Expression of the Rat Arginine
Vasopressin
Gene in Transgenic
Mice
Frederick D. Grant, Jaume Reventos,
Jon W. Gordon*,
Shigeki Kawabata,
Myron Miller, and Joseph A. Majzoub
Divisions of Endocrinology, Children’s Hospital,
Women’s Hospital (F.D.G., J.A.M.)
Departments of Medicine and Pediatrics
Harvard Medical School
Boston, Massachusetts
02115
and Brigham and
Department of Geriatrics and Adult Development
J.W.G., S.K., M.M.)
The Mount Sinai Medical Center
New York. New York
(JR.,
studies to determine
the role of cis-acting
elements
in the physiological
regulation
of VP gene expression in vivo. (Molecular
Endocrinology
7: 659-667,
1993)
A line of mice has been developed
which are transgenie for an 8.2-kilobase
(kb) genomic clone of the
rat vasopressin (VP) gene. Using a polymerase chain
reaction technique,
the rat VP (rVP) transgene
was
shown to have tissue-specific
mRNA expression
in
the hypothalamus,
temporal lobe, parietal cerebral
cortex, cerebellum,
and posterior pituitary, similar to
the tissue distribution
of endogenous
mouse and rat
VP expression.
Expression
of transgenic
rVP mRNA
was also found in the lung and pancreas
of the
transgenic mice, sites of known ectopic expression
of VP. Using two methods, Northern blot analysis
with species-specific
cRNA probes and a quantitative polymerase chain reaction technique, the quantity of rVP transgene
mRNA was shown to regulate
appropriately
in response
to an osmotic stimulus.
After 72 h of water deprivation,
the quantity of transgenie rVP mRNA increased
6.8 + 3.0-fold. This was
not significantly
different than the fold increase in
mouse VP mRNA quantity seen in nontransgenic
mice (4.8 f 1.5) but was significantly
different (P c
0.05) than the 1.2 + 0.03-fold
increase
in rat VP
mRNA seen in normal rats after water deprivation.
In the rat hypothalamus,
VP mRNA poly(A) tail length
increases
with osmotic stimulation,
while in the
mouse it does not. The poly(A) tail of transgenic
rVP
mRNA expressed
in mouse hypothalamus
did not
increase in length after osmotic stimulation.
These
findings suggest that: 1) this 8.2-kb rVP genomic
clone contains information
sufficient for proper tissue-specific
and osmotic regulation of the rVP gene;
and 2) mRNA poly(A) tail length regulation in viva is
dependent
on frans-activating
factors of the host
cell. Mice transgenic
for mutated constructs
of this
8.2-kb rVP gene should be useful for whole animal
0888-8809/93/0659-0667$03.00/O
Molecular Endocrinology
CopyrIght 0 1993 by The Endocrme
INTRODUCTION
Arginine vasopressin(VP) is a nine-aminoacid neuropeptide hormonewhich is important in the regulationof
water metabolism. After synthesis in hypothalamic
magnocellularneurons, VP is secreted from the posterior pituitary and acts on the distal tubule and collecting
ducts of the kidney to increasewater reabsorption(1).
VP is also synthesized in parvocellularneurons of the
hypothalamus,where it is important in the regulationof
ACTH secretion from cells of the anterior pituitary (2,
3) and may be involved in the regulation of circadian
rhythms (4, 5). Although VP is synthesized primarily in
the neurons of the hypothalamus(6), expression has
been reported in other sites of the central nervous
system (7, 8) and elsewherein the body (9).
In the rat, osmotic stimulation results in an increase
in VP mRNA levels in the magnocellularneuronsof the
hypothalamus(1O-l 2). This increase is accompanied
by an increase in the length of the polyadenylate
[poly(A)] tail attached to the 3’-end of VP mRNA. After
24 h of dehydration, the poly(A) tail increasesin length
by approximately 150 nucleotides(13, 14).
Molecularcloning of the genomicsequencefor VP in
several species,includingthe rat (15) and mouse(16)
has allowed the identification of potential &-acting
sequences,which may be requiredfor the regulationof
VP gene expression(17). The functional importanceof
these c&acting DNA sequences has been studied in
vitro usingprimary culture of vasopressinergicneurons
(18) and transfection of the VP gene into heterologous
Society
659
MOL
660
ENDO.
1993
Vol7
cells (19, 20). However, whole animal studies are necessary to demonstrate the role of a c&-acting regulatory
element in the response of the VP gene to physiological
stimuli, such as a change in plasma osmolality.
Development
of transgenic animals by pronuclear
microinjection
provides a method to study genomic
construct in vivo and to determine the role of cis-acting
elements in the regulation of gene expression (21, 22).
