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INTRODUCTION |
Folate cofactors play a key role in the de novo
synthesis of purines and pyrimidines, and folate deficiency has been
associated with megaloblastic anemia (1), neural tube defects (2, 3), cardiovascular disease (4, 5), and predisposition to cancer (6). Hence,
folate absorption and delivery to folate-requiring peripheral tissues
is essential to health. Folate transport in mammalian cells is mediated
by a number of different processes. The reduced folate carrier
(RFC1)1 is a facilitative
anion exchanger that mediates folate delivery into a variety of cells
of different origin (7, 8). RFC1 has a much higher affinity for reduced
folates, including the physiological substrate 5-methyltetrahydrofolate
(~2 µM), than folic acid (~200 µM) (9).
Folate receptors (
and
), which anchor to cell membranes through
a glycosylphosphatidylinositol moiety, transport folates via an
endocytotic process (10, 11). These receptors have a very high affinity
for folic acid (~1 nM) and a lesser, but still high
affinity, for 5-methyltetrahydrofolate (12, 13). In addition, there are
at least two other folate transport systems in murine leukemia cells,
but their molecular basis has not been identified. One mechanism
operates optimally at low pH (14, 15) with comparable affinities for
MTX, folic acid, and reduced folates and properties similar to the
folate transport process in intestine (16). Another poorly
characterized system has been proposed as a transporter for folic acid
(17).
Recently, two of the folate receptors (
and
) were inactivated in
the mouse. Loss of folate receptor-
resulting in early embryonic
death associated with a failure of neural tube closure. However,
receptor-
null animals could be sustained through day 18 of
gestation by oral intubation with administration of
5-formyltetrahydrofolate, but live birth of these animals was not
reported (18). Loss of folate receptor-
was not associated with a
pathological phenotype (18). In this paper, we examine the consequences
of the targeted disruption of the other major folate transporter, RFC1,
in particular, the defects that occur during embryonic development and
in the early neonatal period. This carrier is of particular importance, since (i) RFC1 is a very efficient transporter; the cycling rate of
RFC1 is 2 orders of magnitude faster than that of folate receptor-
(19). (ii) The tissue distribution pattern of RFC1 in normal tissues is
different, in part, from that of folate receptor-
. RFC1 is expressed
in placenta, liver, lung, and small intestine (20, 21), whereas folate
receptor-
has been detected in choroid plexus, lung, thyroid,
kidney, breast, placenta, ovary, and testis (22, 23). (iii) RFC1 and
folate receptor-
may localize to different regions in polarized
cells, i.e. apical versus basolateral membranes,
as demonstrated in mammalian retinal pigment epithelium (24). Hence,
these spatial relationships may play a role in the vectorial transport
of folates. The results of the current study shed further light on the
critical role that RFC1 plays in embryonic development and in meeting
folate demands in hematopoietic tissues.
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MATERIALS AND METHODS |
Construction of RFC1 Targeting Vector--
A mouse genomic
Charon 35, 129/ola phage library was screened with a
PstI/NarI fragment (662 base pairs) of
exon 4 (25). Three positive clones were identified; one contained the
exons 3-6 (25). HindIII fragments of this clone were
subcloned into plasmid Bluescript (Strategene). The targeting construct
was generated by a two-step insertion of HindIII fragments
into plasmid PKGNeo. A 1.3-kb fragment containing only a
small part of exon 3 was treated with Klenow polymerase and cloned into
an EcoRI site, whereas a 7-kb fragment containing exons 4-6
was inserted into a HindIII site of PKGNeo.
Electroporation of Embryonic Stem Cells--
The targeting
vector was linearized at the single XhoI site and
electroporated into 2.0 × 107 WW 6 embryonic
stem cells (26). Neomycin (150 µg/ml)-resistant clones were screened
by PCR using a forward primer (p1) specific for the upstream RFC1 gene,
5'-CAGGACAGAATCGTGAGACAGAAC-3'; a reverse primer (p2) specific for the
downstream RFC1 gene, 5'-CTGCTCCTTGGTGAATTTAC-3'; and a reverse primer
(p3) specific for the neomycin cassette in PKGNeo,
5'-CCCAGAAAGCGAAGGAACAAAG-3'. Positive clones were identified by a
1.6-kb PCR fragment specific for the disrupted RFC1 and a 1.3-kb
fragment specific for the wild-type RFC1 gene. Two positive clones,
RFC1-99 and RFC1-113, were identified from a total of 395 clones. The
correct targeting event was also shown by SacI digestion of
high molecular weight DNA and Southern blot analysis using the
PstI/NarI fragment directed at exon 4 of
RFC1.
