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Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Haartman Institute, University of Helsinki, 00014 Helsinki, Finland;
* Department of Human Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA; and
A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland
1Correspondence: Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Haartman Institute, University of Helsinki, P.O.B. 21, Haartmaninkatu 3, 00014 Helsinki, Finland. E-mail: Kari.Alitalo{at}Helsinki.FI
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: FLT4 • receptor tyrosine kinase • promoter • endothelial cell • lymphangiogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2
).
Two major families consist of vascular endothelial growth factor
receptors (VEGFRs) and the angiopoietin receptors, also called Tie
(tyrosine kinase with immunoglobulin and epidermal growth factor
homology domains) receptors. VEGFRs are distinguished by an
extracellular domain containing seven immunoglobulin (Ig) homology
domains. The VEGFR family has three members: VEGFR-1 (also known as
flt-1), VEGFR-2 (KDR/flk-1), and VEGFR-3 (FLT4)
(3
4
5
6
7)
. In VEGFR-3, the fifth Ig homology domain of the
extracellular part is proteolytically cleaved and the resulting
polypeptides remain linked by two disulfide bonds (8)
. The
ligands binding to VEGFRs belong to the VEGF family of growth factors,
which has five cellular members: VEGF, placenta growth factor (PlGF),
VEGF-B, VEGF-C, VEGF-D (9
10
11
12
13)
, and the recently cloned
viral VEGF homologue VEGF-E (14
15
16)
. VEGFs have specific
sites of expression and show distinct binding patterns to the different
VEGFRs, reflecting the complex biological signaling involved in
endothelial cell functions.
Targeted deletion of genes encoding VEGFRs in mice leads to impaired
development of the vasculature and embryonic death
(17
18
19
20)
. Despite the importance of the VEGFRs in the
development of blood vasculature, VEGFR-3 is unique among these
receptors in that it is found almost exclusively in lymphatic
endothelium in adults. The expression of VEGFR-3 starts at embryonic
day (E) 8.5 of mouse development in the angioblasts of the head
mesenchyme, the cardinal vein, and the allantois (21)
.
During development, however, the strongest VEGFR-3 mRNA expression
becomes gradually restricted first to venous endothelia, and
subsequently to the lymphatic vessels (22)
.
Whereas blood vessel formation has been extensively studied, less is
known about the regulation of lymphatic vessel growth. The development
of lymphatic vessels starts at E12 by centrifugal extension of vessels
from venous sacs in the perimetanephric and jugular regions (20
, 23)
. Thereafter, lymphatic vessels extend to most tissues
growing around major arteries and become particularly abundant in the
mesenterium and mediastinum. VEGFR-3, the first lymphatic endothelial
cell-specific RTK, was cloned as an orphan receptor from human
erythroleukemia cell and placental cDNA libraries (6
, 7)
and the VEGFR3 gene was located to the chromosomal segment
5q33-q35 (7
, 24)
. Subsequently, VEGF-C and
VEGF-D were found to bind to and to activate VEGFR-3 (12
, 13
, 25)
. VEGF-C mRNA is expressed in close proximity to its receptor
VEGFR-3 during mouse development (21)
. Accordingly, VEGF-C
is capable of inducing hyperplasia of lymphatic vessels in transgenic
mice and in chick chorioallantoic membrane (26
, 27)
. These
results suggest that VEGFR-3 plays a role in the generation of the
lymphatic system.
