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Tissue-specific expression of the human renin gene in transgenic mice
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 165, No. 2, 1989
Pages 826-832
December 15, 1989
T i s s u e - s p e c i f i c E x p r e s s i o n o f the Human Renin Gene in T r a n s g e n i c Mice Akiyoshi Fukamizu, Min Seok Seo, T o s h i h i s a Hatae, Minesuke Yokoyama*, T a t s u j i Nomura*, Motoya Katauki #, and g a z u o Murakami Gene E x p e r i m e n t C e n t e r , I n s t i t u t e o f Applied B i o c h e m i s t r y , U n i v e r s i t y o f Tsukuba, Tsukuba Science City, I b a r a k i 305, J a p a n * C e n t r a l I n s t i t u t e f o r E x p e r i m e n t a l Animals, 1430 Nogawa Miyamae, Kawasaki 213, J a p a n #Department
o f DNA Biology, School o f Medicine, Tokai B o h s e i d a i , I s e h a r a , Kanagawa 259-11, J a p a n
University,
Received October 23, 1989
SUMMARY: T r a n s g e n i c mice c a r r y i n g human r e n i n gene w e r e p r o d u c e d by m i c r o i n j e c t i o n o f 15 k i l o b a s e s (kb) DNA m o l e c u l e s with up to 3 kb o f 5 " f l a n k i n g s e q u e n c e and 1.2 kb o f 3 ' - f l a n k i n g s e q u e n c e . The t r a n s g e n e s have b e e n s h o w n to be s t a b l y t r a n s m i t t e d t o p r o g e n y . It w a s r e v e a l e d by RNase p r o t e c t i o n a s s a y t h a t the human r e n i n g e n e in a t r a n s g e n i c m o u s e is e x p r e s s e d p r e f e r e n t i a l l y in t h e k i d n e y . The human r e n i n RNA w a s a l s o d e t e c t e d a t a s m a l l l e v e l in a v a r i e t y o f t i s s u e s such a s b r a i n , h e a r t , lung, p a n c r e a s , s p l e e n , s t o m a c h , t e s t i s , and t h y m u s . The d i r e c t r a d i o i m m u n o a s s a y u s i n g a m o n o c l o n a l a n t i b o d y s p e c i f i c f o r the a c t i v e s i t e o f human renin d e m o n s t r a t e d t h e s y n t h e s i s o f human a c t i v e renin in the t r a n s g e n i c m o u s e k i d n e y . T h e s e r e s u l t s s u g g e s t t h a t t h e human renin gene in the t r a n s g e n i c m o u s e is r e g u l a t a d in a t i s s u e - s p e c i f i c m a n n e r . © 1989 A c a d e m i c
Press,
Renin,
Inc.
an a s p a r t y l
proteinase
(E.C.
3.4.23.15),
mainly in the k i d n e y and p l a y s an i m p o r t a n t role blood p r e s s u r e . rat
(4),
and
determined.
is e x p r e s s e d
in the r e g u l a t i o n o f
The renin g e n e s h a v e been i s o l a t e d from human ( 1 - 3 ) , mouse
Moreover,
recognized
to
have
protection
assay.
(5), rat
and
their
nucleic
acid
sequences
were
( 6 , 7 ) and m o u s e (8) r e n i n mRNAs h a v e been
widespread However,
distribution the
tissue
among
tissues
distribution
of
by
human
RNase renin
mRNb h a s b e e n p o o r l y u n d e r s t o o d b e c a u s e o f limited a v a i l a b i l i t y o f human materials. 0006-291X/89 $1.50 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
826
VoI. 165, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Recent a d v a n c e s of m o l e c u l a r and d e v e l o p m e n t a l b i o l o g i e s p r o v i d e d the techniques for transfer (9). an
of foreign genes
into mouse fertilized eggs
The i n t r o d u c t i o n o f c l o n e d g e n e s i n t o mice by t h i s m e t h o d g a v e us opportunity
to
analyze
r e g u l a t e d g e n e in a l l t i s s u e s method,
genetic
We,
have
been
therefore,
of
a
developmentally
throughout normal development.
many g e n e t i c a n a l y s e s
transgenes
mechanism
of the tissue-specific
reported
(10).
anticipated
that
the creation
With t h i s
expression
of the
of transgenic
mice
c a r r y i n g t h e human r e n i n g e n e w i t h i t s n a t i v e p r o m o t e r m i g h t be u s e f u l to s t u d y t h e t i s s u e - s p e c i f i c In t h e p r e s e n t
study,
expression
of the cloned human renin gene.
we d e s c r i b e t h e c r e a t i o n a n d t h e c h a r a c t e r i z a t i o n
o f t h e t r a n s g e n i c m o u s e b e a r i n g t h e human r e n i n g e n e w h o s e e x p r e s s i o n r e g u l a t e d in a t i s s u e - s p e c i f i c
is
manner.