Generation of Mice with the RFC1 Mutation--
Chimeric mice
were generated by injecting C57BI/6 blastocysts with 8-12 embryonic
stem cells derived from RFC1-99 and RFC1-113 clones. RFC1-99 gave
rise to a male chimeric animal, while RFC1-113 produced a female
chimera. Both mice transmitted the RFC1 mutation through the
germ line. Heterozygous mice were maintained, and mating was initiated
to generate homozygous mutants.
Western Blot Analysis and Measurement of MTX Influx--
Primary
embryonic fibroblasts were established from 15.5-day fetuses and
cultured in Dulbecco's modified Eagle's medium using a standard
protocol (27). The fibroblast cells were trypsinized, washed twice with
cold PBS, and sonicated in cold PBS supplemented with a proteinase
inhibitor mixture (P8340, Sigma, 10% (v/v)). Total lysates (10 µg)
were treated with dithiothreitol-free loading buffer at room
temperature and resolved on a 12% SDS-polyacrylamide gel. Subsequent
blotting and detection were performed according to the ECL Plus Western
blotting detection systems (Amersham Pharmacia Biotech) with an RFC1
antibody directed to the C terminus of the carrier (28). MTX was used
as a model transport substrate for RFC1 (29). MTX influx was assayed in
fibroblasts growing on, and adherent to, the bottom of 20-ml glass
scintillation vials with 0.5 µM [3H]MTX as
described previously (30).
Maternal Folate Supplementation--
Folate supplementation was
administrated by subcutaneous injections of
RFC1+/
dams. Initially, folate supplementation
was begun 1 week before matings; later, supplementation (most of the
animals that received a folic acid dose of 1 mg/day) was started at the
time of appearance of the vaginal plug. The latter supplementation
schedule appeared to improve the mating efficiency. Folate
supplementation continued until pregnant dams were sacrificed or
RFC1-null pups died or were sacrificed. Folic acid and
5-formyltetrahydrofolate were dissolved in water at appropriate
concentrations, neutralized to pH 7.0, sterile-filtered, and injected
with a 26-gauge needle in a volume of 0.1 ml.
Histopathological Examination--
The neonatal mice were
sacrificed before death, and their organs were examined and then
immersed in 10% neutral buffered formalin for fixation. Sections from
all organs were cut at 4-5 µm and stained with hematoxylin and eosin
(HE). The E18.5 embryos were fixed, then serial coronal sections of the
entire embryos were processed and embedded to obtain HE-stained
cross-sections. Serial sectioning of the paraffin-embedded blocks of
tissues from neonates and sectioning of embryos were undertaken, as
warranted, to ensure complete evaluation of representative sections of
all organs.
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RESULTS |
Generation of RFC1+/
Mice--
A targeted disruption
of an RFC1 allele was generated in embryonic stem cells by
homologous recombination. A large part of exon 3, which harbors the
initiation codon ATG, was replaced with a neomycin cassette as
illustrated in Fig. 1a.
Heterozygous progeny of chimeric animals were identified by genomic PCR
and Southern blot analysis of SacI-digested tail DNA (Fig.
1, b and c, respectively). Two breeding colonies
derived from two independently targeted embryonic stem cell clones were
studied and gave the same results.

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Fig. 1.
Targeted inactivation of RFC1
by homologous recombination. a, gene targeting
strategy. b, PCR analysis of mouse tail DNA using three the
primers (p1, p2, p3), indicated in
a. c, Northern blot analysis of tail DNA digested
with SacI and assessed with an exon 4-specific probe.
d, Western blot analysis of total lysate prepared from
embryonic fibroblast cells with an antibody directed to the C terminus
of RFC1 (28). e, MTX influx in embryonic fibroblast cells.