The regulatory regions of six genes sufficient to target expression of
heterologous genes specifically to the endothelial cells in
vivo have been described. These comprise the promoters/enhancers
of the Tie1 (28
29
30)
, Tie2
(30
, 31)
, Vegfr2 (32)
,
intercellular adhesion molecule 2 (ICAM2) (33
, 34)
, von Willebrand factor (vWF) (35
, 36)
, and vascular endothelial cadherin (VE-cadherin)
(37)
genes. These genes are expressed in the endothelium
of blood vessels, whereas the VEGFR3 expression is
relatively specific for lymphatic endothelial cells in adults except it
is also expressed in some fenestrated blood endothelial cells and
angiogenic blood vessels in tumors (38
, 39)
. It is not
clear which transcription factors regulate VEGFR3 expression
or what molecular mechanisms are responsible for the restriction of its
expression to the lymphatic endothelium during development. Here we
have cloned and analyzed the VEGFR3 gene and characterized
its structure as well as the promoter region responsible for its
endothelial cell-specific expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was subcloned as
EcoRI fragments (10, 9.5, 7.0, 4.3 and 3.5 kb) into the
pGEM3 vector (Promega, Madison, Wis.). The genomic clones were ordered
by using 2.5, 1.2, and 0.6 kb EcoRI fragments of
VEGFR3 cDNA (40)
as probes in Southern blotting
analysis. The cosmid subclones were sequenced using primers based on
the published VEGFR3 cDNA (GenBank access. no. X68203 for
VEGFR-3S and S66407 for the carboxyl-terminal tail of VEGFR-3L)
(40)
. The sequence data from the cosmid clone was
independently confirmed by using cDNA primers from the ends of exons to
amplify segments of genomic DNA. We sequenced 
80% of the
genomic DNA using PCR amplimers, cycle sequencing with dRhodamine ready
reactions dye Terminator kit, and running them out on an ABI Prism
Model 377. The sequences were analyzed further and aligned on Sequencer
3.1. (GeneCodes). Polymorphic variation in the VEGFR3 gene
was identified by resequencing of a minimum of 50 chromosomes and
allele frequencies were estimated from the sequence results. Recently,
a genomic region of chromosome 5 containing the VEGFR3 gene
region in unordered fragments was published (AC022095), and a direct
sequence comparison confirmed our results.
Cell culture and transfection assays
To analyze the function and activity of the Vegfr3
promoter, luciferase reporter gene constructs were used in transfection
assays of cultured cells. A 3 kb KpnI/NotI
genomic fragment of the mouse genomic clone comprising the putative
Vegfr3 promoter was cloned into the pGL3 basic reporter
vector. pGL3 basic and pGL3 control vectors contained the firefly
(Photinus pyralis) luciferase gene without promoter and
driven by the SV40 promoter/enhancer, respectively. A restriction
enzyme map of the mouse 3 kb Vegfr3 genomic fragment was
made, deletions were introduced into the promoter fragment with the
restriction enzymes indicated, and DNA fragments were inserted into the
pGL3 basic vector. All constructs were confirmed by sequencing.
Mouse lung (LE-II) and brain endothelial cells (BEND) were grown in minimal essential medium and NIH3T3 murine fibroblasts in Dulbecco’s modified minimum essential medium (DMEM) containing 10% fetal calf serum and antibiotics. Mouse keratinocytes (MK-2) were grown in low (2 mM) CaCl2 DMEM containing 10% fetal calf serum, antibiotics, and epidermal growth factor (4 ng/ml). Cells were transfected using the calcium phosphate method. The minimal amount of plasmid registering full transcriptional activity was titrated prior to the experiments. In subsequent cases, 5 µg of the appropriate reporter construct was transfected along with 0.2 µg of pRL-TK (Promega) to control the variability in transfection efficiency. The pRL-TK vector contains the Herpes simplex virus thymidine kinase promoter region upstream of the Renilla reniformis luciferase gene. Cell extracts were prepared 48 h after transfection in passive lysis buffer (Promega). Luciferase activity was measured using Digene DCR-1 luminometer and Promega Dual Luciferase Assay System. The ratio of firefly luciferase activity to renilla luciferase activity in each sample served as a measure of the normalized luciferase activity, which was divided by the activity of the pGL3 control vector and expressed as relative luciferase activity. Each construct was transfected at least five times and data for each construct are presented as the mean ± SE. Relative luciferase activities among constructs were compared by a factorial analysis of variance, followed by Student’s t test. Statistical significance was accepted at P<0.05.
Production and analysis of Vegfr3 promoter-LacZ
embryos
A HindIII fragment of mouse Vegfr3 genomic
sequence was cloned to the HindIII site of pSDKLacZpA.
Transgenes containing either 3.6 kb HindIII/NotI,
1.6 kb BsaI/NotI, or 0.8 kb
SpeI/NotI fragment of Vegfr3 genomic
sequence, followed by the ß-galactosidase gene and SV40pA, were
excised from the plasmid by digestion with SalI,
ApaI, and BsaI/SpeI, respectively.
