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ORIGINAL PAPER
New lines of GFP transgenic rats relevant for regenerativemedicine and gene therapy
S. Remy • L. Tesson • C. Usal • S. Menoret • V. Bonnamain •
V. Nerriere-Daguin • J. Rossignol • C. Boyer • T. H. Nguyen •
P. Naveilhan • L. Lescaudron • I. Anegon
Received: 24 June 2009 / Accepted: 8 December 2009 / Published online: 22 January 2010
� Springer Science+Business Media B.V. 2010
Abstract Adoptive cell transfer studies in regener-
ative research and identification of genetically mod-
ified cells after gene therapy in vivo require
unequivocally identifying and tracking the donor cells
in the host tissues, ideally over several days or for up
to several months. The use of reporter genes allows
identifying the transferred cells but unfortunately
most are immunogenic to wild-type hosts and thus
trigger rejection in few days. The availability of
transgenic animals from the same strain that would
express either high levels of the transgene to identify
the cells or low levels but that would be tolerant to the
transgene would allow performing long-term analysis
of labelled cells. Herein, using lentiviral vectors we
develop two new lines of GFP-expressing transgenic
rats displaying different levels and patterns of GFP-
expression. The ‘‘high-expresser’’ line (GFPhigh) dis-
played high expression in most tissues, including
adult neurons and neural precursors, mesenchymal
stem cells and in all leukocytes subtypes analysed,
including myeloid and plasmacytoid dendritic cells,
cells that have not or only poorly characterized in
previous GFP-transgenic rats. These GFPhigh-trans-
genic rats could be useful for transplantation and
immunological studies using GFP-positive cells/tis-
sue. The ‘‘low-expresser’’ line expressed very low
levels of GFP only in the liver and in less than 5% of
lymphoid cells. We demonstrate these animals did not
develop detectable humoral and cellular immune
responses against both transferred GFP-positive
splenocytes and lentivirus-mediated GFP gene trans-
fer. Thus, these GFP-transgenic rats represent useful
tools for regenerative medicine and gene therapy.
Keywords Transgenic rats � Lentiviral vectors �Dendritic cells � Neural stem/progenitor cells �Immune tolerance
S. Remy (&) � L. Tesson � C. Usal � S. Menoret �V. Bonnamain � V. Nerriere-Daguin �J. Rossignol � C. Boyer � P. Naveilhan �L. Lescaudron � I. Anegon (&)
INSERM, U643, 30 Bd Jean Monnet,
44093 Nantes cedex 01, Nantes, France
e-mail: [email protected]
I. Anegon
e-mail: [email protected]
S. Remy � L. Tesson � C. Usal � S. Menoret �V. Bonnamain � V. Nerriere-Daguin �J. Rossignol � C. Boyer � P. Naveilhan �L. Lescaudron � I. Anegon
CHU Nantes, Institut de Transplantation et de Recherche
en Transplantation, ITERT, 44093 Nantes, France
S. Remy � L. Tesson � C. Usal � S. Menoret �V. Bonnamain � V. Nerriere-Daguin �J. Rossignol � C. Boyer � P. Naveilhan �L. Lescaudron � I. Anegon
Faculte de Medecine, Universite de Nantes,
44093 Nantes, France
T. H. Nguyen
INSERM, U948, 44093 Nantes, France
T. H. Nguyen
CHU Nantes, Nantes, France
123
Transgenic Res (2010) 19:745–763
DOI 10.1007/s11248-009-9352-2
Introduction
Adoptive cell transfer studies or cell trafficking
analysis in regenerative and transplantation research
requires unequivocally identifying and tracking the
donor cells in the host tissue over a given time period.
In this context, the development of appropriate
animal models is increasingly required. Thus, trans-
genic animals stably expressing at high levels and in
different cell types is an attractive and useful
approach. Nevertheless, it has become increasingly
clear that the transfer of cells expressing a reporter
molecule into immunocompetent hosts triggers an
immune response directed against the labelled cells
(Stripecke et al. 1999; Gambotto et al. 2000; Rosen-
zweig et al. 2001). This problem considerably
hampers the use of cells labelled with any immuno-
genic marker, especially in cell-therapy models
requiring long-term analysis of the fate of the
transferred cells. Thus, the availability of animals
that are tolerant to a given marker protein would be
extremely useful.
The rat is a model of choice in several experi-
mental settings (Aitman et al. 2008) and recent
advances in genetic engineering have resulted in the
development of transgenic rats expressing reporter
proteins such as b-galactosidase (Menoret et al. 2002;
Takahashi et al. 2003) or enhanced-green fluorescent
protein (eGFP) (Hakamata et al. 2001; Ito et al. 2001;
Sawamoto et al. 2001; Lois et al. 2002; van den
Brandt et al. 2004; Cronkhite et al. 2005; Inoue et al.
2005; Itakura et al. 2007; Michalkiewicz et al. 2007).
EGFP, a fluorescent protein derived from the jellyfish
Aequorea Victoria (Prasher et al. 1992), is an ideal
marker for labelling cells, since its expression is
stable in mammalian cells and its visualization in situ
is easy, quantitative and non-invasive (Chalfie et al.
1994). Several previous groups have generated GFP-
transgenic rat strains as tool for organ transplantation
research, adoptive cell transfer experiments or devel-
opment studies. Nevertheless, depending on the
promoter and likely genome integration locus, dif-
ferent pattern and level of GFP expression are
observed. In many GFP-transgenic rat lines, the
expression of the reporter molecule was only assessed
by a macroscopic analysis of various organs/tissues
(Hakamata et al. 2001; Inoue et al. 2005; Mich-
alkiewicz et al. 2007). Other teams have generated
GFP-transgenic rats by using lentiviral vectors to
demonstrate the feasibility and the efficiency of this
method but, information concerning phenotypic
characteristics of these animals is scarce (Lois et al.
2002; Hamra et al. 2002; van den Brandt et al. 2004).
Other lines have been described to express GFP in
specific cell types, such as male and female germline
(Cronkhite et al. 2005), mesencephalic precursor cells
(Sawamoto et al. 2001) or pituitary folliculo-stellate
cells (Itakura et al. 2007), restricting their utility to
specific studies.
In the present study, we have generated new lines
of GFP-transgenic rats displaying different expres-
sion patterns and levels of the reporter molecule, and
so might be useful for different experimental settings.