In the present study, transgenic mice that express the
normal rat VP (rVP) gene were developed. The 8.2kilobase (kb) genomic clone of the rVP gene used in
this study includes 3 kb each of upstream and downstream sequence flanking the structural portion of the
VP gene. Heterozygous
offspring of a transgenic founder were studied to determine if the 8.2-kb VP construct
contained sufficient information to direct tissue-specific
expression as well as appropriate osmotic regulation of
rVP mRNA content and poly(A) tail length in the mouse.
rVP Genomic
Clone (8.2 kb):
lntron
Amplification
and
Taq I
rVP gene
rVP cDNA
A/148
+=
Pl
for
Two lines of transgenic mice, designated VP3 and VP6,
were produced using an 8.2-kb rVP genomic fragment
which included 3 kb each of upstream and downstream
flanking sequences (Fig. 1). Both lines contained at
least five copies of the transgene per cell, as determined
by comparison of blotting intensities with control rVP
DNA. All expected internal restriction endonuclease
fragments were present in both lines, which indicated
integration without rearrangement
of the 8.2-kb genomit rVP fragment (data not shown). Although line VP3
appeared to express the transgene at a higher level,
line VP6 was a more successful breeder, and therefore
the data presented are from the study of heterozygous
male animals from line VP6.
of rVP Transgene
Expression
Due to the high degree of evolutionary
conservation
between the rat and mouse VP coding sequences (15,
16), it was necessary to develop methods which could
distinguish expression of the rVP transgene from endogenous
mouse VP (mVP) transcripts.
A technique
which used the polymerase chain reaction (PCR) (23)
and which detected differences in restriction digest sites
in the sequences of the two species was used to
characterize
rat and mouse VP mRNA expression in
the tissues of transgenic mice (Fig. 1). Total RNA was
extracted from individual organs, and cDNA was synthesized by reverse transcription
using random hexamer primers. The cDNAs were amplified by PCR using
two synthetic oligonucleotide
primers with DNA sequences common to both mouse and rat VP. Amplification of either rat or mouse VP cDNA resulted in
production
of a 296-base pair (bp) fragment, while
4
Taq I
$
Taq I
148 +
148
L-T:”
P2
296
+=
Pl
of Mice Transgenic
II
Digestion:
mVP cDNA
Analysis
lntron
I (1.2 kb)
RESULTS
Production
and Breeding
the rVP Gene
No. 5
P2
Fig. 1. The 8.2-kb rVP Genomic Clone Introduced into Transgenie Mice (Upper Panel)
The construct contained the complete VP structural sequence, including 3 exons (A, B, and C) and two introns (I and
II), with 3 kb each of 5’- and 3’-flanking sequences. Lower
pane/, Amplification
of mVP and rVP mRNA into cDNA using
PCR. Total RNA was reverse
transcribed
into cDNA using
random
hexamer
oligonucleotide
primers
and amplified
by
PCR using primers Pl and P2 (see Materials and Methods) to
cDNA fragments
of 296 bp (mVP and rVP mRNAs) and 526
bp (genomic
VP DNA). +, The position
of mRNA splicing
between
exons A and B. Digestion with Taql produced DNA
fragments
of 296 bp (mVP), 148 bp &VP), and 247, 131, and
148 bp (genomic
DNA). A radiolabeled
oligonucleotide
probe
@) detected,
by Southern
blot, cDNA bands of 296 bp (mVP),
148 bp (rVP), and 247 bp (genomic VP DNA).
amplification of genomic VP DNA produced a 526-bp
fragment.
Rat and mouse VP cDNAs differ by the presence of
a Taql restriction site, which is present in the rat and
not the mouse sequence (15, 16). Therefore, amplified
rat and mouse sequences can be distinguished
from
each other and can also be distinguished from amplified
genomic DNA. After digestion with Taql, rat cDNA
(which was cut into two 148-bp fragments) and mouse
cDNA (which was not cut by Taql) fragments were
analyzed by Southern blot using a radiolabeled oligonucleotide probe complementary
to both the rat and
mouse VP sequences. Digestion of amplified genomic
VP DNA with Taql resulted in a 248-bp fragment on
Southern blot (Fig. 1).
Tissue-Specific
rVP mRNA
Expression
of Transgenic
Animals from three separate litters which had descended from founder VP6 were analyzed. In each litter,
Rat VP Gene
Expression
in Transgenic
Mice
661
tissue-specific expression of the rVP transgene was
determined in an animal shown by Southern blot analysis to carry the transgene, while a littermate not carrying
the transgene served as a negative control. Expression
of the transgene in an organ was confirmed by demonstrating expression of transgenic rVP mRNA in tissue
obtained from other transgenic littermates. Normal
expression of the rVP gene was also studied by analysis
of tissue from normal rat.
The transgenic mice expressed rVP mRNA in the
hypothalamus and the temporal lobe of the brain (Fig.
2). Low level expression of the rVP transgene was also
demonstrated in the parietal cerebral cortex, cerebellum, and lung of the transgenic mice. Even lower levels
of transgenic rVP mRNA were detectable in the posterior pituitary and pancreas after overexposure of the
Southern blot (Fig. 3). There was no expression of the
rVP transgene in anterior pituitary, adrenal, kidney,
testes, heart, liver, or spleen of the transgenic mice.