Transgenic DNA fragments were microinjected into fertilized oocytes of
the FVB/N strain of mice and the injected zygotes were transplanted
into oviducts of pseudopregnant C57BL/6xDBA/2J hybrid mice. Embryos
were dissected from the decidua on day E15.5 and stained for
ß-galactosidase expression as described previously (29)
.
The embryos were genotyped by PCR analysis of the amnion DNA using
primers 5'-TCTGTCGATCCTT-3' and 5'-GCTGGATGCGGCGTGCGGT-3' specific for
the LacZ gene.
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RESULTS |
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, 42)
. Because VEGFR3
is one of a handful of receptor tyrosine kinase genes involved in human
hereditary disease, we wanted to characterize this gene and its
sequence variation. A cosmid clone containing the human
VEGFR3 genomic sequences was analyzed by Southern blotting
and hybridization with subcloned VEGFR3 cDNA fragments as
probes, and suitable EcoRI fragments were subcloned into
plasmid vectors. To characterize the exon–intron boundaries, these
subclones were sequenced with primers based on the published human
VEGFR3 cDNA sequence (40)
. The
VEGFR3 gene was found to consist of 31 exons (Fig. 1A
), the largest of which (exon 13) is only 363 bp in length
(Table 1
). The 3' exons 30a and 30b are alternatively spliced into the mRNA,
producing polypeptide isoforms differing by 65 amino acid residues
(43)
. The nucleotide sequences of VEGFR3
exon–intron junctions were determined in their entirety; the lengths
of most of the introns were estimated by using genomic PCR and further
confirmed by comparing the lengths of the PCR products to the genomic
sequence covering most of the VEGFR3 gene (Table 1)
. All
exon–intron boundaries were found to conform to the 5'-GT-AG-3' splice
site rule. The first intron of the VEGFR3 was only partially
sequenced. Its length is at least 13.5 kb whereas the rest of the
coding region from the second exon to in exon 30b spans more than 29 kb
of genomic DNA.
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The overall genomic organization of the VEGFR3 gene is
highly similar to the previously published organization of the
Vegfr1 and VEGFR2 genes (44
, 45)
.
The coding capacity of the different exons is indicated in Fig. 1A
in relation to the domain structure of the mature VEGFR-3
receptor protein, which is presented in Fig. 1B
.
Resequencing of VEGFR3 in a minimum of 25 individuals
identified 22 intragenic single nucleotide polymorphisms (SNPs) and an
intronic (CA)n repeat element. Six SNPs occurred
in exons, two synonymous substitutions (G507T and T3198C), and four
predicted to cause amino acid substitutions in the coding sequence
(N149D, T494A, P641S, and R1146H). The intragenic polymorphic sites and
allele frequency estimates are indicated in Table 2
.
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Mouse and human VEGFR3 promoters contain two
distinct regions of homology
Sequences upstream of the mouse Vegfr3 gene were cloned
from a mouse genomic DNA library. Sequencing of clone F2N2.7,
encompassing a 2.7 kb fragment upstream of the NotI site in
the 5' leader sequence of the first coding exon, revealed that its
5'-end contains 1.3 kb of repetitive sequences, followed by 1.4 kb
of sequences not homologous to known genes (Fig. 1C
).
These sequences had two distinct regions of nonrepetitive sequence
homology to the human promoter region that were separated by an
Alu-like element in the human clone. These conserved regions, called
homology regions 1 and 2 (HR1 and HR2 in Fig. 1C
), showed
70% identity between the mouse and human sequences. HR1 contained
conserved putative transcription factor binding sites, including a
putative site for myocyte enhancer factor 2 (MEF-2) and several
possible binding motifs for transcription factors for members of the
Ets family, marked PEA3 and E1A-F (Fig. 1C
). HR2 was
immediately upstream of the NotI site and contained
multiple conserved binding sites for Sp1 like factors, as well as
sequences that resemble the transcription start sites found in genes
which do not contain a TATA box (Fig. 1C
; 46
).