One of them, identified as the ‘‘high-expresser’’
line (GFPhigh), strongly expressed eGFP in multiple
tissues and in important cell types, especially in
mature neurons and in leukocytes subtypes including
myeloid and plasmacytoid dendritic cell (DCs),
populations never characterised in existing GFP-
transgenic rat lines. We also demonstrated a strong
accumulation of GFP in stem cells of neural or
mesenchymal origin. Such animals could potentially
be used as a source of GFP-positive cells in
regenerative medicine or immunological studies.
The other line, identified as the ‘‘low-expresser’’
line (GFPlow), expressed only very low levels of
eGFP in the liver and in cells from peripheral blood
or lymphoid organs. Nevertheless, we demonstrated
that GFPlow-animals did not develop any humoral and
cellular immune responses against both transferred
GFP-positive splenocytes from GFPhigh-animals and
lentivirus-mediated GFP gene transfer. GFPlow-ani-
mals could thus be used as recipients of GFPhigh- cells
or tissues as well as in gene therapy studies, allowing
for the long-term analysis of the outcome of GFP-
labelled cells. Such transgenic rats tolerant toward
GFP have never reported in the literature.
Materials and methods
Generation of eGFP-transgenic rats using
lentiviral vectors and analysis of DNA integration
A lentiviral vector expressing eGFP driven by the
ubiquitous PGK promoter (kindly provided by
D. Trono, Lausanne, Switzerland) was used to gen-
erate the eGFP transgenic rats (Fig. 1a). This vector
746 Transgenic Res (2010) 19:745–763
123
contained self-inactivating long terminal repeats
(SIN-LTR), a rev responsive element (RRE), a central
polypurine tract (cPPT), the PGK promoter, the eGFP
cDNA and a woodchuck hepatitis virus posttranscrip-
tional regulatory element (WPRE) (pRRL.SIN-
cPPT.PGK/GFP WPRE).
Single-cell embryos from Sprague–Dawley (SD)
rats (Charles River) were injected with 1.8 9 1010
infectious units/ml in the perivitelline space (sub-
zonal microinjection), then immediately implanted in
the oviduct of pseudo-pregnant females and allowed
to develop to full term in order to provide the founder
transgenic rats (F0). Offspring were obtained by
crossing founders with wild-type rats.
Transgenic animals were identified by PCR anal-
ysis of genomic DNA isolated from tail biopsies. PCR
amplification was performed using the primer pair
hGFP-For 50-GCCGACCATTATCAACAGAACA-30
and hGFP-Rev 50-GCAGCGGTCACAAACTCCA-30.The PCR was performed under the following condi-
tions: 5 min at 94�C followed by 35 cycles of 30 s at
55�C, 30 s at 72�C, 30 s at 94�C and a final extension
at 72�C for 3 min.
The lentiviral transgene copy number in the
founders and their progeny was determined by
Southern blot. DNA was digested with BamHI
(New England Biolabs), which cuts the transgene at
a unique site between the PGK promoter and the
eGFP cDNA. After digestion, the DNA was sepa-
rated, transferred to a nylon membrane (Hybond N?,
GE Healthcare), hybridised to a 3.7 kb SphI-KpnI
fragment of the pRRL.SINcPPT.PGK/GFP WPRE
plasmid, labelled with a32P-dCTP, washed and
subjected to autoradiography.
The insertion sites of the transgene into the
genome were identified by linker-mediated PCR
(LM-PCR) adapted from the BD GenomeWalker kit
(BD Biosciences Clontech). Briefly, 2.5 lg of trans-
genic rat’s genomic DNA was digested by blunt-
ended restriction enzyme (EcoRV, PvuII, SspI). Each
batch of digested genomic DNA was linked to an
adaptor, created by annealing two oligonucleotides to
built separate libraries. After that, the protocol
consists of two PCR amplifications using long-
distance proofreading Taq DNA polymerase (Platinum
Taq DNA polymerase high fidelity, Invitrogen).
Fig. 1 Southern blot and
LM-PCR analysis of the
genomic DNA of GFPhigh
and GFPlow-transgenic rats.
a Schematic representation
of the lentiviral vector used
to generate the transgenic
rats. Note the BamHI
restriction site used to digest
the genomic DNA and the
HIV-GFP probe from the
construct used to analyse the
DNA. b Southern blot
analysis of both lines
showing only two bands for
each line corresponding to
the hybridisation of the
HIV-GFP probe to both parts
of the transgene linked to
upstream and downstream
genomic DNA cut by the
closest BamHI restriction
sites. c Definition by
LM-PCR of the
chromosome, genomic
region and nucleotide
sequence of transgene
integration into the genome
Transgenic Res (2010) 19:745–763 747
123
The major PCR product were then cloned using
pCRII TOPO plasmid (TOPO TA cloning, Invitro-
gen), sequenced using M13 forward and M13 reverse,
and further analysed on BLAST Rat Sequences
(www.ncbi.nlm.nih.gov).
Flow cytometry analysis
Leukocytes were isolated from whole blood, spleen,
thymus, lymph nodes and bone marrow of GFP-
transgenic or wild-type SD rats and analysed using a
FACSCalibur flow cytometer and CellQuest software
(Becton–Dickinson, Pont de Claix, France). EGFP-
expressing cells were directly identified in the FL-1
channel. The level of GFP expression in each specific
leukocyte subset was determined by staining with
PE-conjugated monoclonal antibodies (mAbs) spe-
cific for CD11b/c-positive monocytes/macrophages
(OX42), NK cells (3.2.3), CD45R-positive B cells
(His24), T cell receptor (TCR)? ab cells (R7.3),
CD4? cells (OX35) and CD8? cells (OX8) (all
antibodies from BD Pharmingen).
Bone marrow-derived DCs were analysed by
staining cells with PE-conjugated mAbs specific for
MHC class II (OX6), CD80 (B7.1) and CD86 (B7.2)
(all antibodies from BD Pharmingen).
Bone marrow-derived mesenchymal stem cells
(MSCs) were analysed with PE-conjugated mAbs
specific for CD90 (OX7), MHC class I (OX18),
CD106 (MR106), CD73 (5F/B9), CD44 (OX49),
MHC class II, CD45 (OX1 ? OX30), CD11b (WT.5)
and CD31 (TLD-3A12) (all antibodies from BD
Pharmingen).
PE-labelled mouse anti-IgG1 (Immunotech) anti-
body was used as a negative control.
Isolation of splenic myeloid DCs (mDCs)
and plasmacytoid DCs (pDCs)
Spleen fragments were digested in 2 mg/ml collage-
nase D (Roche Diagnostics, Meylan, France) for
30 min at 37�C followed by addition of EDTA at
10 mM for 5 min. Cells were separated into high-
density cells (containing most of the pDCs) and low-
density cells (containing most of the myeloid OX62?
