorescence presents in some oviductal epithelial cells (indicated
by arrows in (k)). (a), (d), (g), and (j) Photographs taken under light
(Light); (b), (e), (h), and (k): photographs taken under UV illumination using
filters for detection of green fluorescence (EGFP); (c), (f), and (i)
photographs taken under UV illumination using filters for detection of red
fluorescence (HcRed1). (B) Luciferase reporter gene activity in oviduct sampled 1
day after in vivo gene delivery
to oviducts. Oviducts were in vivo transfected with pCRTEIL alone (negative control),
pCRTEIL and pCAG/NCre (experiment), pCAG/NCre alone (negative control), or pCL
(positive control). Oviducts were isolated from each mouse at 2 days following
electroporation to measure luc activity. From a total of 8 females injected
with DNA, 4 oviducts were subjected to measurement of luc activity for each
transfection group. Note also that the vertical axis of the figure is
interrupted and has one scale. Different letters indicate statistically
significant differences (P<.001).Successful in Vivo Cre-Mediated Recombination in pCETRIZ System
Cells carrying pCETRIZ are expected to exhibit HcRed1 and
lacZ after Cre-mediated excision. For testing the feasibility of pCETRIZ-based
Cre-loxP system in vivo, tissues that never exhibit
endogenous β-galactosidase
activity must be chosen. In this occasion, skin epidermis appeared to be
suitable for this purpose, because it does not exhibit such kind of activity
[12] and has been considered as an attractive target to study gene delivery due
to its easy accessibility and visualization [13]. Since particle-mediated
devices have been studied to deliver genes to skin efficiently [12, 14, 15], we
introduced pCETRIZ (control) or pCETRIZ + pCAG/NCre (experiment) into mouse
skin using Helios gene gun. Skin biopsies were taken after 48 hours for
inspection of fluorescence and lacZ staining. In the samples bombarded with
single pCETRIZ, green fluorescence was detected over the area gene-transfected
(Figure 4(b)). However, neither red fluorescence nor lacZ activity was noted in
the area exhibiting green fluorescence (Figures 4(c) and 4(d)). This was also
observed in the other 2 injected mice. Samples bombarded with pCETRIZ +
pCAG/NCre showed coexpression of green and red fluorescence (arrows in Figures
4(f) and 4(g)), suggesting successful Cre-mediated excision of the introduced
pCETRIZ. As expected, lacZ staining revealed expression of lacZ over the area
cotransfected (Figure 4(h)). These patterns of gene expression were also seen
in the other 2 injected mice.Figure 4
Gene gun-mediated skin transfection with pCETRIZ (a)–(d) and pCETRIZ +
pCAG/NCre (e)–(h). After shaving
of the abdominal area of mice, plasmid DNA coated with gold particles was
transfected with a 300-psi
helium gas pulse using the Helios gene gun. Two days after gene delivery, skin
was dissected and inspected for fluorescence under a UV illuminator. After
observation for fluorescence, the same sample was then subjected to
histochemical staining for lacZ activity. Figures (a)–(c) and (e)–(g) are
photographs of the same portion. Figure (d) is a different portion from those
in Figures (a)–(c). Similarly,
Figure (h) is a different portion from those in Figures (e)–(g). (a), (d), (e),
and (h) taken under light; (b) and (f): taken under UV illumination for
detection of GFP fluorescence; (c) and (g): taken UV illumination for detection
of red fluorescence. Bar: 10μm.