MATERIALS AND ~{ETHODS C o n s t r u c t i o n o f human r e n / n g e r m - An 8 kb BamHI f r a g m e n t c o n t a i n i n g t h e 3 kb 5 ' - f l a n k i n g r e g i o n , e x o n 1, 2, and 3 w a s e x c i s e d from IHRn88 ( 1 1 ) , c o n v e r t e d to b l u n t e n d s , a n d l i g a t e d i n t o BglII linker. The 3 kb BglII-gpnI f r a g m e n t ( f r a g m e n t A) w a s o b t a i n e d by d i g e s t i n g t h e 8 kb f r a g m e n t w i t h BglII a n d gpnI. An 11 kb EpnI f r a g m e n t ( f r a g m e n t B) w a s f r o m IHRn72 ( 1 ) . A 1.8 kb S t u I f r a g m e n t w a s from pHRg57 ( 1 ) , l i g a t e d i n t o ClaI l i n k e r , and d i g e s t e d with gpnI and ClaI to obtain the 1.2 kb gpnI-ClaI fragment ( f r a g m e n t C). The Sinai a n d gpnI s i t e s in pUC19 w e r e c o n v e r t e d to BglII and C/aI sites, respectively, to generate pUCGC19. F r a g m e n t s A, B, and C w e r e l i g a t e d i n t o Bg/II and ClaI s i t e s o f pUCGC19 to c n s t r u c t phRNTM15. The r e s u l t i n g c o n s t r u c t w a s s e p a r a t e d from v e c t o r sequences and m i c r o i n j e c t e d into male pronuclei of f e r t i l i z e d C57BL/6J m o u s e e g g s a s d e s c r i b e d ( 9 ) . RNA p r e p a r a t i o n and RNase p r o t e c t i o n a s s a y - T o t a l RNA w a s p r e p a r e d by t h e g u a n i d i n i u m t h i o c y a n a t e m e t h o d ( 1 2 ) . Poly(A)+RNA w a s e n r i c h e d by o l i g o ( d T ) - c e l l u l o s e c h r o m a t o g r a p h y . A 278 bp gpnI-DraI f r a g m e n t ( f r a g m e n t D) c o n t a i n i n g 144 bp o f t h e 5 " - f l a n k i n g r e g i o n and 134 bp o f e x o n 1 w a s e x c i s e d f r o m pUX77 (11). A 196 bp RsaI f r a g m e n t c o n t a i n i n g 38 bp o f e x o n 1, 151 bp o f e x o n 2, a n d 7 bp o f e x o n ] w a s f r o m pHRn321 ( 1 3 ) , l i g a t e d i n t o BamI l i n k e r , and d i g e s t e d w i t h BamHI and DraI t o r e c o v e r t h e 163 bp DraI-BamHI f r a g m e n t ( f r a g m e n t E). F r a g m e n t s D and E w e r e l i g a t e d i n t o t h e KpnI a n d BamHI s i t e s o f pGEM4Z to c o n s t r u c t pGEMhRN444. 827
Vol. 165, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The p l a s m i d l i n e a r i z e d w i t h EcoRI w a s u s e d a s a t e m p l a t e f o r RNase protection assay. The RNase p r o t e c t i o n a s s a y w a s p e r f o r m e d by h y b r i d i z i n g t o t a l o r poly(A)+RNAs a t 60°C f o r 14 h to t h e a 2 P - l a b e l e d p r o b e , p r e p a r i n g u s i n g T7 p o l y m e r a s e , f o l l o w e d by d i g e s t i n g w i t h RNase A and T1 a s d e s c r i b e d ( 1 4 ) . The RNAs p r o t e c t e d a g a i n s t R N a s e s d i g e s t i o n w e r e a n a l y z e d by e l e c t r o p h o r e s i s t h r o u g h 8 M u r e a / S Z p o l y a c r y l a m i d e g e l . The g e l s w e r e d r i e d and a l l o w e d to e x p o s e t o x - r a y film a t -70oC. R e n i n a s s a y - The e a c h k i d n e y from h u m a n , m o u s e , a n d t r a n s g e n i c m o u s e w a s h o m o g e n i z e d in f o u r v o l u m e s o f s a l i n e c o n t a i n i n g 1 mM EDTA ( 1 5 ) . A f t e r c e n t r i f u g a t i o n a t 100,000 x g , t h e s u p e r n a t a n t w a s s u b j e c t e d to t h e d i r e c t r a d i o i m m u n o a s s a y (16) ( I n s t i t u t P a s t e u r de L y o n ) . The protein concentration was determined using bovine serum albumin as a standard (17).