DCs) using a 14.5% Nycodenz (Nycomed, Oslo,
Norway) gradient centrifugation as previously described
(Voisine et al. 2002).
Low-density cells were stained with TCRab-biot
followed by streptavidin-PerCpCy5.5, CD4-PE, and
CD103-Alexa Fluor 647 (OX62) mAbs. OX62high
CD4- and OX62lowCD4? cells were then analysed
on a FACS LSRII (Becton–Dickinson) after excluding
TCR? cells.
pDCs were isolated from high-density spleen cells
after removal of red blood cells. T and partial B cell
depletions were performed by incubating cells with
anti-TCRab and TCRcd(V65), CD45RA (OX33),
OX12 and OX8 mAbs, followed by a mixture of anti-
mouse coated magnetic beads (Dynal Biotech, Oslo,
Norway) as previously described (Hubert et al. 2004).
Cells were then stained with TCRab-biot followed by
streptavidin-PerCpCy5.5, CD45R-PE and CD4-Alexa
Fluor 647 mAbs. CD45R?CD4high were analysed on
a FACS LSRII after excluding TCR? cells.
Isolation and expansion of bone marrow-derived
DCs (BMDCs)
BMDCs were obtained as previously described
(Peche et al. 2005). Briefly, bone marrow cells
isolated from tibias and femurs of GFPhigh-transgenic
or wild-type rats were cultured for 8 days in medium
supplemented with rat IL4 or murine granulocyte–
macrophage colony-stimulating factor (GM-CSF).
Medium plus cytokines were renewed on days 3
and 6. At day 8, adherent immature BMDCs were
harvested and matured by a 48 h treatment with
0.5 lg/ml LPS (E. coli 0111:B4 strain; Invitrogen,
San Diego, CA). Non adherent mature BMDCs were
collected for analysis.
Isolation and enrichment of bone marrow-derived
MSCs
MSCs were obtained as previously described (Azizi
et al. 1998). Briefly, bone marrow cells were
collected by flushing the femurs and tibias of
GFPhigh-transgenic or wild-type rats. These cells
were cultured in a-MEM medium supplemented with
20% fetal calf serum (FCS, Invitrogen). After 24 h,
the nonadherent cells were removed and fresh
medium was added to the adherent cells. The cells
were further propagated for 4 passages. The pheno-
type of GFP-positive or wild-type MSCs was char-
acterized after 4 passages. Osteogenic differentiation
was achieved by addition of phosphate derivates
748 Transgenic Res (2010) 19:745–763
123
(10 mM) and dexamethasone (10-8 M) to the culture
medium. After 14 days, differentiation into osteo-
cytes was confirmed by alkaline phosphatase
staining.
Isolation and culture of neural stem/progenitor
cells (NSCs)
Primary cultures of neural stem cells (NSCs) were
established from the whole brain of 15-day-old
GFPhigh transgenic rat foetuses as previously
described (Sergent-Tanguy et al. 2006). Briefly,
tissues freed of meninges were incubated with
0.05% trypsin for 15 min at 37�C. Following addition
of 10% FCS, tissues were exposed to 100 lg/ml of
DNase I prior to mechanical trituration. Cells were
plated in medium composed of Dulbecco’s modified
Eagle medium (DMEM)/Ham’s F12 (1/1, v/v),
supplemented with 10% FCS, 33 mM glucose,
5 mM HEPES (pH 7.2), 100 lg/ml streptomycin
and 100 U/ml penicillin. The next day, the floating
cells were harvested, washed, plated in uncoated
dishes and expanded as neurospheres for 10 days in a
serum-free medium supplemented with N2 (Invitro-
gen, Cergy Pontoise, France) in the presence of
25 ng/ml fibroblast growth factor-2. Neurospheres
were then collected, enzymatically dissociated and
either transplanted or differentiated in vitro.
In vitro differentiation of GFP-positive NSCs
GFP-positive neurospheres were plated at a concen-
tration of 2 9 105 cells/cm2 onto poly-L-ornithine-
coated coverslips in serum-supplemented medium.
The next day, the medium was changed and the cells
were grown for 3 days in N2-supplemented medium.
Cells were then fixed with 4% paraformaldehyde
(PFA) for 10 min, washed in PBS and kept in PBS
containing 0.1% sodium azide at 4�C until processed
for immunocytochemistry.
In vivo differentiation of GFP-positive NSCs
The transplantation was performed as previously
described (Remy et al. 2001). Deeply anesthetized
male SD rats were placed in a stereotaxic frame
(Stoelting, Wood Dale, IL, USA) and 2 ll of
dissociated GFP-positive NSCs (2 9 105 cells/ll)
were transplanted unilaterally into the striatum (AP,
?0.7; L -2.8; V -5.4 and -5.8 mm) at a rate of
0.8 ll/min using a 10-ll Hamilton syringe The
needle was left in place for 4 min and then slowly
withdrawn to avoid aspiration of the transplanted
cells. Transplanted rats did not receive any form of
immunosuppressive treatment. Hundred twenty days
after transplantation, animals were transcardially
perfused with cold 4% PFA in 0.1 M phosphate
buffer (PB). Brains were then removed from the skull
and cryoprotected in two successive solutions of 15
and 30% sucrose in 0.1 M PB, then embedded in
cryomount (Tissue-Tek, Elkhart, IN) and serially
sectioned into 16 lm sections on Superfrost slides.
Slides were stored at -80�C until processed for
immunohistochemistry.
Analysis of GFP expression in transgenic rat
tissues
The GFP-transgenic rats were sacrificed at 8–
12 weeks of age. Animals were anaesthetized and
transcardially perfused with 4% PFA in 0.1 M PB.
Tissues were cryoprotected successively in 15 and
30% sucrose in 0.1 M PB, then embedded in cryo-
mount and cryosectioned into 10–14 lm sections on
Superfrost slides. Slides were stored at -80�C until
ready to be viewed and then thawed at room
temperature. Upon thawing sections were rehydrated
with PBS buffer. Fluorescent tissues were examined
using a Zeiss microscope (Thomwood, NY), and
images were captured with a digital camera (Axio-
CamHRC, Zeiss) driven by AxioVision Release 4.2
software.