## 4. Discussion

The Cre-loxP system permits the generation of mouse models, in which the
fate of a cell can be followed through time. In such models, a cell-type-specific
promoter that is turned on at a given stage of differentiation regulates Cre-expression. When
combined with a reporter system, regulated expression of Cre may lead to
specific expression of inductive reporter molecules and permanent tagging of
those cells, which have completed a defined genetic program. An essential
component of such strategy is the development of appropriate reporter strains
of mice in which the inducible reporter molecule is ubiquitously expressed.Several reporter strains carrying
different floxed transgenes and exhibiting switching of gene expression upon
Cre-mediated recombination have been reported in mice (i.e., CAG-CAT-Z [4],
Rosa26R (R26R) [16], Z/AP [17], Z/EG [18], ROSA26flox [19], CMV-floxed
stop-lacZ [20], ACZL [21], and CETZ-17 [6]). Almost all have employed a single
fluorescent marker gene, alkaline phosphatase, (ALP) and/or lacZ gene. The most
commonly utilized strain appears to be Rosa26R mice [16], which are generated
by targeting a reporter construct to the proviral ROSA26 locus that is
constitutively expressed in all tissues. In these reporter strains carrying
lacZ or ALP, detection of these reporters following Cre-mediated recombination
should always be performed for fixed samples, thus hampering continuous
observation of activated gene products in living cells or embryos. There are
only a few reports on reporter strains capable of exhibiting noninvasive
fluorescent markers upon Cre-mediated excision of a “stop sequence.” Such
strains carry a single fluorescent marker (EGFP for Schwenk et al. [22], Mao et
al. [23], Motoike et al. [24], and Kawamoto et al. [25], DsRed-T3 for Vintersten
et al. [26], and ECFP or EYFP for Srinivas et al. [27]), placed downstream of
the floxed stop sequence. In our CRTEIL system, the reporter plasmid contains
two fluorescent markers (i.e., floxed HcRed1 cDNA and EGFP placed downstream of
the floxed gene) enabling monitoring of gene switching based on changes from
red to green fluorescence (see Figure 1(a)). This type of Cre excision reporter
construct was first reported by Yang and Hughes [28], who adopted two
fluorescent markers (EGFP and DsRed1 in their reporter plasmid, called Cre stoplight) and
demonstrated that cells carrying Cre stoplight as transgene can be converted from red to green by
transient  expression of Cre. However, DsRed1 protein has displayed toxicity in
murine embryos [29]. For these reasons, we used HcRed, a dimeric far-red
fluorescent protein variant [30] isolated from the coral reef Heteractis crispa, which exhibits a
spectral profile (exc. 588 nm, em. 618 nm) that does not overlap with those of
other existing fluorescent proteins. HcRed1 has already been used as a safe
noninvasive protein marker in a transgenic system [31].Recently, Muzumdar et al. [32] generated  mT/mG; a double-fluorescent Cre
reporter mouse that expresses membrane-targeted tandem dimer Tomato (mT)  prior
to Cre-mediated excision and  membrane-targeted green fluorescent protein (mG) after excision. The
use of a double-fluorescent marker system by Muzumdar et al. [32] is the same
as ours. They observed that this strain works well in vivo after mating with the pre-existing Cre expressor
strains. This encouraged us to generate transgenic mouse strains carrying
CRTEIL/CETRIZ transgenes. The advantage of our system over that of Muzumdar et
al. [32], may be the employment of IRES-luc and IRES-lacZ. These elements will
be useful when tissue samples are after Cre-mediated excision subjected to
biochemical and histochemical analyses, since we can measure the level of luc
or lacZ quantitatively using commercially available kits (e.g., the Luminescent
β-galactosidase
Detection Kit II (Clontech Laboratories, Inc., Palo Alto, Calif, USA)) after
observation for fluorescence. In this study, ca. 630-bp IRES placed between
EGFP and luc sequences was useful for simultaneous expression of the two
proteins, though the level of luc placed downstream of the IRES appeared to be
greatly decreased (approximately 1/400 of luc activity in the oviducts
transfected with pCL alone; see Figure 3(B)). This appears to be
due to the insertion of IRES between EGFP and luc, since the level of luc in
the oviducts transfected with pCTL (composed of CAG promoter, loxP-flanked CAT
sequence, and luc gene) + pTC (carrying an NCre gene controlled by Herpes
simplex virus-derived thymidine kinase promoter) was only 1/4 of that in the oviducts
transfected with pCL alone [33]. In this sense, use of 81-bp 2A sequence, which
is known to yield synthesis of two different proteins from a single mRNA
[34, 35], may be appropriate in our system, since similar levels of each of the
proteins have been reported to be produced in cells transfected with the
2A-containing construct [36].In this study, we have described in vitro and in vivo switching of gene expression from red to green or green
to red fluorescence by the Cre-loxP system
when pCRTEIL and pCETRIZ plasmids are used as tester plasmids. These events
occurred when tester plasmid and Cre-expression plasmid are cotransfected (see Figures 2–4). In our
LF2000-based transfection system, approximately 20% of NIH3T3 cells could
incorporate two constructs simultaneously, of which more than 50% exhibited
Cre-mediated gene switching. This rate appears to be too low, since in our
previous experiments using F9 embryonal carcinoma cell line stably transfected
with loxP-containing reporter plasmid
pCETZ-17, all clones exhibited gene switching within one day after transient
expression of pCAG/NCre [6]. This means that 100% gene switching is possible if
stable cell lines carrying a reporter construct are subjected to Cre-mediated
transfection. The low efficiency in Cre-mediated recombination in the present
study may be due solely to the cotransfection method itself, since we cannot
strictly control the number of copies of exogenous gene introduced into cells.The present Cre-loxP system based on two fluorescent markers appeared to have great
advantages over the previous systems, since the process of change in gene
expression can be easily monitored in noninvasive fashion. Furthermore, it is
possible to trace the fate of the descendants that express fluorescence under physiological
conditions. For finer analysis of the fate of cells transfected with exogenous Cre-expression vector, it
will be convenient to use transgenic mouse lines carrying the CRTEIL or pCETRIZ
transgene as generalized reporter strains. This experiment is now underway.



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*Source: 985140-2009-04-01.xml*