The data are the mean ± S.E. from three separate
experiments.
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Effect of RFC1 Inactivation on Embryonic Development--
Mice
heterozygous for RFC1 were mated to generate F2 offspring.
From these matings 284 pups from 37 litters were obtained and
genotyped. One-hundred and eighty-four pups were heterozygous for
RFC1, and 100 were wild-type (Table
I). No homozygous RFC1-null pups were identified, indicating that the mutation was embryonic lethal. RFC1+/
mice were physically
indistinguishable from their wild-type littermates.
Studies were conducted to determine at which stage embryos ceased to
develop. At day 11.5 no RFC1
/
embryos were identified from a total number of 49 (Table
II). However, embryonic tissues could be
isolated from two small implants and were homozygous for
RFC1 disruption. At day 9.5, 11 smaller and markedly
deformed nonviable RFC1
/
embryos
were identified among 95 fetuses; examples are illustrated in Fig.
2. Additionally, there were 10 (10%)
completely reabsorbed implants (Table II). Hence, the
RFC1-null embryos died before day 9.5.

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Fig. 2.
Comparison of
RFC1 / and
RFC1+/+ embryos harvested at E9.5.
The RFC1-null embryos are smaller and morphologically
deformed as compared with the well developed wild-type embryo
(upper left).
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Rescue of Embryonic Lethality by Maternal Folate
Supplementation--
In an attempt to circumvent the folate transport
defect in RFC1-null embryos, RFC1+/
dams were injected subcutaneously with folic acid at doses of 10, 25, 100, 250, 500, or 1000 µg/day beginning 1 week before conception and
continuing during gestation and for 2 weeks after the pups were born.
Folic acid is a very poor substrate for RFC1 (8, 9), and at the blood
levels generated with the high doses utilized, it is likely that
appreciable quantities of folic acid may be delivered into cells by
passive diffusion as well as other secondary transport routes (14, 15,
17). There were no live births of
RFC1
/
pups until a dose of 1 mg
of folic acid/day was administrated to RFC1+/
dams. Eighteen (9.3%) RFC1
/
mice were obtained from 194 live births in 39 litters (Table I). No
RFC1
/
neonates were identified
when RFC1+/
dams were injected with 1 mg/day
5-formyltetrahydrofolate, a reduced folate with a high affinity for
RFC1, but a much lower affinity for folate receptors than folic acid
(9, 12, 13).
Characterization of RFC1-null Neonatal Mice Rescued by Folic Acid
Supplementation--
RFC1
/
mice born from
RFC1+/
dams supplemented with folic acid,
aside from being pale, appeared normal during the first few days of
life but then showed growth retardation and increasing weakness dying
at or before day 12. Four RFC1
/
mice were euthanized for pathological studies at postpartum days 9, 10, 11, and 12, along with four RFC1+/
and four
RFC1+/+ littermates. The body weights of these
RFC1
/
animals (3.1 ± 0.2 g) were significantly lower (paired t test, p < 0.0025) than the weight of the
RFC1+/+ (6.9 ± 0.4 g) or
RFC1+/
(6.7 ± 0.4 g) neonates (Fig.
3a). There were no significant
pathologic differences between wild-type and heterozygous mice.
However, the spleen and thymus of RFC1-null mice were
extremely small, and these organs and the liver were very pale (Fig.
3b). Histologic examination indicated a marked absence of
hematopoiesis in the bone marrow (Fig. 3, c
versus d) of
RFC1
/
mice, and extramedullary
hematopoiesis was also absent in spleen (Fig. 3, e
versus f) and liver. The lymphocyte population in
the white pulp of the spleen (Fig. 3, e versus
f) and in the cortex of the thymus was markedly diminished
(Fig. 3, g versus h) in the
RFC1
/
mice. Postnatal
development of renal medullary tubules and the seminiferous tubules in
the testis was also impaired. There were no abnormalities in other
organs, including stomach, small intestine, proximal and distal colon,
cecum, heart, lungs, uterus, urinary bladder, eye, brain, pancreas, or
salivary gland.