Analysis of the mouse Vegfr3 promoter activity in
cultured cells
To analyze whether the genomic sequences 5' of the
VEGFR3 open reading frame confer endothelial cell-specific
promoter activity, a 3 kb KpnI-NotI
fragment upstream of the first coding exon of the mouse
Vegfr3 gene was cloned into a luciferase reporter vector and
transfected into cultured endothelial and nonendothelial cells. Cell
extracts were assayed for luciferase activity (Fig. 2
). As the lymphatic vessels originate from the vascular endothelium and
no lymphatic endothelial cell lines are currently available, blood
vascular endothelial cells were used to study the Vegfr3
promoter activity and cell type specificity in vitro. The
Vegfr3 expression in the endothelial cell lines used in the
study was checked by Northern blot analysis. Mouse endothelial cells
isolated from the lung (LE-II) did not express Vegfr3
whereas the brain endothelial cells did (data not shown). In LE-II and
BEND cells, the 3 kb Vegfr3 promoter fragment showed 13%
and 27% of the activity, respectively, of the SV40 promoter/enhancer
used as a positive control. In NIH3T3 fibroblasts and MK-2 epithelial
cells, the promoter showed much weaker activity (3% and 2%,
respectively), indicating specificity for endothelial cells. However,
the transcriptional activity of the 3 kb Vegfr3 promoter
fragment in endothelial cells was weaker than the activity of a 1.1 kb
HindIII-NcoI Tie promoter fragment
(47)
.
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Progressive 5'-deletions were made to the Vegfr3 promoter to
characterize sequences critical for the promoter activity. Figure 2
shows schematically the deleted Vegfr3 promoter fragments
used in the transfection experiments. As only background promoter
activity was detected in NIH3T3 and MK-2 cells, no conclusions about
the effects of mutations to Vegfr3 promoter activity were
made based on the results obtained from these cell lines. The deletion
of upstream sequences leaving only a 1.6 kb Vegfr3 promoter
fragment increased significantly (57%) the promoter activity in BEND
cells, whereas in LE-II cells, the promoter activity was slightly
reduced (Fig. 2)
. Further deletions leaving only 858 or 819 bp of the
sequences upstream of the NotI site further increased the
promoter activity in both LE-II and BEND cells. However, upon further
deletion, luciferase activity driven by the 453 bp fragment was reduced
by 40% in LE-II cells and 50% in BEND cells. These results suggest
that the KpnI-ArvII Vegfr3 promoter
fragment upstream of the HR1 contains DNA sequences inhibitory for
transcription whereas the HR1 itself contains transcriptional enhancer
elements.
Activity of the Vegfr3 regulatory sequences in
transgenic mouse embryos
To analyze whether the Vegfr3 regulatory region
identified in vitro is functional and specific to lymphatic
as opposed to blood vascular endothelium in vivo, we
generated transgenic mouse embryos having the 3.6 kb
HindIII-NotI promoter fragment upstream of the
LacZ reporter gene. The embryos were stained and analyzed
for ß-galactosidase expression at E15.5. At this developmental stage,
the lymphatic vessels are developing, while the skin is still permeable
to the ß-galactosidase staining reagents. Of 11 embryos positive for
the 3.6 kb Vegfr3 promoter/LacZ DNA,
endothelial-specific transgene expression was observed in only one
(Fig. 3A
). Three other embryos with staining in a subset of
endothelial cells also showed some ectopic expression. Two DNA-positive
embryos showed only ectopic staining and five did not stain at all. As
the 3.6 kb Vegfr3 promoter fragment could occasionally
target expression of the LacZ reporter gene to the
endothelium of mouse embryos, we tested whether the 1.6 kb
BsaI-NotI promoter fragment could drive
the reporter gene expression specifically to the lymphatic endothelium.
Based on whole mount analysis, of the 22 embryos positive for the 1.6
kb Vegfr3 promoter/LacZ transgene DNA
endothelial-specific transgene expression was observed in two, while
seven embryos had transgene expression also in nonendothelial cells.