RESULTS AND DISCUSSION The human
renin
(hRN) gene
that w e
have used
to generate
transgenic mice consists of I0 exons and 9 introns spanning 15 kb, which includes the 3 kb 5"-flanking region with its native promoter. t r a n s g e n i c mice (hRN8-3, 8 - 5 , a n d 8 - 1 2 ) w e r e o b t a i n e d . o f t h e i n t r o d u c e d hRN g e n e in a l l t h r e e or more (data not shown). mice, genes
and p r o g e n y w e r e by
Southern
The copy n u m b e r
f o u n d e r mice w a s found t o be 2
T h e s e f o u n d e r s w e r e c r o s s e d w i t h C57BL/6J
tested
blot
Three
for transmission
analysis.
All
of the
three
mice
i n t r o d u c e d hRN transmitted
the
hRN g e n e s in a m e n d e l i a n f a s h i o n (~50~ p o s i t i v e ) . To e x a m i n e t h e e x p r e s s i o n o f t h e t r a n s g e n e
in p r o g e n y ,
the kidney
RNAs from hRNS-3 and 8-12 l i n e s w e r e a n a l y z e d by RNase p r o t e c t i o n a s s a y (Fig. l-A).
As i n d i c a t e d
expression
than
transcription
hRNS-3.
in t h e s e
site of the promoter line
for
further
in F i g . l - B ,
mice
F i g . 1-R is correctly
o f hRN g e n e
(11).
mainly
also
demonstrated
initiated Thus,
higher
from
the
levels of that
the
known
cap
we h a v e c h o s e n hRNS-12
analysis.
Renin mNNAs in human ( 1 1 , 1 3 ) , identified
hRNS-12 s h o w e d
in t h e
rat
( 1 8 ) , and m o u s e (19) h a v e b e e n
k i d n e y by N o r t h e r n
blot
analysis.
With t h e
d e v e l o p m e n t o f h i g h l y s p e c i f i c and s e n s i t i v e RNase p r o t e c t i o n a s s a y , 828
rat
Vol, 165, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
M 1 2 3 4 5
4 5 1 0 nt
4 3 0 0 nt
A.
Probe
510 nt
Protected fragment
300 nt
F i ~ . l . Renal e x p r e s s i o n of human r e n i n mRNA in t r a n s g e n i c mice. (A) S t r a t e g y f o r RNase p r o t e c t i o n a s s a y . Arrowhead i n d i c a t e s the s t a r t s i t e of t r a n s c r i p t i o n , hRN, human r e n i n ; TT, T7 p r o m o t e r ; n t , n u c l e o t i d e s . (B) RNase p r o t e c t i o n a s s a y . A ~2P-labeled probe was h y b r i d i z e d to 50 #g of t o t a l RNA fron k i d n e y s . Lane 1, p r o b e ; lane 2, c o n t r o l mouse; lane 3, human ( r e n o v a s c u l a r k i d n e y ) ; lane 4, hRNS-3; lane 5, hRN8-12; M, ~x174////ncII m a r k e r . Lanes 1, 2, 3, 5, and M were exposed for 18 h, and lane 4 f o r 96 h.
(6,7)
and mouse
tissues,
(8)
r e n i n mRNAs h a v e
although the level of the extra-renal
To i n v e s t i g a t e
the tissue-specific
12, t h e l e v e l o f RNA in v a r i o u s t i s s u e s assay.
been detected
As s h o w n in F i g . 2 - A ,
in t h e t r a n s g e n i c
expression
testis,
in h e a r t ,
pancreas,
transcript
detected
in s u b m a x i l l a r y
hRN mRNA in t h e k i d n e y w a s (data
not
active
(Fig.2-B),
and spleen.
and stomach.
hRN mRNA
Furthermore,
On t h e o t h e r
gland and
16S i d e n t i c a l t o t h a t
expressed
liver.
hand,
The s i z e
it no of
f o u n d in h u m a n k i d n e y
shown).