Mouse VP mRNA was detected in the hypothalamus
and temporal lobe of the brain (Fig. 2) and also was
seen at lower levels in the parietal cerebral cortex,
Ad
AP
PP
Cb
TRANSGENIC
Sp
Lv
At
Lg
Cx
TL
lit
Ht
MOUSE
Kd
cerebellum, and posterior pituitary (Fig. 3) of the transgenie and nontransgenic mice. No mouse VP mRNA
was present in anterior pituitary, adrenal, pancreas,
kidney, testes, lung, heart, liver, or spleen of either
transgenic or nontransgenic mice. In the normal rat,
there was expression of the rVP gene in the hypothalamus and the temporal lobe of the brain and low level
expression in the parietal cerebral cortex and the cerebellum (Fig. 2). No VP mRNA was detected in the
lung, pancreas, adrenal, kidney, testes, or heart of the
rat. Therefore, in neuronal tissues of the transgenic
mice, the pattern of expression of the rVP transgene is
similar to the expression observed in normal rat and
nontransgenic mouse. In nonneuronal tissues, the
transgene was expressed at very low levels in lung and
pancreas, sites where no rat or mouse VP mRNA was
detected in nontransgenic animals.
Osmotic Regulation of Transgenic
mRNA Content
The regulation of transgenic rVP mRNA and endogenous mVP mRNA in transgenic mice in response to an
TL
Cx
Cb
NON-TRANSGENIC
Te
Pa
Pa
Hypothalamic VP
Te
Kd
Lg
PP
AP
Ad
MOUSE
At
Lv
sp
Kd
Lg
At
lit
Ht
RAT
Ht
TL
Cx
Cb
Ad
Pa
Fig. 2. Organ Survey of VP mRNA Expression
in Transgenic
and Nontransgenic
Mice and in Normal Rat
Southern
blot analysis of mRNA which had been reverse
transcribed,
amplified
by PCR, digested
with Taql, and probed as
described
in Fig. 1. Results were confirmed
in animals from three litters of mice which had descended
from founder
VP6. The
upper and lower bands on the Southern
blots are derived
from mouse and rat VP mRNA,
respectively.
Mouse VP mRNA is
detected
in the hypothalamus
and temporal
lobe of both transgenic
and nontransgenic
mice. Transgenic
rVP mRNA is seen in the
hypothalamus
(Ht) and temporal
lobe (TL) of the transgenic
mouse. There is also low-level expression
of transgenic
rVP mRNA in
parietal cerebral cortex (Cx), cerebellum
(Cb), and lung (Lg) of the transgenic
mouse. There is no expression
of rVP in anterior
pituitary
(AP), adrenal (Ad), testes (Te), kidney (Kd), atrium (At), liver (Li), or spleen (Sp). Low levels of VP mRNA in posterior
pituitary
(PP) and pancreas
(Pa) are not visible on this exposure
of the Southern
blot. In the lung sample from the transgenic
mouse, amplification
of genomic
VP DNA is detected
as a band located between
the expected
positions
of mouse and rat VP
cDNA. In normal rat (bottom panel), VP mRNA is detected
in hypothalamus,
temporal
lobe, panetal cerebral cortex, and cerebellum.
There is no expression
of VP mRNA in adrenal, pancreas,
kidney, lung, or atrium of normal rat.
MOL ENDO. 1993
Vol7 No. 5
662
PP
Cb
Cx
TL
Ht
Ht
Cx
TL
Cb
PP
Lg
Kd
Te
Pa
mVP RNA >
VP DNA >
rVP RNA >
TRANSGENIC
MOUSE
NON-TRANSGENIC
MOUSE
TRANSGENIC
MOUSE
3. Organ Survey of VP mRNA Expression in Transgenic and Nontransgenic Mice in an Overexposure of the Southern Blot
In addition to the expression seen in Fig. 2, mVP mRNA is detected in parietal cerebral cortex (Cx), cerebellum (Cb), and posterior
pituitary (PP) in both transgenic and nontransgenic mice. Transgenic rVP mRNA is detected in the posterior pituitary and pancreas
(Pa) of the transgenic mouse. Amplification of genomic VP DNA is seen in the samples from the hypothalamus (Ht), temporal lobe
(TL), parietal cerebral cortex, cerebellum, and lung (Lg) of the transgenic mouse.
Fig.
osmotic stimulus was studied using two techniques:
quantitative PCR and Northern blot analysis with species-specific probes. Water-deprived
animals received
no water for 72 h, while control animals received water
ad libitum. Expression of hypothalamic VP mRNA was
assessed with the technique of quantitative PCR which
used coamplification
of a competitive synthetic sense
RNA standard (seeMaterials and Methods).
After 72 h of dehydration, total (mouse + rat) hypothalamic VP mRNA increased approximately
8.5fold
compared with water-replete
conditions. Analysis of the
PCR reaction products after Taql digestion showed that
mouse and rat VP mRNA were expressed at approximately equal levels and that both were regulated in
response to an osmotic stimulus (data not shown).