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Fig. 3.
Pathological changes in
RFC1 / mice. a, reduced
growth of an RFC1 / mouse
(left) compared with wild-type (middle) and
heterozygous (right) littermates at day 9 postpartum.
b, marked reduction in the size of thymus (left)
and spleen (right) and pale color of these two organs and
the liver (middle) from 9-day-old RFC1-null mice
(bottom) compared with organs from wild-type animals
(top) are noted. Sections of bone marrow of an
RFC1-null mouse demonstrating severe reduction in
hematopoietic cells (c) as compared with the densely
populated marrow (arrow) in a wild-type littermate
(d). The spleen in RFC1-null mice (e)
shows almost complete loss of erythropoiesis in the red pulp
(RP) compartment and marked reduction in lymphoid cells in
the white pulp (WP), whereas wild-type mice (f)
show well developed white pulp (WP) and large numbers of
nucleated differentiating red blood cell series in the red pulp
(RP). The cortex (C) of the thymus in
RFC1-null mice is markedly reduced in thickness and
virtually depleted of small lymphocytes with dark staining nucleus
(g). The thymus of the wild-type mice at this stage shows a
dense lymphocytic population in the cortex (C) with distinct
delineation from the medulla (M) (h).
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Characterization of E18.5 Embryos Rescued by Folic Acid
Supplementation--
E18.5 embryos were examined histologically to
determine the extent to which development was sustained prior to birth
by folic acid supplementation of dams at a dose of 1 mg/day of folic
acid. The percentage of RFC1-null fetuses (20%) was close
to the predicted 25% for the homozygous genotype (Table II).
Histologic examination on RFC1-null (n = 6),
heterozygous (n = 4), and wild-type (n = 4) embryos indicated almost complete protection from adverse effects of RFC1 inactivation by folic acid supplementation.
Erythropoeisis was active in liver (Fig.
4, a versus
b) and bone marrow (Fig. 4, c versus
d), with comparable numbers of nucleated red cells as
compared with wild-type and heterozygous littermates. Splenic erythropoiesis was mildly to markedly impaired in only two of the six
RFC1
/
embryos (Fig. 4,
e versus f). The thymus (Fig. 4,
g versus h) and kidneys in
RFC1-null embryos were not different from
RFC1+/
and RFC1+/+
embryos.

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Fig. 4.
Histopathological examination of
RFC / embryos at E18.5 rescued by maternal folic acid
supplementation. Nucleated red cells indicating active
erythropoiesis (arrow) in liver (a), bone marrow
(c), and hematopoiesis and lymphopoiesis in the spleen
(e) of RFC1-null E18.5 embryos compared with
wild-type embryos (b, d, f,
respectively). Development of the thymus of
RFC1 / E18.5 embryos
(g) is comparable with that of the thymus in wild-type
embryo (h), with well developed cortex and medulla. The dams
received 1 mg/day folic acid supplementation.
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RFC1 Expression and MTX Influx in Embryonic Fibroblast
Cells--
Three independent embryonic fibroblast cell lines were
established for each genotype from three 15.5-day fetuses from dams that received folic acid supplementation to confirm that RFC1 had been
completely inactivated in mice (Table II). An antibody against the C
terminus of RFC1 detected a 58-kDa protein in wild-type fibroblast
cells, the same size as found in murine leukemia L1210 cells (Fig.
1d) (28). The level of expression of RFC1 in heterozygous fibroblasts was decreased as compared with that of wild-type cells. No
RFC1 protein was detected in RFC1-null fibroblasts. As
indicted in Fig. 1e, MTX influx was decreased by 28 and 82%
in RFC1± and
RFC1
/
fibroblasts, respectively,
as compared with wild-type cells. The small residual transport activity
in RFC1-null cells was apparently due to alternative folate
transport route(s) in the fibroblasts.