A direct the
w a s v e r y low.
o f hRN g e n e in hRN8-
F o r long e x p o s u r e
lung, thymus,
other
w a s a n a l y z e d by RNase p r o t e c t i o n
was detectable was
expression
t h e hRN mRNA w a s p r e f e r e n t i a l l y
mouse kidney.
w a s f o u n d in b r a i n ,
in s e v e r a l
site
hRNS-12 k i d n e y .
radioimmunoassay o f hRN w a s
with a monoclonal antibody specific for
used
As i n d i c a t e d
to
monitor
in T a b l e I,
k i d n e y w a s s h o w n to be e x p r e s s e d
hRN in t h e
at 7.5-fold 829
expression
of the
hRN in
transgenic
mouse
higher than that of human
Vol. 165, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A. M 1
2 3
4
5
6 7 8 9 1011 121314
510 nt=,
300 nt,
R M 1
2 3
4
5
6
7
8
9 10 11 1213 14
510 n t ,
300 nt,
F i g . 2 . T i s s u e d i s t r i b u t i o n o f human renin mRNA in hRNS-12 mouse. RNase p r o t e c t i o n a s s a y was c a r r i e d o u t as d e s c r i b e d in Fig. l , (A) 12 h e x p o s u r e . Lane 1, p r o b e ; lane 2, c o n t r o l mouse kidney, 50 ~g (T); lane 3, human kidney ( r e n o v a s c u l a r ) 2.5 Jg (T); lane 4, kidney, 50 ~g (T); lane 5, l i v e r , 10 ~g (P); lane 6, b r a i n , 10 ~g (P); lane 7, lung, 6 gg (P); lane 8, h e a r t , 50 ~g (T); lane 9, s p l e e n , 50 j g (T); lane 10, thymus, 50 gg (T); lane 11, t e s t i s , 2.5 ~g (P); lane 12, s u b m a x i l l a r y gland, 2 ~g (P); lane 13, p a n c r e a s , 2.5 #g (P); lane 14, s t o m a c h , 3 ~g (P); M, ~x174/H/ncll m a r k e r ; T, t o t a l RNA; P, poly(A)+RNA. (B) Lanes 1 to 7, 72 h e x p o s u r e ; l a n e s 8 to 14, 2 w e e k s e x p o s u r e .
kidney,
whereas
suggest
that
translated human specific
to
renin
c o n t r o l m o u s e s h o w e d no s u c h e x p r e s s i o n . the
hRN
its
product,
gene
in
mRNA
the
in
the
renin.
transgenic Therefore,
transgenic
mouse
was
These results
mouse we
kidney
concluded
regulated
can that
in a
be the
tissue-
manner. The
pathophysiology hypertension,
renin-angiotensin of many
several
system
types
inhibitors
to
of
been
hypertension.
human 830
has
renin
have
implicated To been
in
the
control
the
extensively
Vol. 165, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table I :~, : Direct radioimnamoassay of human renin in kidneys Samples
Human renin concentration (ng/mg protein)
Human Transgenic mouse (hRN8-12) Mouse
O. 14 -+ 0.01 1.06 +- 0.08 N.D.
Results are expressed as mean + 8.D.
investigated
(20).
Recent works
(21-23) and x-ray can provide renin.
the
crystal basis
However,
evaluation
potent
believe
that
excellent
there
is
classes the
no
of human
not
animal renin
renins
model
are
inhibitors the
investigate
renin
inhibitors
in
for
for
(25).
data human
/n
insensitive
hRN g e n e the
vivo
to many
Therefore, may
we
provide
tissue-specific
but also to develop animal models for screening
human
models
and these
inhibitors
animal
carrying to
(24),
of specific
ideal
mouse only
the three-dimensional
of human renin
design
because
transgenic
occasions
expression, specific
the
of inhibitors,
of the
revealed
structure
for
N.D. ; not detectable.
new types of
future.
ACKNOWLEDGMENTS We w o u l d helpful Ministry Cement
like to thank
discussions. of Co.
Education,
This
Drs.
work
Science,
Hitoshi Hori and Masazumi was
and
supported Culture
of
by
grants
Japan,
and
Tada for from
the
Chichibu
Ltd.