However, the quantitative PCR method did not have a
high degree of precision in quantifying the individual
changes in the levels of expression of mouse and rat
VP mRNA. Therefore, the osmotic regulation of rat and
mouse VP mRNA levels was studied by Northern blot
analysis using species-specific
probes. Although there
is a high degree of similarity between the rat and mouse
VP cDNA sequences, in the 50-bp region between base
2194 and base 2243 of the third exon of the rVP
genomic sequence (15), 8 bp differ from the corresponding mouse sequence (16) (16% dissimilarity). Using oligonucleotide
primers specific to this region, PCR
was used to amplify this 50-bp sequence of the rat and
mouse genes. The downstream
primers also included
the sequence encoding a T3 RNA polymerase recognition site and the amplified sequences served as templates for the synthesis of species-specific
radiolabeled
cRNA probes (see Materials and Methods).
Using Northern blot analysis with these rat- and
mouse-specific VP cRNA probes, rVP transgenic mRNA
expression could be distinguished
from expression of
endogenous
mouse VP mRNA in the hypothalamus of
transgenic mice. This technique was used to compare
regulation of rVP mRNA size and quantity in rat and in
transgenic mouse and regulation of mouse VP mRNA
in transgenic and nontransgenic
mouse (Fig. 4). In rat
hypothalamus,
there was a highly significant (P =
0.0001) 1.2 f 0.03-fold increase in VP mRNA quantity
Rat
---+
Rat
Transgene
-+
Rat-Specific
Mouse
Rat
+
--+
Probe
Transgenic
Mouse
Mouse
-+
-+
Mouse-Specific
Probe
Fig. 4. Northern Blot Analysis of Transgenic and Nontransgenie Hypothalamic RNA with Rat- and Mouse-Specific cRNA
Probes
Animals were water deprived for 72 h (-), and control
animals received water ad libitum (+). Total hypothalamic RNA
(5 pg) was analyzed by Northern blot using either a rat-specific
or a mouse-specific cRNA probe. The rat-specific probe (/eff
panel) does not hybridize to mVP mRNA (Mouse), and there
is little hybridization of the mouse-specific probe (right panel)
to rVP mRNA (Rat). The rat-specific probe shows that levels
of transgenic rVP mRNA (Rat Transgene) in water-replete (+)
transgenic
mice
are less than
half the
levels
in normal
rat.
However, the mouse-specific probe shows that basal (+) mVP
mRNA levels are increased 3.6 f O.Cfold in transgenic mouse
compared to nontransgenic mouse.
in response to 72 h of water deprivation (Fig. 5). When
rVP mRNA expression in transgenic mouse hypothalamus was studied with the rat-specific probe, osmotic
stimulation caused a significant (P c 0.05) 6.8 f 3.0fold increase in rVP mRNA quantity. This fold increase
was significantly (P < 0.05) greater than the response
in normal rat but similar to the response detected with
a mouse-specific
probe in nontransgenic
mice, which
also had a significant (P < 0.001) 4.8 + 1.5-fold increase
in hypothalamic VP mRNA after water deprivation.
In transgenic mice, water deprivation caused a significant (P < 0.05), 1.8 f 0.5fold increase in mVP
mRNA. This incremental response of the endogenous
mVP gene in the transgenic mice was attenuated compared to the response seen in nontransgenic
mice. The
attenuated response was not due to a difference in the
absolute response of mVP mRNA levels to osmotic
stimulation (85 + 18 arbitrary units in nontransgenic,
105 f 25 arbitrary units in transgenic), but because
Rat VP Gene Expression in Transgenic Mice
c
663
tion of poly(A) tail
mouse VP mRNA.
cies differences in
are determined, at
solely by &-acting
8.0
0
.?j 6.0
length was similar for both rat and
These results demonstrate that spemRNA poly(A) tail length regulation
least in part, by the host cell and not
gene sequences.
E
z 4.0
z
2
DISCUSSION
2.0
0.0
+
-
normal
rat
+
-
rat
transgene
+
-
+
transgenic
mouse
-
normal
mouse
Fig. 5. Response of Hypothalamic VP mRNA Levels to an
Osmotic Stimulus in Normal Rat, Nontransgenic Mice, and
Transgenic Mice
Northern blots in Fig. 4 were quantitated by phosphorimager
analysis. Dehydration causes a 1.2 + 0.03-fold increase in VP
mRNA in normal rat and a 4.8 + 1.5 fold increase in VP
mRNA in nontransgenic mice 0. In the transgenic mice @),
the rat transgene responds to dehydration with a 6.8 f 3.0fold increase in rVP mRNA levels, while mVP mRNA increases
only 1.8 f 0.6-fold after water deprivation.
basal levels of mVP mRNA in the transgenic mouse
were 3.6 f 0.4 times greater than levels in nontransgenie mice. Mouse and rat VP mRNA levels could not
be directly compared, as the levels had to be determined using different cRNA probes. Therefore, Northern blot analysis with species-specific probes and analysis by quantitative PCR both demonstrate that there is
appropriate
osmotic
regulation
of rVP transgene
expression in the mouse hypothalamus.