Immunostaining
For immunostaining, tissues or cells were incubated in
PBS containing 10% (v/v) normal goat serum (NGS)
and 0.3% (v/v) Triton X-100 for 1 h at room temper-
ature. Sections or cells were then incubated overnight
at 4�C with one of the following primary antibodies:
rabbit anti-GFP (Molecular Probes), monoclonal
anti-NeuN (Chemicon), monoclonal anti-DARPP32
(Chemicon), monoclonal HuC/D (Molecular Probes),
mouse anti-GFAP (Sigma–Aldrich), mouse anti-
Nestin (rat 401; Developmental Studies Hybridoma
Bank (DHSB), Iowa City, IA), mouse anti-b-tubulin
isotype III (Tuj-1; Sigma–Aldrich), or mouse anti-RIP
(DHSB). After washing with PBS, sections or cells
Transgenic Res (2010) 19:745–763 749
123
were incubated in Alexa488-conjugated anti-rabbit
secondary antibody or Alexa568-conjugated anti-
mouse secondary antibody (Jackson Immunoresearch,
Cambridgeshire, UK) for 1 h at room temperature.
Nuclear staining was performed with DAPI for 5 min.
Slides or glass coverslips were mounted in Dabco-
mounting medium (Fluka).
Adoptive transfer studies
Spleens were removed from adult GFPhigh-transgenic
rats and mechanically disrupted by passage through a
wire mesh. Single-cell suspensions were depleted of
erythrocytes and the remaining leukocytes were
washed and resuspended in HBSS. One hundred
million splenocytes were injected into the lateral tail
vein of adult GFPhigh, GFPlow-transgenic or wild-type
rats. Serum samples and spleens from recipient rats
were collected 14 days after transfer.
Detection of anti-GFP antibodies
Serum samples were analysed for the presence of
antibodies to GFP by an indirect ELISA. Briefly,
microtiter plates were coated with 2 lg/ml of
recombinant GFP (rGFP; Upstate, Temecula, CA)
in PBS for 1 h at 37�C. Plates were washed three
times in PBS/0.05% Tween 20 (PBST) and blocked
with 1% BSA in PBS for 1 h at 37�C. Serum samples
serially diluted in PBST were then added for 2 h at
room temperature. After washing with PBST, plates
were incubated with peroxidase-conjugated donkey
anti-rat IgG (Jackson ImmunoResearch Laboratories,
Inc) for 1 h at room temperature. After three washes,
TMB substrate reagent (Becton–Dickinson) was
added, and the reaction was stopped after 10 min
by the addition of 1 M H3PO4. Absorbance was read
at 490 nm.
Spleen lymphocyte proliferation studies
Spleens were removed from adult recipient rats and
mechanically disrupted by passage through a wire
mesh. After depletion of erythrocytes, the remaining
leukocytes were cultured in medium supplemented
with 40 lg/ml rGFP and proliferation was deter-
mined 3 days later by uptake of 3H-thymidine
(0.5 lCi/well; Amersham, Orsay, France) during the
last 8 h of culture.
Lentiviral transduction of GFPlow-transgenic rats
hepatocytes
High-titer GFP-lentiviral vector stocks (pRRL.SIN-
cPPT.PGK/GFP WPRE) were generated as previ-
ously described by transient transfection of three
plasmids: the transfer GFP vector plasmid, the
packaging plasmid psPAX2encoding virus proteins
and the VSVG envelope protein-coding plasmid
pMD.G (Nguyen et al. 2005).
Transduction of hepatocytes of wild-type or
GFPlow-transgenic rats (8 weeks of age) was achieved
by portal vein injection of 0.5 9 107 infectious units
of virus/gram. Animals were sacrificed 65 days after
gene transfer and GFP expression was analysed in the
liver (as described above). In wild-type rats, this
protocol of gene transfer results in an anti-GFP
immune response leading to the clearance of GFP?
liver transduced cells.
Statistical analysis
Statistical significance was assessed using a one-way
ANOVA test (Newman-Keuls Multiple Comparison
Test). Differences were considered significant for p
values \ 0.05.
Results
Generation of eGFP-transgenic rat lines
eGFP-transgenic rat lines were generated by perivi-
telline injection of a recombinant lentiviral vector
(Fig. 1a) into 109 fertilized rat eggs. Of the 10 pups
born, PCR analysis showed that 6 were transgenic
founders. FACS analysis revealed that 4 founders
expressed low-to-high levels of GFP in peripheral
blood leukocytes. No fluorescence was detected in
the 2 others. The founder with the highest levels of
fluorescence (referred to as GFPhigh-transgenic) in
peripheral blood cells was selected and its GFP
expression pattern characterized more extensively.
The founder with low expression (referred to as
750 Transgenic Res (2010) 19:745–763
123
GFPlow-transgenic) was chosen to assess the immune
response towards GFP.
The Southern blot analysis of the GFPhigh and
GFPlow-transgenic lines showed that each of them
harboured only one copy of the transgene (Fig. 1b)
and the precise definition of the transgene insertion
into the genome was obtained using LM-PCR method
(Fig. 1c).
F1 and following generations resulted from
respective crossing of male founders or F1–F2-
transgenic animals with wild-type female rats. The
level and pattern of eGFP expression were evaluated
over three generations.
GFP is highly expressed in multiple leukocyte
subtypes
The level of GFP expression in total leukocytes derived
from peripheral blood, spleen, lymph nodes, thymus
and bone marrow of GFPhigh-transgenic rats was
analysed by flow cytometry (Fig. 2a). High levels of
fluorescence (85–99%) were detected in these animals.
Moreover, as exemplified for the spleen, cells of almost
all lineages analysed, including macrophages, NK, B
and T cells, expressed GFP (Fig. 2b). The relative and
absolute numbers (data not shown) of these various cell
types did not differ between wild-type and transgenic
Fig. 2 GFP expression in the leukocyte subtypes of GFPhigh-
transgenic rats. a Flow cytometry analysis of GFP expression
in leukocytes derived from peripheral blood, spleen, lymph
nodes, thymus and bone marrow in GFPhigh-transgenic rats.
Data indicate the percentage of GFP-positive cells. b Single
cell suspensions were prepared from the spleen of GFPhigh-
transgenic or wild-type rats, stained with PE-conjugated
monoclonal antibodies specific for CD11b/c-positive mono-
cytes/macrophages (OX42), NK cells (3.2.3), B cells (His24),
TCRab-positive cells (R7.3), T CD4? cells (OX35) or T CD8?
cells (OX8) and analysed by flow cytometry. The percentage of
GFP? lineage? and GFP- lineage? cells in transgenic rats and
the percentage of lineage? cells in wild-type rats are indicated
in the corresponding quadrant
Transgenic Res (2010) 19:745–763 751
123
752 Transgenic Res (2010) 19:745–763
123
rats, indicating that expression of GFP in these cells did
not affect cell development.