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Pages 826-832
December 15, 1989
T i s s u e - s p e c i f i c E x p r e s s i o n o f the Human Renin Gene in T r a n s g e n i c Mice Akiyoshi Fukamizu, Min Seok Seo, T o s h i h i s a Hatae, Minesuke Yokoyama*, T a t s u j i Nomura*, Motoya Katauki #, and g a z u o Murakami Gene E x p e r i m e n t C e n t e r , I n s t i t u t e o f Applied B i o c h e m i s t r y , U n i v e r s i t y o f Tsukuba, Tsukuba Science City, I b a r a k i 305, J a p a n * C e n t r a l I n s t i t u t e f o r E x p e r i m e n t a l Animals, 1430 Nogawa Miyamae, Kawasaki 213, J a p a n #Department
o f DNA Biology, School o f Medicine, Tokai B o h s e i d a i , I s e h a r a , Kanagawa 259-11, J a p a n
University,
Received October 23, 1989
SUMMARY: T r a n s g e n i c mice c a r r y i n g human r e n i n gene w e r e p r o d u c e d by m i c r o i n j e c t i o n o f 15 k i l o b a s e s (kb) DNA m o l e c u l e s with up to 3 kb o f 5 " f l a n k i n g s e q u e n c e and 1.2 kb o f 3 ' - f l a n k i n g s e q u e n c e . The t r a n s g e n e s have b e e n s h o w n to be s t a b l y t r a n s m i t t e d t o p r o g e n y . It w a s r e v e a l e d by RNase p r o t e c t i o n a s s a y t h a t the human r e n i n g e n e in a t r a n s g e n i c m o u s e is e x p r e s s e d p r e f e r e n t i a l l y in t h e k i d n e y . The human r e n i n RNA w a s a l s o d e t e c t e d a t a s m a l l l e v e l in a v a r i e t y o f t i s s u e s such a s b r a i n , h e a r t , lung, p a n c r e a s , s p l e e n , s t o m a c h , t e s t i s , and t h y m u s . The d i r e c t r a d i o i m m u n o a s s a y u s i n g a m o n o c l o n a l a n t i b o d y s p e c i f i c f o r the a c t i v e s i t e o f human renin d e m o n s t r a t e d t h e s y n t h e s i s o f human a c t i v e renin in the t r a n s g e n i c m o u s e k i d n e y . T h e s e r e s u l t s s u g g e s t t h a t t h e human renin gene in the t r a n s g e n i c m o u s e is r e g u l a t a d in a t i s s u e - s p e c i f i c m a n n e r . © 1989 A c a d e m i c
Press,
Renin,
Inc.
an a s p a r t y l
proteinase
(E.C.
3.4.23.15),
mainly in the k i d n e y and p l a y s an i m p o r t a n t role blood p r e s s u r e . rat
(4),
and
determined.
is e x p r e s s e d
in the r e g u l a t i o n o f
The renin g e n e s h a v e been i s o l a t e d from human ( 1 - 3 ) , mouse
Moreover,
recognized
to
have
protection
assay.
(5), rat
and
their
nucleic
acid
sequences
were
( 6 , 7 ) and m o u s e (8) r e n i n mRNAs h a v e been
widespread However,
distribution the
tissue
among
tissues
distribution
of
by
human
RNase renin
mRNb h a s b e e n p o o r l y u n d e r s t o o d b e c a u s e o f limited a v a i l a b i l i t y o f human materials. 0006-291X/89 $1.50 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
826
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Recent a d v a n c e s of m o l e c u l a r and d e v e l o p m e n t a l b i o l o g i e s p r o v i d e d the techniques for transfer (9). an
of foreign genes
into mouse fertilized eggs
The i n t r o d u c t i o n o f c l o n e d g e n e s i n t o mice by t h i s m e t h o d g a v e us opportunity
to
analyze
r e g u l a t e d g e n e in a l l t i s s u e s method,
genetic
We,
have
been
therefore,
of
a
developmentally
throughout normal development.
many g e n e t i c a n a l y s e s
transgenes
mechanism
of the tissue-specific
reported
(10).
anticipated
that
the creation
With t h i s
expression
of the
of transgenic
mice
c a r r y i n g t h e human r e n i n g e n e w i t h i t s n a t i v e p r o m o t e r m i g h t be u s e f u l to s t u d y t h e t i s s u e - s p e c i f i c In t h e p r e s e n t
study,
expression
of the cloned human renin gene.
we d e s c r i b e t h e c r e a t i o n a n d t h e c h a r a c t e r i z a t i o n
o f t h e t r a n s g e n i c m o u s e b e a r i n g t h e human r e n i n g e n e w h o s e e x p r e s s i o n r e g u l a t e d in a t i s s u e - s p e c i f i c
is
manner.