Regulation of Transgenic
Tail Length
rVP mRNA Poly(A)
Previous studies have demonstrated
an increase in the
length of the VP mRNA poly(A) tail in response to
osmotic stimulation in the hypothalamus of the rat (13,
14) but this increase has not been seen in the mouse
(24). This species difference could be the result of either
&-acting
DNA sequences in the VP genes or transacting host cell differences. This line of transgenic animals, with expression of the rVP gene in the mouse
hypothalamus, provides an opportunity to examine the
relative importance of these two factors in the regulation of VP mRNA poly(A) tail length.
Northern blot analysis with species-specific
probes
demonstrated
the previously described
(13, 14) increase in rat hypothalamic VP mRNA size with dehydration (Fig. 4). In the transgenic mice, 72 h of water
deprivation did not cause an increase in the size of the
transgenic rVP mRNA but did result in the appearance
of both shorter rat and mouse VP mRNA (Fig. 4). This
differed from nontransgenic mice, which had no change
in VP mRNA size with osmotic stimulation (24) (Fig. 4).
Thus, in the transgenic mouse hypothalamus,
regula-
The transgenic mouse system has proven extremely
useful for demonstrating
the presence of &-acting
promoter/enhancer
elements that direct tissue-specific
and developmentally regulated expression of genes (25,
26). We have previously demonstrated
that genes expressed in several specific tissues may have discrete
enhancer elements responsible for expression in each
site (27). When the complete complement of promoter/
enhancer elements is not present on the donor gene,
then integration
site may be important in transgene
expression.
However, when a transgene includes all
important promoter/enhancer
elements, integration site
may be unimportant in tissue specificity and regulation
of gene expression (28).
The VP gene normally is expressed primarily in the
neurons of the mammalian hypothalamus
(12, 29). In
addition, VP immunoreactivity
has been detected in the
temporal lobes of the rat brain (7, 8) and has been
reported in a number of tissues outside of the central
nervous system, including ovary (30), testis (31) pancreas (32-34) and adrenals (35). VP mRNA has been
demonstrated
in cells of the medial amygdala in the
temporal lobe of the rat brain (36). VP mRNA levels in
the posterior pituitary (37) diminish after stalk section,
which suggests that the VP mRNA is axonally transported from the hypothalamic magnocellular
neurons to
the posterior pituitary (36). Alternatively spliced transcripts of the VP gene have been found in rat testis
(39) but similar transcripts could not be isolated from
mouse (40) or human (41) testes.
Transfection
experiments
in cultured cells using a
fusion gene construct which included the metallothionein-l (MTl) promotor driving the rVP structural gene
resulted in secretion of properly processed VP peptide
from pituitary (AtT-20) and pancreatic (RIN-1046-38)
cell lines, but not from a fibroblast (BHK) or another
pituitary (GH4) cell line (42). This suggested that some
pituitary and pancreatic cells express the proper enzymes required for processing the VP prohormone.
Habener et al. (43) introduced
this same MTl-VP
gene construct
into transgenic
mice. The MTl-VP
transgene was expressed,
as expected, in liver and
kidney but also was expressed in brain and pancreas
(43). VP peptide was detected in the latter two tissues,
possibly because of the presence of appropriate processing enzymes. It is not possible to confidently attribute this pattern of expression to VP-specific enhancer
elements because the rVP construct included only 36
bases upstream to exon A. However, brain and pancreas are not typical tissues for MTl-directed
expres-
MOL
664
ENDO.
1993
sion, and either the short sequence of 5’-flank or the
2.2-kb structural sequence of the VP gene could contain
an important tissue-specific
element of the rVP gene
(43).
Expression of the promotor sequence of the VP gene
was demonstrated
in pancreatic islets and pituitary in
studies of a mouse line which was transgenic for a
fusion gene containing only 1.25 kb of the rVP promotor
driving the coding sequence for the SV-40 T antigen.
Tumors of the anterior pituitary and of the pancreatic
islet 8-cells were found in these animals, but no abnormalities were seen in the hypothalamus
or posterior
pituitary (44). Therefore,
1.25 kb upstream flanking
sequence of the VP gene do not contain sufficient
information to direct appropriate tissue-specific expression.
The line of transgenic
mice in the present study
carries the structural sequence of the rVP gene flanked
by 3 kb upstream and 3 kb downstream
sequences.
Tissue-specific expression of transgenic VP mRNA was
identified with a PCR technique. In transgenic mice it
previously has been shown that genes expressed in a
specific tissue of a donor species may also be expressed in the corresponding
site of the transgenic
recipient (27). Our findings show that the 8.2-kb VP
construct
directs tissue-specific
expression
of VP
mRNA to the hypothalamus and the temporal lobe, with
low level expression
in the parietal cerebral cortex,
cerebellum, and posterior pituitary. The pattern of VP
expression seen in the neuronal tissues of the transgenie mice is similar to the distribution in both nontransgenie mice and normal rat. Therefore, the 8.2-kb VP
genomic construct contains sufficient c&acting
elements to direct tissue-specific expression of VP mRNA
in the neuronal tissues of transgenic mice.