GFP is highly expressed in immature mDCs,
pDCs and BMDCs as well as mature BMDCs
DCs in lymphoid organs comprise two cell popula-
tions with different functions, mDCs and pDCs
(Voisine et al. 2002; Hubert et al. 2004). Rat mDCs
are OX62? and are divided in two subtypes,
OX62lowCD4? and OX62highCD4-, with different
functions. GFP expression was analysed in splenic
freshly isolated mDCs and pDCs (Fig. 3a). We
observed that over 95% of both OX62lowCD4? and
OX62highCD4- mDCs expressed GFP. Similarly,
more than 95% of pDCs, defined as CD4?CD45R?,
expressed GFP.
Lymphoid organs DCs are scarce cells and studies
requiring large number of cells for in vitro or in vivo
studies largely rely in the use of expanded BMDCs. In
order to determine whether BMDCs from GFPhigh-
transgenic rats retain their fluorescence after differ-
entiation and maturation in vitro, we derived DCs
from bone marrow by culturing them for 8 days in the
presence of GM-CSF and IL-4, and then assessed their
fluorescence after 2 days in the absence or presence of
LPS to induce their maturation. Flow cytometry
analysis showed that LPS-induced DC maturation
had no effect on GFP expression (Fig. 3b). Moreover,
the cell surface phenotype of these cells did not differ
significantly between immature GFP?-BMDCs and
wild-type cells (Fig. 3b, upper histogram), suggesting
that differentiation of these cells is not affected by
GFP expression. In addition, no statistical difference
in phenotypic maturation was observed between GFP-
expressing BMDCs and wild-type upon LPS activa-
tion (Fig. 3b, bottom histogram). These data suggest
that GFP expression does not affect DC maturation.
Characterization of GFP-positive bone marrow-
derived MSCs
MSCs were isolated from bone marrow of GFPhigh-
transgenic rats and expanded over a period of 21 days
(four passages). Flow cytometry analysis showed that
nearly 80% of MSCs were GFP-positive (Fig. 4a). We
further characterized these cells by analysing the
expression of different cell-surface markers, known
to be expressed or not by in vitro expanded MSCs.
More than 90% of cells expressed CD90 (Thy-1),
suggesting that they were in an undifferentiated state.
These cells also expressed CD106 (VCAM-1), MHC
class I and CD73 (SH3), but not CD11b, MHC class II
or CD44. Finally, GFP?-MSCs were negative for
hematopoietic and endothelial markers such as CD45
and CD31 (PECAM-1) (Fig. 4a). These GFP-positive
cells presented similar phenotypic characteristics to
those derived from wild-type rats. We also showed that
GFP-positive MSCs exhibited a spindle-shaped fibro-
blastic morphology following expansion (Fig. 4b, left
panel) and maintained their potential to undergo
osteogenic lineage differentiation under appropriate
conditions, as indicated by alkaline phosphatase
staining (Fig. 4b, right panel).
These data suggest that GFP expression does not
affect the morphology, phenotype or differentiation
potential of MSCs.
GFP expression in adult non-neural tissues
Tissue sections obtained from the non-neural organs
of adult GFPhigh-transgenic rats were examined for
native GFP expression with direct fluorescence under
a 488-nm excitation light (data are summarized in
Table 1).
In the heart, diffuse expression of GFP was
detected in cardiomyocytes, and high levels were
observed in parenchyma-isolated cells with leuko-
cyte-like morphology (Fig. 5a). The kidney also
expressed high levels of GFP in tubules, whereas no
fluorescence was detected in glomeruli (Fig. 5b). In
pancreatic tissue, strong expression was observed in
islets of Langherans with lower expression in exocrine
acini (Fig. 5c). The testis showed strong expression of
GFP in the seminiferous tubules (Fig. 5d). The gut
and stomach also expressed moderate levels of GFP,
whereas a weak expression was observed in the liver
Fig. 3 GFP expression in splenic mDCs and pDCs and in
bone marrow-derived DCs. a Flow cytometry analysis showing
GFP expression in OX62highCD4- mDCs (upper left histo-gram) and OX62lowCD4? mDCs (upper right histogram), and
in CD45R?CD4? pDCs (bottom histogram), derived from the
spleen of GFPhigh-transgenic rats. b Flow cytometry analysis
showing the expression levels of GFP and various cell surface
markers (MHC class II, CD80 and CD86) in immature (upperhistograms) and LPS-matured (bottom histograms) BMDCs
derived from the bone marrow of GFPhigh-transgenic or wild-
type rats. The numbers within the graph indicate the percentage
of positive cells
b
Transgenic Res (2010) 19:745–763 753
123
and skin (Table 1). No fluorescence was detected in
skeletal muscle (Table 1).
GFP expression in adult CNS tissues and in neural
precursors
Sections obtained from the neural tissues of GFPhigh-
transgenic adult rats were examined for native GFP
expression with direct fluorescence under a 488-nm
excitation light (data are summarized in Table 2).
In the olfactory system, GFP was strongly
expressed in the granular cell layer of the accessory
olfactory bulb (Fig. 6a) and also in the internal
granular layer (data not shown) but in no other
structures (data not shown). In the cortex, numerous
neurons expressed GFP (Fig. 6b). Analysis of the
hippocampus also revealed high levels of GFP
expression in most cells of the granular layer of the
dentate gyrus (Fig. 6c) and in the pyramidal layer of
the CA1-3 fields of the Ammon’s Horn (Fig. 6d and
data not shown). Interestingly, NeuN immunostaining
showed an almost total coexpression of GFP positive
cells with cell bodies of neurons from the dentate
gyrus (Fig. 6g–i) and with cell bodies and axonal
Fig. 4 Characterization of GFP-positive bone marrow-derived
MSCs. a Passage 4 GFP-positive or wild-type MSCs were
analysed by FACS. Nearly 80% of cells expressed GFP. The
phenotype of GFP-positive or wild-type MSCs was assessed by
staining cells with PE-conjugated control isotype IgG or anti-
rat monoclonal antibodies directed against the following cell
surface markers: CD90, CD106, MHC class I, CD73, CD44,
MHC class II, CD11b, CD45 and CD31. b Morphology of
GFP?-MSCs during proliferation (left panel) and during
differentiation (right panel). After 4 passages in proliferative
conditions, MSCs exhibited a spindle-shaped fibroblastic
morphology. Upon culture for 3 weeks in the appropriate
differentiation medium containing phosphate derivates
(10 mM), GFP?-MSC differentiated into nodules of osteo-
blasts expressing alkaline phosphatase (red staining)
754 Transgenic Res (2010) 19:745–763
123
processes from CA3 neurons (Fig. 6j–l). GFP was
also widely expressed in the caudate/putamen area
(Fig. 6e) with a restricted expression to the neuronal
population, in particular GABAergic neurons revealed
by DARPP32 immunostaining (Fig. 6m–o). In the
cerebellum, only the granular layer was GFP-positive.