MATERIALS AND ~{ETHODS C o n s t r u c t i o n o f human r e n / n g e r m - An 8 kb BamHI f r a g m e n t c o n t a i n i n g t h e 3 kb 5 ' - f l a n k i n g r e g i o n , e x o n 1, 2, and 3 w a s e x c i s e d from IHRn88 ( 1 1 ) , c o n v e r t e d to b l u n t e n d s , a n d l i g a t e d i n t o BglII linker. The 3 kb BglII-gpnI f r a g m e n t ( f r a g m e n t A) w a s o b t a i n e d by d i g e s t i n g t h e 8 kb f r a g m e n t w i t h BglII a n d gpnI. An 11 kb EpnI f r a g m e n t ( f r a g m e n t B) w a s f r o m IHRn72 ( 1 ) . A 1.8 kb S t u I f r a g m e n t w a s from pHRg57 ( 1 ) , l i g a t e d i n t o ClaI l i n k e r , and d i g e s t e d with gpnI and ClaI to obtain the 1.2 kb gpnI-ClaI fragment ( f r a g m e n t C). The Sinai a n d gpnI s i t e s in pUC19 w e r e c o n v e r t e d to BglII and C/aI sites, respectively, to generate pUCGC19. F r a g m e n t s A, B, and C w e r e l i g a t e d i n t o Bg/II and ClaI s i t e s o f pUCGC19 to c n s t r u c t phRNTM15. The r e s u l t i n g c o n s t r u c t w a s s e p a r a t e d from v e c t o r sequences and m i c r o i n j e c t e d into male pronuclei of f e r t i l i z e d C57BL/6J m o u s e e g g s a s d e s c r i b e d ( 9 ) . RNA p r e p a r a t i o n and RNase p r o t e c t i o n a s s a y - T o t a l RNA w a s p r e p a r e d by t h e g u a n i d i n i u m t h i o c y a n a t e m e t h o d ( 1 2 ) . Poly(A)+RNA w a s e n r i c h e d by o l i g o ( d T ) - c e l l u l o s e c h r o m a t o g r a p h y . A 278 bp gpnI-DraI f r a g m e n t ( f r a g m e n t D) c o n t a i n i n g 144 bp o f t h e 5 " - f l a n k i n g r e g i o n and 134 bp o f e x o n 1 w a s e x c i s e d f r o m pUX77 (11). A 196 bp RsaI f r a g m e n t c o n t a i n i n g 38 bp o f e x o n 1, 151 bp o f e x o n 2, a n d 7 bp o f e x o n ] w a s f r o m pHRn321 ( 1 3 ) , l i g a t e d i n t o BamI l i n k e r , and d i g e s t e d w i t h BamHI and DraI t o r e c o v e r t h e 163 bp DraI-BamHI f r a g m e n t ( f r a g m e n t E). F r a g m e n t s D and E w e r e l i g a t e d i n t o t h e KpnI a n d BamHI s i t e s o f pGEM4Z to c o n s t r u c t pGEMhRN444. 827
Vol. 165, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The p l a s m i d l i n e a r i z e d w i t h EcoRI w a s u s e d a s a t e m p l a t e f o r RNase protection assay. The RNase p r o t e c t i o n a s s a y w a s p e r f o r m e d by h y b r i d i z i n g t o t a l o r poly(A)+RNAs a t 60°C f o r 14 h to t h e a 2 P - l a b e l e d p r o b e , p r e p a r i n g u s i n g T7 p o l y m e r a s e , f o l l o w e d by d i g e s t i n g w i t h RNase A and T1 a s d e s c r i b e d ( 1 4 ) . The RNAs p r o t e c t e d a g a i n s t R N a s e s d i g e s t i o n w e r e a n a l y z e d by e l e c t r o p h o r e s i s t h r o u g h 8 M u r e a / S Z p o l y a c r y l a m i d e g e l . The g e l s w e r e d r i e d and a l l o w e d to e x p o s e t o x - r a y film a t -70oC. R e n i n a s s a y - The e a c h k i d n e y from h u m a n , m o u s e , a n d t r a n s g e n i c m o u s e w a s h o m o g e n i z e d in f o u r v o l u m e s o f s a l i n e c o n t a i n i n g 1 mM EDTA ( 1 5 ) . A f t e r c e n t r i f u g a t i o n a t 100,000 x g , t h e s u p e r n a t a n t w a s s u b j e c t e d to t h e d i r e c t r a d i o i m m u n o a s s a y (16) ( I n s t i t u t P a s t e u r de L y o n ) . The protein concentration was determined using bovine serum albumin as a standard (17).