One of the two embryos having endothelial cell-specific expression is
shown in Fig. 3B
. As can be seen from the Fig. 3A
, B
, the ß-galactosidase marker was expressed
in a loose branching network pattern in the skin in both cases. This
pattern was particularly evident in the dorsal parts of the developing
forelimbs and in the neck region. The morphology of these structures
suggested that they represent developing lymphatic vessels that form in
the skin at this stage of mouse development by an (lymph)angiogenic
mechanism. A similar kind of staining pattern, although much stronger
and more extensive, was seen in the Vegfr3 LacZ knock-in
embryos (Fig. 3C
). No blue staining could be found in
similarly treated transgene-negative embryos.
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The vascular specificity of the staining patterns was confirmed by
analyzing tissue sections. Skin sections of the 3.6 kb
Vegfr3 promoter/LacZ transgenic embryo revealed
that although the ß-galactosidase staining was very faint, it was
clearly restricted to the developing lymphatic endothelium (Fig. 3D
). Analysis of the sections from the 1.6 kb
Vegfr3 promoter/LacZ transgenic embryos confirmed
that this fragment was sufficient to drive ß-galactosidase expression
largely to the lymphatic vessels (Fig. 3E
), although some
veins were also weakly positive. However, the other transgenic embryo
for the 1.6 kb Vegfr3/LacZ construct, which showed staining
only in the lymphatics, had some weak staining in the chondrocytes.
Analysis of tissue sections of the Vegfr3/LacZ
knock-in embryo confirmed the endogenous pattern of expression and
weaker activity of the promoter fragments (Fig. 3F
).
Staining of blood capillaries or arteries was not observed in either
case.
As the 1.6 kb promoter fragment was able to drive the gene expression
to the lymphatic endothelium in some of the embryos, we continued to
study the reporter gene expression targeted by the 0.8 kb
SpeI-NotI Vegfr3 promoter fragment,
which showed stronger promoter activity in cultured cells. In the
transgenic analysis of the 0.8 kb Vegfr3 promoter/LacZ
construct, 20 embryos screened were transgene positive; of these, four
embryos had staining in endothelial cells. Three also had ectopic
staining in other cell types. However, even in the embryos showing
endothelial cell-specific expression of the transgene, the staining
pattern was not restricted to the developing lymphatic endothelium.
Strong promoter activity was found also in the developing
capillary-sized vessels, e.g., in the head (data not shown).
Table 3
summarizes the results from the Vegfr3
promoter/LacZ transgenic studies.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 45)
. In our analyses, we also identified many
polymorphisms of the VEGFR3 sequence. The data concerning
the exon–intron boundaries and polymorphisms is important and useful
from the clinical point of view as we have recently demonstrated that
VEGFR3 missense mutations are a cause of familial early
onset lymphedema (41
, 42)
. Due to the complexity and
heterogeneity of this disease, it is likely that several other
VEGFR3 mutations may also be found in different lymphedema
families. Using the information about the VEGFR3 genomic
structure, intragenic polymorphisms and linkage studies in families
with lymphedema or other inherited abnormalities of the lymphatic
system should allow rapid screening for mutations in
VEGFR3-linked families. Since the endothelial
cell-associated genes are candidate loci for vascular dysmorphogenesis
syndromes and hereditary angiogenic disorders, analysis of
VEGFR3 mutations from patients suffering from lymphatic
disorders might reveal the molecular basis of such phenotypes.
We have also isolated and partially characterized the genomic regions
upstream of the mouse and human VEGFR3 genes. The upstream
sequences showed 70% identity in two distinct regions, which
represented the putative 5' enhancer/promoter and 3' minimal promoter
elements, respectively. No classical TATA or CAAT boxes were found in
VEGFR3. The 3 kb fragment of the 5'-flanking sequence of the
mouse Vegfr3 demonstrated activity in cultured endothelial
cells but only minimal activity in keratinocytes and fibroblasts.
Deletion studies of the Vegfr3 promoter indicated that both
homology elements were needed for activity. The 5' homology element had
conserved putative binding sites for transcription factors conforming
to the MEF-2, NF-1, AP-2, GATA, and Ets families. Two Ets family
transcription factor binding sites, PEA3 and E1A-F, were conserved in
the mouse and human VEGFR3 promoters. Sites for Ets-related
transcription factors are also found in all other known promoters
showing endothelial specificity in vivo, such as in the
promoters of genes encoding Tie-1, Tie-2, VE-cadherin, ICAM-2, and vWF
(29
30
31
, 33
, 35
36
37)
. The 3' homology region contains
multiple conserved binding sites for Sp1 like factors as well as
sequences that resemble the transcription start sites found in genes
that do not contain a TATA box. The transcription factors controlling
the VEGFR3 gene expression remain unknown and further
studies are needed to determine which consensus binding sites are
critical for the VEGFR3 promoter activity.