No expression was detected in the Purkinje cell layer
or in the molecular layer (Fig. 6f). We also analysed
GFP expression in several other brain regions and in
the spinal cord (Table 2). Double labelling experi-
ments with GFP and anti-GFAP or anti-RIP antibod-
ies, to detect astrocytes and oligodendrocytes,
respectively, revealed no GFP expression by these
glial cells (data not shown).
We analysed GFP expression in neural stem/
progenitor cells (NSCs) derived from embryonic
whole brain. In culture, NSCs grow in suspension in
defined medium supplemented with mitogens where
they form spherical aggregates called neurospheres
(Gritti et al. 1996). Neurospheres consist mainly of
progenitor cells with a restricted proliferative and
phenotypic potential, and also of a small number of
multipotent neural stem cells.
After 5 days of culture, floating aggregates shown
in Fig. 7a displayed neurosphere-like morphology as
expected, and most of them strongly expressed GFP
(Fig. 7b). FACS analysis of a single-cell suspension
after dissociation of the neurospheres revealed that
nearly 80% were GFP-positive (Fig. 7c). GFP-
positive neurospheres expressed nestin (Fig. 7g),
which is a marker of neural stem/progenitor cells in
the central nervous system (Lendahl et al. 1990).
When GFP-expressing neurospheres were dissociated
and plated onto an adherent substrate in appropriate
culture conditions, a fraction of the cells differenti-
ated into oligodendrocytes (Fig. 7h), astrocytes
(Fig. 7l) and neurons (Fig. 7p), as identified with
cell-type-specific antibodies. Dual immunostaining
revealed that all oligodendrocytes, astrocytes and
neurons expressed GFP (Fig. 7k, o, s, respectively).
Thus, although only neurons expressed GFP in the
CNS, astrocytes and oligodendrocytes as well as
neurons derived from neurospheres expressed GFP.
To investigate the in vivo differentiation potential
of GFP-positive NSCs, we transplanted single cell
suspension of dissociated NSCs generated from E15
Fig. 5 GFP expression patterns in non-neural tissues in adult
GFPhigh-transgenic rats. GFP expression was visualized by direct
fluorescence in a the heart, b kidney, c pancreas and d testis of
GFPhigh-transgenic rats. Each inset depicts the corresponding
background signal of wild-type tissue. Magnification = 940.
Abbreviations: G glomeruli; I islets of Langherans
Transgenic Res (2010) 19:745–763 755
123
GFPhigh-transgenic rats into the striatum of wild-type
SD rats. Four months after transplantation, GFP-
positive cells were differentiated in Hu-positive
neurons (Fig. 8). No GFP-positive/GFAP, OX42 or
RIP—positive cells were observed in transplanted
brains.
These data suggest that NSCs derived from these
GFPhigh-transgenic rats could be a useful tool for
studying the development and fate of transplanted
neural cells in replacement strategies.
Low-expresser GFP-transgenic rats are tolerant
to GFP
It is well documented that when cells expressing a
reporter molecule such as GFP are transferred into
immunocompetent hosts, they are often eliminated,
even in syngeneic combinations, as a result of GFP
immunogenicity (Stripecke et al. 1999; Gambotto
et al. 2000; Rosenzweig et al. 2001). Thus, the
availability of marker protein tolerant animals would
be very useful for the long-term analysis of labelled
cells or tissues, in particular in transplantation and
immunological studies.
Table 1 GFP expression in non-neural tissues in adult
GFPhigh-transgenic rats
Tissues and cell types Fluorescence intensity
Heart
Cardiomyocytes ?
Isolated parenchymal cells ???
Kidney
Tubules ???
Pancreas
Islets of langerhans ???
Exocrine acini ?
Testis
Seminiferous tubules ???
Ovary ??
Stomach ??
Gut ?
Liver ?
Skin ?
Skeletal muscle Undetectable
GFP expression was examined by direct fluorescence in PFA-
perfused non-neural tissues in adult (8–12 weeks old) GFPhigh-
transgenic rats. Plus signs (?) indicate the degree of relative
fluorescence intensity: ?, weak expression; ??, moderate
expression; ???, strong expression
Table 2 GFP expression in neural tissues in adult GFPhigh-
transgenic rats
Neural tissues Fluorescence intensity
Olfactory bulb
Granular cell layer of the AOB ???
Internal granular layer of the OB ???
Cortex ??
Hippocampus
CA1-3 regions ???
Dentate gyrus ???
Corpus callosum Undetectable
Caudate/putamen ???
Septum
Lateral septal nucleus ??
Amygdala ??
Substantia nigra (pars compacta) Undetectable
Cerebellum
Granular layer ???
Purkinje cells Undetectable
Molecular layer Undetectable
Spinal cord ?
GFP expression was examined by direct fluorescence in PFA-
perfused non-neural tissues in adult (8–12 weeks old) GFPhigh-
transgenic rats. Plus signs (?) indicate degree of relative
fluorescence intensity: ?, weak expression; ??, moderate
expression; ???, strong expression
AOB accessory olfactory bulb; OB olfactory bulb
cFig. 6 GFP expression in the central nervous system of adult
GFPhigh-transgenic rats. a–f GFP expression was visualized by
direct fluorescence in a the accessory olfactory bulb, b cortex,
c dentate gyrus of the hippocampus, d the CA3 field of the
Ammon’s Horn of the hippocampus, e the caudate/putamen
and f the cerebellum of GFPhigh-transgenic rats. Magnifica-
tion = 910 (a, c, e, f) and 9 20 (b, d). g–l Dual GFP/NeuN
(neuronal marker) immunostaining was performed to demon-
strate GFP (green) and NeuN (red) coexpression (merged) in
(g–i) the dentate gyrus of the hippocampus and in (j–l) the CA3
field of the hippocampus. Magnification = 940. (m–o) Dual
GFP/DARPP32 (specific marker of GABAergic neurons)
immunostaining was performed to demonstrate GFP (green)
and DARPP32 (red) coexpression (merged) in the caudate/
putamen. Magnification = 940. Abbreviations: AOB, acces-
sory olfactory bulb; GrA, granular cell layer of the AOB; Cx,
cortex; DG, dentate gyrus; CA, cornu amonis; CPu, caudate
putamen; cc, corpus callosum; Cb, cerebellum; Gr, granular
layer; Mol, molecular layer
756 Transgenic Res (2010) 19:745–763
123
Transgenic Res (2010) 19:745–763 757
123
Fig. 7 Neural Stem/progenitor cells cultured as neurospheres
from the whole brain of 15-day-old GFPhigh-transgenic rat
fetuses. a Phase image of neurospheres after 5 days of culture.