Previous studies have shown that magnocellular neurons of the hypothalamus are the source of VP mRNA
in the posterior pituitary (38). During the process of
axonal transport from the hypothalamus
to pituitary,
VP mRNA length is shortened. Northern blot analysis
with a rat-specific cRNA probe demonstrated
that, in
the transgenic mice, rVP mRNA was shorter in the
posterior pituitary than in the hypothalamus (our unpublished observation). This finding is consistent with hypothalamic magnocellular
expression of the VP transgene.
Low level expression of the rat transgene was also
demonstrated
in lung and pancreas of the transgenic
mouse, but similar expression of VP mRNA was not
seen in normal rat or nontransgenic
mouse. In humans,
lung tissue is a common site of ectopic expression of
vasopressin with small cell carcinoma and other pulmonary diseases (45-47).
Vasopressin
peptide has
been detected in human pancreas and in the islets of
the rat pancreas (32-34). Detection of low levels of rVP
mRNA in the pancreas and lung could represent decreased specificity of the transgene. However, the increased copy number per cell of the transgene may
result in a higher level of detectable expression of VP
Vol7
No. 5
mRNA in tissues which would normally express only
extremely low levels of VP mRNA.
A band detected in lung (Fig. 2) hypothalamus, temporal lobe, parietal cerebral cortex, and cerebellum (Fig.
3) has a size consistent with a 247-bp fragment of
amplified genomic DNA as diagramed in Fig. 1. Because
there are multiple copies per cell of the transgene
present, amplification of a small amount of contaminating genomic DNA may be more easily detected in RNA
isolated from transgenic animals. Amplification of unspliced VP mRNA could also produce a similar band on
the Southern blot.
The 8.2-kb rVP construct was also shown, by two
different methods, to be appropriately
regulated in the
hypothalamus
of transgenic animals exposed to the
osmotic stimulus of water deprivation. The magnitude
of the response of the transgene was similar to that
observed in nontransgenic
mice and was greater than
the response in normal rats. With expression of the rat
transgene, the response to an osmotic stimulus of the
endogenous
mVP gene was attenuated from approximately 5-fold to 2-fold. This apparent attenuation was
the result of an increased basal level of endogenous
mVP mRNA in the transgenic mice. The increased basal
levels of mVP mRNA, despite low basal levels of rVP
mRNA in the transgenic mice, suggests that there may
be an interaction between the exogenous and transgenie VP genes, most likely mediated by cell-specific
factors. The difference in mRNA quantities may also be
related to differences in the regulation of poly(A) tail
length, which could affect mRNA half-life. These findings suggest that regulation of mRNA levels in response
to an osmotic stimulus may be in part dependent upon
frans-activating
factors specific to the host cell.
The function of the 3’-poly(A) tail of mRNA is not
clear. It may have a role in the stability or in the
translatability of eukaryotic mRNA (48, 49). Changes in
either of these characteristics
could alter the effective
efficiency of an mRNA transcript in expressing its protein product. In magnocellular
neurons of the rat hypothalamus, osmotic stimulation results in an increase in
the poly(A) tail length of VP mRNA from 250 to 400
adenylate bases. With withdrawal
of the stimulus, the
poly(A) tail returns to the basal length of 250 bases
(13). The absence of regulation of VP mRNA poly(A) tail
length in nontransgenic
mice could be a result of differences in the &-acting
elements of the mVP gene or in
ffans-activating
factors in the mouse hypothalamic
cells. When the rVP gene was expressed in the hypothalamus of the mouse, osmotic stimulation did not
cause an increase the rVP mRNA poly(A) tail. However,
with osmotic stimulation, there was a broadening
of
both rat and mouse VP mRNA on the RNA blot, suggesting the appearance of mRNA with shorter poly(A)
tails. This demonstrated
that regulation of poly(A) tail
length is, at least in part, cell specific and requires both
appropriate &-acting
sequences in the gene and cellspecific trans-activating
factors.
In summary, we have developed a mouse line which
is transgenic for the rVP gene. Using a PCR technique
Rat VP Gene Expression
in Transgenic
which can easily distinguish between the similar endogenous mouse and transgenic rat gene products, we
have demonstrated
tissue-specific expression of transgenie rVP mRNA in the mouse. Mouse and rat VP
mRNA levels in the hypothalamus were shown by two
methods to be appropriately
regulated in response to
an osmotic stimulus. Regulation
of the VP mRNA
poly(A) tail was shown to be dependent
on host cell
factors in addition to any c&acting elements contained
in the rVP gene. The tissue-specific
expression and
appropriate
regulation of the transgene suggest that
mice transgenic for mutated constructs of the 8.2-kb
genomic clone will be useful in studies to better determine the role of various c&-acting sequences in the
regulation of VP gene expression.