RESULTS AND DISCUSSION The human
renin
(hRN) gene
that w e
have used
to generate
transgenic mice consists of I0 exons and 9 introns spanning 15 kb, which includes the 3 kb 5"-flanking region with its native promoter. t r a n s g e n i c mice (hRN8-3, 8 - 5 , a n d 8 - 1 2 ) w e r e o b t a i n e d . o f t h e i n t r o d u c e d hRN g e n e in a l l t h r e e or more (data not shown). mice, genes
and p r o g e n y w e r e by
Southern
The copy n u m b e r
f o u n d e r mice w a s found t o be 2
T h e s e f o u n d e r s w e r e c r o s s e d w i t h C57BL/6J
tested
blot
Three
for transmission
analysis.
All
of the
three
mice
i n t r o d u c e d hRN transmitted
the
hRN g e n e s in a m e n d e l i a n f a s h i o n (~50~ p o s i t i v e ) . To e x a m i n e t h e e x p r e s s i o n o f t h e t r a n s g e n e
in p r o g e n y ,
the kidney
RNAs from hRNS-3 and 8-12 l i n e s w e r e a n a l y z e d by RNase p r o t e c t i o n a s s a y (Fig. l-A).
As i n d i c a t e d
expression
than
transcription
hRNS-3.
in t h e s e
site of the promoter line
for
further
in F i g . l - B ,
mice
F i g . 1-R is correctly
o f hRN g e n e
(11).
mainly
also
demonstrated
initiated Thus,
higher
from
the
levels of that
the
known
cap
we h a v e c h o s e n hRNS-12
analysis.
Renin mNNAs in human ( 1 1 , 1 3 ) , identified
hRNS-12 s h o w e d
in t h e
rat
( 1 8 ) , and m o u s e (19) h a v e b e e n
k i d n e y by N o r t h e r n
blot
analysis.
With t h e
d e v e l o p m e n t o f h i g h l y s p e c i f i c and s e n s i t i v e RNase p r o t e c t i o n a s s a y , 828
rat
Vol, 165, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
M 1 2 3 4 5
4 5 1 0 nt
4 3 0 0 nt
A.
Probe
510 nt
Protected fragment
300 nt
F i ~ . l . Renal e x p r e s s i o n of human r e n i n mRNA in t r a n s g e n i c mice. (A) S t r a t e g y f o r RNase p r o t e c t i o n a s s a y . Arrowhead i n d i c a t e s the s t a r t s i t e of t r a n s c r i p t i o n , hRN, human r e n i n ; TT, T7 p r o m o t e r ; n t , n u c l e o t i d e s . (B) RNase p r o t e c t i o n a s s a y . A ~2P-labeled probe was h y b r i d i z e d to 50 #g of t o t a l RNA fron k i d n e y s . Lane 1, p r o b e ; lane 2, c o n t r o l mouse; lane 3, human ( r e n o v a s c u l a r k i d n e y ) ; lane 4, hRNS-3; lane 5, hRN8-12; M, ~x174////ncII m a r k e r . Lanes 1, 2, 3, 5, and M were exposed for 18 h, and lane 4 f o r 96 h.
(6,7)
and mouse
tissues,
(8)
r e n i n mRNAs h a v e
although the level of the extra-renal
To i n v e s t i g a t e
the tissue-specific
12, t h e l e v e l o f RNA in v a r i o u s t i s s u e s assay.
been detected
As s h o w n in F i g . 2 - A ,
in t h e t r a n s g e n i c
expression
testis,
in h e a r t ,
pancreas,
transcript
detected
in s u b m a x i l l a r y
hRN mRNA in t h e k i d n e y w a s (data
not
active
(Fig.2-B),
and spleen.
and stomach.
hRN mRNA
Furthermore,
On t h e o t h e r
gland and
16S i d e n t i c a l t o t h a t
expressed
liver.
hand,
The s i z e
it no of
f o u n d in h u m a n k i d n e y
shown).