The corresponding fluorescence signal in b shows native GFP
expression. c Flow cytometry analysis of isolated cells
from dispersed neurospheres. Data indicate the percentage of
GFP-positive cells. d–g GFP-positive neurospheres express
nestin (red), a specific marker of stem/progenitor cells and
DAPI stains the nuclei. h–s Phenotype of NSCs after 3 days of
differentiation, DAPI stains the nuclei. Certain GFP-positive
cells express markers of: (h–k) oligodendrocytes (RIP, red);
l–o astrocytes (GFAP, red); and p–s neurons (Tuj1, red)
758 Transgenic Res (2010) 19:745–763
123
We generated a line of transgenic rats that
expressed low levels of GFP in leukocytes derived
from peripheral blood, spleen, lymph nodes and
thymus or from bone marrow cells (Fig. 9a). More-
over, analysis of native GFP expression by direct
fluorescence in organs and tissues revealed a very
weak signal only in the liver (data not shown).
The low levels of GFP detected in lymphoid
organs suggest that these animals could potentially
display GFP-specific immune tolerance. To verify
this hypothesis, we transferred GFP-positive spleno-
cytes isolated from GFPhigh-transgenic rats, into
syngeneic GFPlow-animals, and analysed the immune
response 14 days later. Wild-type animals were used
as control recipient groups. Splenocytes from low-
expresser rats showed a significantly decreased
proliferation against GFP, equivalent to those of
non-immunized animals, as compared to those from
wild-type littermates (Fig. 9b, left graph). Moreover,
GFPlow-transgenic rats did not develop anti-GFP IgG
antibodies whereas the non-transgenic group devel-
oped high levels (Fig. 9b, right graph). As expected,
GFPhigh-transgenic rats adoptively transferred with
splenocytes from GFPhigh-transgenic rats did not
show anti-GFP immune responses (Fig. 9b).
To confirm a long-term absence of anti-GFP
immune responses in GFPlow-transgenic rats, we
analyzed the GFP-expression level in the liver of
these animals after in vivo transduction by using a
GFP-lentiviral vector. Nearly 50% of cells with
features of hepatocytes from GFPlow-transgenic rats
displayed a persistent expression of GFP at 65 days
after gene transfer (Fig. 9c, right panel). In contrast,
no GFP-expression was detected in transduced wild-
type rats (Fig. 9c, left panel) while expression was
high at 14 days after gene transfer (data not shown).
These findings demonstrate immune tolerance
towards GFP in GFPlow-transgenic rats.
Discussion
In this study, we characterized two transgenic rat
lines carrying an eGFP gene under the control of the
ubiquitous PGK promoter, generated by using the
lentiviral microinjection technique. As reported by
Lois’s group (Lois et al. 2002) and others (Pfeifer
et al. 2002; van den Brandt et al. 2004; Pfeifer 2006),
this method is highly efficient, since we obtained six
founders among the ten pups born.
We found the pattern and intensity of GFP
expression to vary among the lines derived from
each founder (data in this report and data not shown).
This is likely explained by the different chromosomal
location of the different insertion sites that we
identified by the LM-PCR method (data in this report
and data not shown). These results confirm the
description that up to a third of lentiviral individual
transgenes are subject to epigenetic silencing through
hypermethylation in transgenic pigs generated with
the same lentiviral vector used in this study, which
contains the PGK promoter and deletions of LTRs,
which are prone to hypermethylation (Hofmann et al.
2006). Another publication did not find epigenetic
modifications of lentiviral transgenic rats generated
with a lentiviral vector with the CAG promoter and
also mutated LTRs (Michalkiewicz et al. 2007).
Further studies will be needed to define whether
promoter or species differences result in epigenetic
regulation of lentiviral transgenic animals. Trans-
genes derived from plasmid DNA microinjection are
also subject to complete silencing as well as in
changes in the profile of expression of the promoter
(Karpen 1994; Grieshammer et al. 1995). Silencing of
Fig. 8 In vivo differentiation of GFP-positive NSCs trans-
planted into the striatum of wild-type rats. Representative image
of GFP-positive NSCs stained with the Hu-neuronal marker at
120 days post-cell transplantation. Magnification = 940
Transgenic Res (2010) 19:745–763 759
123
Fig. 9 Analysis of anti-GFP immune response in low-
expresser transgenic animals. a GFP expression in the leukocyte
subtypes of GFPlow-transgenic rats. Flow cytometry analysis of
GFP expression levels in leukocytes of peripheral blood, spleen,
lymph nodes, thymus and bone marrow in low-expresser GFP-
transgenic (GFPlow-transgenic) rats. Data indicate the percent-
age of GFP-positive cells. b Adoptive transfer experiments. One
hundred million GFP-positive splenocytes were injected into
the tail vein of adult GFPhigh, GFPlow-transgenic or (wild-type)
wt rats. Two weeks later, spleen and sera were collected.
(Left graph) Proliferation of splenocytes from wt (n = 5),
GFPhigh-transgenic (n = 5), GFPlow-transgenic (n = 5), or
naive (did not receive splenocytes; n = 3) rats against 40 lg/ml
of GFP. (Right graph) Anti-GFP antibodies detected in serum
from wt (n = 5), GFPlow-transgenic (n = 5), GFPhigh-transgenic
(n = 5) or naive (non-immunized; n = 3) rats by ELISA.
Statistical significance of the GFPlow and GFPhigh groups
compared to the wild-type group is indicated by a P value. cAnalysis of GFP expression in lentiviral-transduced liver of
GFPlow-transgenic rats. GFP-lentiviral vector was injected in
8 weeks GFPlow-transgenic (n = 3) or wt rats (n = 3). Analysis
of GFP expression in liver sections from GFPlow-transgenic
(right image) or wt (left image) rats at day 65 after gene
transfer. Nearly 50% of cells with features of hepatocytes from
GFPlow-transgenic rats displayed a persistent GFP expression,
whereas no expression was detected in transduced wt rats. The
insets show non-transduced controls in each group of animals
760 Transgenic Res (2010) 19:745–763
123
transgene expression depend on the location of
transgene insertion (heterochromatin higher than
euchromatin), the degree of transgene methylation
and genetic background. It is likely that the different
GFP expression levels between cells of the same
organ, as is the case in kidney where tubule epithelial
cells express GFP whereas glomerular cells are
negative, are also explained by epigenetic regulation.