MATERIALS
665
Mice
C to allow hybridization
between
the RNA and the hexamer
primers. The reverse transcription
reaction was then allowed
to proceed
at 42 C for 45 min, after which the reverse
transcriptase
was heat inactivated
at 95 C for 10 min.
PCR
For PCR amplification
(23) the reaction vol was increased
to
100 ~1 and adjusted
to 16 mM Tris-HCI,
45 mM KCI, 1.5 mM
MgCh,
and 4 mM dithiothreitol.
An additional
1 nmol each of
dATP, dCTP, dGTP, and dTTP, and 30 pmol each of the two
oligonucleotide
primers,
Pl and P2. were added to the reaction. Pl encodes nucleotides
1780-l
801 of the rVP gene, and
P2 is complementary
to nucleotides
2287-2308
of the rVP
gene (15). After addition
of 5 U Amplitaq
DNA polymerase
(Perkin-Elmer
Cetus), the reaction
mix was denatured
for 3
min at 94 C. Twenty-five
cycles of amplification
were performed with 30 set denaturation
at 96 C, 30 set annealing at
55 C, and 3 min extension
at 72 C. The reaction was then
maintained
at 72 C for an additional 10 min to allow completion
of extension.
AND METHODS
Analysis
Production
of Transgenic
A rat genomic EcoRl X-Charon
4A library (kindly provided
by
J. Bonner,
California
Institute of Technology,
Pasadena,
CA)
was used to isolate the rVP genomic
clone. The 8.2-kb VP
genomic
clone, containing
3 kb upstream
flanking
DNA, 2.2
kb VP structural
gene, and 3 kb downstream
flanking
DNA,
was subcloned
into the EcoRl site of pBluescribe
(Stratagene,
La Jolla, CA) and gel-purified
before zygote
micro-injection.
B6D2F/l
mice were obtained
from the Jackson
Laboratory
(Bar Harbor
ME). CD-l mice were purchased
from Charles
River Laboratories
(Wilmington,
MA). Transgenic
mice were
produced
as previously
described
(21, 26) with 2000 copies/
pl rVP genomic DNA in 10 mM Tris-HCI, 0.2 mM EDTA injected
into each zygote.
Founder
animals were screened
by extraction of DNA from tail tissue according
to the method
of Blin
and Stafford
(50). Samples were digested
with HindIll, which
recognizes
two sites within the genomic rVP clone, and analyzed by Southern
blot using a radiolabeled
genomic rVP probe
(51). Mice heterozygous
for the rVP transgene
were further
analyzed.
RNA Preparation
Animals were killed by decapitation,
were dissected.
These dissected
in liquid nitrogen and stored at -70
from individual organs. RNA was
thiocyanate
and a cesium chloride
of RNA was assessed
by analysis
ethidium bromide-stained
agarose
Reverse
of PCR Products
Mice
and the organs of interest
tissues were rapidly frozen
C until RNA was prepared
prepared
using guanidinium
gradient
(52). The integrity
of 28s and 18s rRNA on
gels.
Transcription
Two and one-half
micrograms
of total RNA and 100 pmol
random
hexamer
oligonucleotide
(BRL,
Gaithersburg,
MD)
were mixed in a total vol of 7 pl water in a 0.5ml
PCR tube
(Perkin-Elmer
Cetus, Norwalk,
CT). In a programmable
thermal
controller
(M. J. Research,
Watertown,
MA) this mixture
was
denatured
at 75 C for 5 min and then rapidly cooled to room
temperature.
In the same reaction
vial, reverse
transcription
of the RNA (53) was performed
in a total vol of 20 ~1 with the
followina
reaction conditions:
50 mM Tris-HCI (DH 8.3). 75 mM
KCI, 3 I-& MgCh, 20 mM dithiothreitol,
2 mM‘each
of deoxyATP (dATP), dCTP, dGTP, and dTTP (Pharmacia
LKB Biotechnology,
Piscataway,
NJ), 25 U human placental
ribonuclease inhibitor (Amersham
Corp., Arlington
Heights,
IL), and
200 U Moloney
murine leukemia
virus reverse
transcriptase
(BRL). The reaction mix was first incubated
for 10 min at 23
PCR products
were digested
for 2 h with Taq 1 restriction
enzyme
and then 40% of the PCR reaction was analyzed
on
a 1% Nu-sieve/l%
agarose gel (FMC Bioproducts,
Rockland,
ME). The DNA fragments
were transferred
to a nylon membrane (Gene-Screen,
NEN Research
Products,
Boston, MA),
by the method of Southern
(51). The blot was probed with a
31-nucleotide
synthetic
oligonucleotide
complementary
to nucleotides
181 O-l 840 of the rVP gene (15) (Fig. l), which was
radioactively
labeled using T4 polynucleotide
kinase and [y=P]ATP (Amersham,
Chicago, II) (54). The hybridized
blot was
exposed
to XAR film (Eastman
Kodak Co., Rochester,
NY) at
-80 C. Overexposure
of the Southern
blot allowed detection
of low-level expression
of VP mRNA.