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DISCUSSION |
Rapidly proliferating tissues require an adequate supply of folate
substrate to meet cellular demands for one-carbon moieties for a
variety of cellular biosynthetic processes. RFC1 is a well characterized folate transporter with a high affinity for the physiological folate substrate 5-methyltetrahydrofolate and has been
the subject of intensive studies because of its critical role in
antifolate transport and resistance (7, 8). However, it was unclear as
to its role in the delivery of folates to a diverse spectrum of tissues
due to the presence of multiple folate transport routes in mammalian
cells. To address the role of the RFC1 gene in mouse embryonic
development and neonatal life, the gene was disrupted by homologous
recombination. The complete absence of carrier in
RFC1
/
mice was verified by the lack of RFC1
protein and a marked decrease in MTX influx in embryonic fibroblast cells.
RFC1
/
embryos died before E9.5. Embryos
lacking folate receptor-
die at a similar development stage (18).
Thus, RFC1 and folate receptor-
simultaneously play distinct roles
in embryonic folate homeostasis, and the presence of one does not
compensate for the loss of the other. This is likely related to their
different tissue expression patterns (20-23) or different localization
if expressed in the same cell type (24). These findings are consistent with the critical requirement for folates in normal embryogenesis (31).
In both cases high doses of folate administered to dams rescued
embryos. However, the amounts of folate used were 3 orders of magnitude
higher than that recommended for pregnant women (0.4 mg/day) to prevent
neural tube abnormalities (2, 3).
It is of interest that folic acid, but not 5-methyltetrahydrofolate,
prolonged the life of RFC1-null mice up to 12 days postpartum, while
the latter extended the viability of folate receptor-
-null embryos
to day 18. At the high blood levels of folic acid generated in these
animals, ~100 µM, adequate folate delivery could have been achieved via a transport route that has a higher affinity for
folic acid than 5-formyltetrahydrofolate (17). Also, because 5-formyltetrahydrofolate is a racemic mixture, and the D
isomer does not have biologic activity, it could compete with the
L isomer and inhibit its utilization. Hence, transport is
not fully stereospecific (32), and limited data suggest mammalian
folylpolyglutamate synthetase can utilize both 6R and
6S diastereomers of folates (33, 34).
While folic acid supplementation can maintain near-normal embryonic
development up to at least E18.5 in RFC1
/
embryos and some animals go on to live birth, the life of animals beyond postpartum day 12 cannot be sustained. Prior to birth, high
folate concentrations achieved by folic acid injection, with transport
or diffusion across the placenta barrier where high levels of folate
receptor are expressed (23), provides adequate folates for the
developing embryo. However, possibly due to low concentrations of folic
acid in the mother's milk and inactivation of intestinal RFC1 that is
involved in folate absorption across the pup's small intestine (21,
35), where folate receptors are not expressed (36), there is apparently
insufficient folate available to sustain the life of pups long after birth.
The major pathological changes occurred in tissues of hematopoietic
origin in RFC1-null neonatal mice, suggesting that cells of other
origins can receive adequate folate via transport route(s) other than
RFC1. The lack of any pathological changes in intestine of neonatal
animals, which normally express high levels of RFC1, raises the
possibility that this carrier may not be the only folate transporter in
this tissue although folate receptor is not expressed in intestine. It
is possible that the small intestine of the neonatal mouse can absorb
sufficient folate to support proliferation and maturation of crypt
cells while not delivering enough folate to support the growth of
peripheral tissues.
While folate deficiency in humans is associated with megaloblastic
anemia (1), cardiovascular disease (4, 5), and neoplasia (6), aplasia
anemia was observed in RFC1-null mice, a phenomenon that is unexplained
but may be related to the severity of the folate deficiency (37). It is
of interest that RFC1+/
mice manifest no apparent
pathological changes, although the level of RFC1 protein in
RFC+/
embryonic fibroblasts was decreased as compared
with that of wild-type cells, and transport was reduced by ~30%.
Hence, a mild decease in RFC1 activity is well tolerated at least in
mice on the usual folate-rich diet. It is therefore likely that
mutations or polymorphisms in RFC1 that markedly impair function in the homozygous state would not have physiological consequences when only
one allele is affected and the wild-type allele is expressed. Recently,
a polymorphism (G80A) in RFC1 has been identified, but its role in
folate transport or folate homeostasis remains to be established
(38).