Despite silencing of some integrated transgenes,
lentiviral vectors compare very favourably with
oncoretroviral vectors which are all uniformly
silenced (Jahner et al. 1982; Pfeifer 2006). A potential
solution to epigenetic silencing could be the use of
sequences that shield the lentiviral transgene, as
recently used in cells in vitro (Zhang et al. 2007).
We thoroughly characterized the GFPhigh-line of
rats that would be useful for regenerative medicine
and transplantation studies because of their high
levels of GFP expression in multiple tissues and
major cell types. Analyses of GFP expression over
three generations in the line described here, revealed
the pattern and intensity of transgene expression to be
stable. Moreover, we observed no significant varia-
tion of fluorescence intensity neither between indi-
vidual rats nor between male and female individuals
(data not shown).
Rats from the GFPhigh-line expressed very high
levels of GFP in all major leukocyte subtypes,
especially in TCRab-positive cells and in both
CD4?CD25? and CD8?CD45RClow regulatory T cell
populations (data not shown). In addition, we observed
similar relative proportions of each cell type between
transgenic animals and wild-type littermates, suggest-
ing that GFP does not affect the development of
the major leukocyte lineages, a finding which is
consistent with the observations of Manfra’s group
(Manfra et al. 2001).
Interestingly, GFP was also expressed by DCs, a
minor subtype of leukocytes with a very important
function in the immune system and never character-
ised in previous transgenic rats. DCs are potent
professional antigen-presenting cells that play a key
role in initiating immune responses or maintaining
self-tolerance and continuously circulate through
lymphoid and non lymphoid tissues in response to a
variety of stimuli. mDCs and pDCs display distinct
anatomical distributions and migration patterns as
well as functions, as exemplified by the predominant
production of IL-12p70 by mDCs and interferon-a by
pDCs (Blanco et al. 2008). Thus, GFPhigh-rats may be
particularly useful for the purification of mDC or
pDC populations, as well as for the expansion of
BMDCs for adoptive cell transfer trafficking studies,
and the analysis of DC-T cells interactions in vivo.
MSCs, most commonly isolated from bone mar-
row, are non-hematopoietic cells with the capacity to
self-renew and to differentiate, depending on the
microenvironment in which they find themselves,
into multiple lineages such as osteoblasts, adipocytes,
chondrocytes, endothelial cells and neural cells
(Barry and Murphy 2004). The growing interest in
the potential use of MSCs in regenerative medicine
supports the broadening field of investigation into the
biology of these cells. In this context, the possibility
of labelling MSCs for their in vivo tracking is a
considerable step forward. Here, we showed that
MSCs derived from the bone marrow of our line of
GFPhigh-transgenic rats, not only expressed high
levels of GFP, but also exhibited the same morpho-
logic, phenotypic and functional properties as wild-
type MSCs. These results suggest that such cells
could be used as donor cells in transplantation
models. MSC-derived from these GFPhigh-transgenic
rats have recently been used in a allotransplantation
model (Rossignol et al. 2009).
A detailed analysis of the organs and tissues of the
GFPhigh-line revealed that almost all of them
expressed GFP, as expected with an ubiquitous
promoter such as PGK, albeit different intensities
could be noted between tissues and between different
cell types within a given tissue, likely as a result of
interactions between the transgene insertion site and
epigenetic regulation. Interestingly, we observed a
strong expression in several cerebral structures, with
the majority of GFP fluorescence observed in certain
subsets of mature neurons, and a relative lack of
native fluorescence in glial cells (data not shown).
These animals have a major advantage compared to
existing lines, in which the majority of neuronal
subtypes of the brain areas analysed were negative for
GFP (Francis et al. 2007). In addition, we showed
that about 80% of neural stem/progenitor cells
isolated from embryonic whole brain and expanded
in vitro expressed GFP and continued to express it
after their differentiation into all CNS cellular types.
When transplanted into the striatum of wild-type rats,
the majority of GFP-positive neural stem/progenitor
cells differentiated into mature neurons. Surprisingly,
Transgenic Res (2010) 19:745–763 761
123
and in apparent contradiction with the in vitro results,
no GFP expression was detected in astrocytes or
oligodendrocytes, suggesting that the PGK promoter
is inserted in a locus under epigenetic control
resulting in either activation of expression by the in
vitro culture conditions or in suppression of its
expression in vivo. Thus, neural stem/progenitor cells
derived from this GFPhigh-transgenic rats may be very
useful for neuronal replacement strategies, unlike
those obtained from existing lines (Mothe et al. 2005;
Francis et al. 2007) rather interesting for oligoden-
drocytes replacement.
Immune responses to GFP-positive cells and
tissues have been reported in several studies
(Stripecke et al. 1999; Gambotto et al. 2000; Inoue
et al. 2005; Hakamata et al. 2006). This has
considerably hampered the use of GFP-transgenic
rats as a model for long-term cell tracking, both in
adoptive cell transfer and in regenerative and trans-
plantation studies. In this investigation, we developed
a second line of transgenic rats that expressed very
low levels of GFP in lymphoid organs, suggesting that
these animals could potentially display GFP-specific
immune tolerance. Our hypothesis was confirmed by
the absence of both humoral and cellular responses
against both transferred GFP-positive splenocytes and
lentivirus-mediated GFP gene transfer. These findings
demonstrate immune tolerance towards GFP in
GFPlow-transgenic rats. To date, such rat strain has
never been described.
In summary, we have generated two new lines of
GFP-transgenic rats which are of valuable interest to
the scientific community. The ‘‘high-expresser’’ GFP-
transgenic rat line reported in this study might
provide an available source of donor leukocytes, as
well as several other important cell types such as
mesenchymal stem cells, neurons or neural progen-
itor cells, whose traffic and fate could be easily
monitored in vivo. In contrast, the rats of the ‘‘low-
expresser’’ line, described here as being tolerant
towards GFP, could be used as recipients of GFP-
positive cells or tissues for the long-term tracking of
labelled cells in regenerative medicine.
Acknowledgments We wish to thank Dr. Xian Liang Li,
Dr. Laetitia Gautreau and Dr. Thomas Condamine for their
technical assistance (INSERM U643, Nantes, France). This
work was supported by funding from, La Region Pays de la
Loire through the IMBIO program, Biogenouest and Fondation
Progreffe.
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