Quantitative
PCR
The quantitative
PCR method of measuring
mRNA quantity
uses coamplification
of a competitive
sense RNA standard
(55). The RNA standard
was obtained
by in vitro transcription
of a rat genomic
VP construct,
which had been altered by
restriction
digestion
and religation to include a 30-bp duplication of sequence.
Therefore,
it had a high degree of similarity
to both mouse and rat VP sequences
but produced
a cDNA
fragment
which could be distinguished
from mouse or rat.
Aliquots
of hypothalamic
total RNA were titrated
against a
dilution series of the synthetic sense RNA standard.
The RNAs
were then reverse transcribed,
amplified by PCR as described
above, and size fractionated
on an ethidium
bromide-stained
agarose
gel. The quantity
of VP mRNA was determined
by
identifying
the concentration
of RNA standard
where
the
standard
and hypothalamic
sample would produce
bands of
equal intensity on the gel.
Species-Specific
cRNA
Probes
The template
for the rat specific probe was obtained
by PCR
amplification
of bases 2194-2243
of exon C of the rVP gene
(15) using 21-nucleotide
oligonucleotide
primers
specific to
this region. The downstream
antisense
primer also included,
at the 5’-end, 23 bases encoding
the antisense
sequence
of
the T3 RNA polymerase
recognition
site (56). When amplified
by PCR, this sequence
of the primer
placed the doublestranded
T3 recognition
site on the 3’-end
of the doublestranded
DNA template.
Five additional deoxyadenosine
bases
were present on the 5’end
of the upstream
sense primer and
were incorporated
into the double-stranded
cDNA template.
As a result, during in vitro transcription
of the template
with
T3 RNA polymerase
in the presence
of [32P]UTP to produce a
MOL
666
Vol7
ENDO.
radiolabeled
cFiNA (57) a five-base
polyuridine
sequence
was
incorporated
which increased the specific activity of the probe.
A mouse-specific
cRNA probe complementary
to bases
3238-3287
of the mouse VP gene (16) (corresponding
to the
same region of exon C recognized
by the rat-specific
probe)
was created in a similar manner.
However,
because a small
degree of cross-reactivity
with rVP mRNA remained,
a singlebase pair mutation
(G to C at nucleotide
3258 of the mVP
gene; 16) was incorporated
into the upstream
primer which
was used to synthesize
the mouse template.
The mVP cRNA
probe synthesized
with this template
was specific
to mVP
mRNA with minimal hybridization
to rVP mRNA.
Northern
Blot Analysis
of Hypothalamic
Species-Specific
cRNA VP Probes
RNA Using
Hypothalami
were dissected
from transgenic
mice, nontransgenie mice, and normal Sprague-Dawley
rats (Charles
River
Laboratories).
Some of the animals were water deprived
for
72 h, while control animals received
water ad libifum. Total
RNA was isolated using guanidinium
thiocyanate
and a cesium
chloride gradient (52) and analyzed
on a 1.4% agarose,
0.6 M
formaldehyde
gel (58). After transfer
to a nylon membrane
(Gene-Screen),
RNA was hybridized
with a species-specific
probe (57) at 68 C in a hybridization
solution which included
50% formamide
with 0.25 M NaCl (rat) or 0.20 M NaCl (mouse).
After washing, hybridization
was quantified
with a digital phosphorimager
(Molecular
Dynamics,
Sunnyvale,
CA) as a measure of VP mRNA quantity.
Variations
in sample loading were
corrected
for by laser densitometric
analysis (Pharmacia
LKB
Biotechnology,
Piscataway,
NJ) of the 18s and 28s ribosomal
RNA bands on a photograph
of the ethidium bromide-stained
agarose gel. Data were analyzed
by analysis of variance
with
P < 0.05 considered
significant.
Permanent
images obtained
on XAR Film (Eastman-Kodak)
were used for analysis of mRNA
length.
Acknowledgments
We thank
Jack Arbiser
for assistance
in cloning
the rVP gene.
Received
December
16, 1992. Revision received
February
5, 1993. Accepted
February
5, 1993.
Address
requests
for reprints to: Dr. Joseph A. Majzoub,
Division of Endocrinology,
Children’s
Hospital,
Boston, Massachusetts
02115.
This work was supported
by NIH Grants
HD-20484
(to
J.W.G.), ROl -DK-40170
and ROl -NS-29384
(to J.A.M.),
NIH
Training Program in Hypertension
Grant HL-07609
(to F.D.G.),
a fellowship
award
from the American
Heart Association,
Massachusetts
affiliate (to F.D.G.),
NIH Mental
Retardation
and Developmental
Disabilities
Research
Program
Grant P30HD-18655-11
(to J.A.M.),
and funds from the Yamanouchi
Pharmaceutical
Co. (Tokyo,
Japan) (to J.W.G.,
M.M.,
and
S.K.). Presented
in part at the 73rd and 74th Annual Meetings
of The Endocrine
Society,
Washington,
DC, 1991, and San
Antonio, TX, 1992.
* Mathers
Professor
of Geriatrics
and Adult Development.
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