A direct the
w a s v e r y low.
o f hRN g e n e in hRN8-
F o r long e x p o s u r e
lung, thymus,
other
w a s a n a l y z e d by RNase p r o t e c t i o n
was detectable was
expression
t h e hRN mRNA w a s p r e f e r e n t i a l l y
mouse kidney.
w a s f o u n d in b r a i n ,
in s e v e r a l
site
hRNS-12 k i d n e y .
radioimmunoassay o f hRN w a s
with a monoclonal antibody specific for
used
As i n d i c a t e d
to
monitor
in T a b l e I,
k i d n e y w a s s h o w n to be e x p r e s s e d
hRN in t h e
at 7.5-fold 829
expression
of the
hRN in
transgenic
mouse
higher than that of human
Vol. 165, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A. M 1
2 3
4
5
6 7 8 9 1011 121314
510 nt=,
300 nt,
R M 1
2 3
4
5
6
7
8
9 10 11 1213 14
510 n t ,
300 nt,
F i g . 2 . T i s s u e d i s t r i b u t i o n o f human renin mRNA in hRNS-12 mouse. RNase p r o t e c t i o n a s s a y was c a r r i e d o u t as d e s c r i b e d in Fig. l , (A) 12 h e x p o s u r e . Lane 1, p r o b e ; lane 2, c o n t r o l mouse kidney, 50 ~g (T); lane 3, human kidney ( r e n o v a s c u l a r ) 2.5 Jg (T); lane 4, kidney, 50 ~g (T); lane 5, l i v e r , 10 ~g (P); lane 6, b r a i n , 10 ~g (P); lane 7, lung, 6 gg (P); lane 8, h e a r t , 50 ~g (T); lane 9, s p l e e n , 50 j g (T); lane 10, thymus, 50 gg (T); lane 11, t e s t i s , 2.5 ~g (P); lane 12, s u b m a x i l l a r y gland, 2 ~g (P); lane 13, p a n c r e a s , 2.5 #g (P); lane 14, s t o m a c h , 3 ~g (P); M, ~x174/H/ncll m a r k e r ; T, t o t a l RNA; P, poly(A)+RNA. (B) Lanes 1 to 7, 72 h e x p o s u r e ; l a n e s 8 to 14, 2 w e e k s e x p o s u r e .
kidney,
whereas
suggest
that
translated human specific
to
renin
c o n t r o l m o u s e s h o w e d no s u c h e x p r e s s i o n . the
hRN
its
product,
gene
in
mRNA
the
in
the
renin.
transgenic Therefore,
transgenic
mouse
was
These results
mouse we
kidney
concluded
regulated
can that
in a
be the
tissue-
manner. The
pathophysiology hypertension,
renin-angiotensin of many
several
system
types
inhibitors
to
of
been
hypertension.
human 830
has
renin
have
implicated To been
in
the
control
the
extensively
Vol. 165, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table I :~, : Direct radioimnamoassay of human renin in kidneys Samples
Human renin concentration (ng/mg protein)
Human Transgenic mouse (hRN8-12) Mouse
O. 14 -+ 0.01 1.06 +- 0.08 N.D.
Results are expressed as mean + 8.D.
investigated
(20).
Recent works
(21-23) and x-ray can provide renin.
the
crystal basis
However,
evaluation
potent
believe
that
excellent
there
is
classes the
no
of human
not
animal renin
renins
model
are
inhibitors the
investigate
renin
inhibitors
in
for
for
(25).
data human
/n
insensitive
hRN g e n e the
vivo
to many
Therefore, may
we
provide
tissue-specific
but also to develop animal models for screening
human
models
and these
inhibitors
animal
carrying to
(24),
of specific
ideal
mouse only
the three-dimensional
of human renin
design
because
transgenic
occasions
expression, specific
the
of inhibitors,
of the
revealed
structure
for
N.D. ; not detectable.
new types of
future.
ACKNOWLEDGMENTS We w o u l d helpful Ministry Cement
like to thank
discussions. of Co.
Education,
This
Drs.
work
Science,
Hitoshi Hori and Masazumi was
and
supported Culture
of
by
grants
Japan,
and
Tada for from
the
Chichibu
Ltd.
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