We also studied the ability of three Vegfr3 promoter
fragments to target gene expression in the developing lymphatic
endothelium in vivo. Our results from transgenic embryos at
E15.5 indicate that the 3.6 kb and 1.6 kb Vegfr3 promoter
fragments contained specific elements sufficient to direct gene
expression to the lymphatic endothelium. However, as only few of the
transgenics showed a similar staining pattern to that of the
Vegfr3/LacZ knock-in embryos, the gene expression driven by
these promoter fragments is apparently very sensitive to interference
by the transgene integration site and further enhancer elements needed
for reproducible endothelium-specific reporter gene expression are
likely to be missing from these promoter fragments. Many
tissue-specific gene regulatory elements are located within the first
two introns, although such elements occasionally can be found at a
great distance, even in the 3' parts of genes. Vegfr2 and
Tie2 are examples of endothelial cell-specific genes whose
activity is partly regulated by enhancers located in their first
introns (31
, 32)
. Therefore, we are currently analyzing
the large first introns of the mouse and human Vegfr3 genes
to locate putative enhancer elements. We have sequenced the first
intron of the mouse Vegfr3 consisting of 15 kb of genomic
DNA. When compared with the human VEGFR3 first intron, which
has recently become available in the GenBank, the sequences were found
to be 70% homologous. Several short regions showing high homology
were found, but so far no additional enhancers could be identified in
transfection studies using various DNA fragments from the mouse intron
(unpublished data of the authors).
Lymphatic vessels are quite different from arteriae, veins, and
capillaries in structure. The lymphatics are characterized by an
extremely permeable, thin endothelial lining devoid of a basal lamina.
In addition, the small lymphatic vessels typically lack supporting
cells, such as pericytes or smooth muscle cells (21
, 48)
.
These differences indicate that the lymphatic endothelial cells
represent a differentiated form of endothelial cells. Differentiation
of endothelia to arterial, venous, and lymphatic lineages has not been
understood at a molecular level. Recent evidence suggests that arteries
and veins are genetically determined at the earliest stages of
vasculogenesis (49
, 50)
. It is unclear at precisely what
stage lymphatic vessels are determined. One possibility is that VEGF-C
and VEGF-D expressed by smooth muscle cells of arteries and veins would
be involved in directing the formation of lymphatic vessels around them
(38)
. The isolation of the VEGFR3 promoter now
makes it possible to identify the transcription factors that contribute
to lymphatic development using techniques of mouse molecular genetics.
The relevant transcription factor binding sites in addition to the
coding sequence may also be targets of mutations in human lymphedema.
Previous studies have aimed to identify promoter elements necessary for
uniform vascular endothelial cell-specific gene expression. However, a
major obstacle in gene therapy directed to endothelial cells is the
fact that leakage of catheters and blood flow rapidly washes away
delivery vehicles, resulting in undesired gene expression in distant
organs, particularly in the liver (51)
. Therefore,
promoters that restrict expression of transgenes to particular subsets
of endothelial cells may be more desirable in such settings. In
hereditary lymphedema with reduced VEGFR3 signaling in
heterozygous affected individuals (41
, 42)
, genes that
would induce VEGFR-3 signaling specifically in the lymphatic
endothelium might improve the growth and function of lymphatic vessels
without side effects in other tissues. Another potential use for the
Vegfr3 promoter would be in ‘partial’ rescue experiments,
as null mutations in several endothelial-specific genes of mice are
known to lead to embryonic lethality at an early stage. Although the
transcriptional activity of the Vegfr3 promoter is
relatively weak, identification of additional endogenous enhancers or
the use of enhancers increasing promoter activity without losing its
cellular specificity might increase the efficacy of Vegfr3
promoter in the potential applications described above
(52)
.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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