From the Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7525
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ABSTRACT |
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We have generated mice having a single copy of
the human haptoglobin gene (Hp2), driven by its
natural promoter, and a neomycin resistance gene (Neo),
driven by a herpes simplex thymidine kinase promoter with polyoma
enhancers, inserted into two defined chromosomal locations, the Hprt
locus on the X-chromosome and the apolipoprotein (apo) AI-CIII gene
cluster on chromosome 9. The haptoglobin promoter is highly specialized
in its tissue of action; the viral promoter has few restrictions. The
apoAI-CIII gene is naturally active in only two tissues, whereas the
Hprt gene region is ubiquitously active. Expression of both
transgenes at substantial levels was achieved only (a) when
the transgenes were inserted into the genome close to a known
tissue-specific enhancer/locus control region in the apoAI-CIII gene
cluster, and (b) when known conditions for function of
their promoters were met. The specificities of the two chromosomal
regions and of the two promoters are preserved, but their interactions
are not specific. We conclude that transgenes are affected by
locus-related enhancers in the same manner as nearby endogenous genes.
Our experiments reinforce the usefulness of using gene targeting to
direct single-copy transgenes to appropriate chromosomal locations.
Transgenic mouse technology has been widely and effectively used
for studying the physiological consequences of expressing exogenous
gene products or analyzing promoter function and protein structure-function relationships in vivo (1). However,
analysis with transgenic mice produced by pronuclear microinjection
requires multiple founders because a foreign gene incorporated into the mouse genome is often expressed differently, both in amount and in
tissue specificity in one founder versus in another.
Integration of the exogenous DNA into random chromosomal locations and
in unpredictable numbers of copies, together with rearrangements in the
introduced DNA or in the sites of incorporation associated with the
integration event, are among the factors accounting for these
variations (2, 3).
We have previously described a general way of introducing single-copy
transgenes into the mouse germ line via embryonic stem (ES)1 cell technology as a
means of comparing the expression of different transgenic sequences
without the complications of variation in copy number and insertion
site (4). In this earlier work, we tested the method by inserting into
the hypoxanthine phophoribosyltransferase (Hprt) locus on
the X chromosome a single copy of a murine Bcl-2 cDNA
driven either by a chicken A gene cluster on mouse chromosome 9, which contains genes, coding for
apolipoprotein (apo) A-I, apoC-III, and apoA-IV is a potential target
for such studies. The region is relatively easily targeted (7), and
alteration of a single copy of any of the three genes in the locus has
physiologically insignificant consequences that do not affect the
development or overall health of the resulting mice (8). All three
apolipoprotein genes are expressed highly in the liver and small
intestine, and the promoter/enhancer elements controlling the
expression of these genes are well characterized in humans.
In the present study, we tested the expectation that the choice of the
site of transgene insertion will cause a predictable influence on the
tissue-specific expression of an inserted transgene. To do this, we
introduced a transgene cassette into two different chromosomal
locations in mice: the apoAI-CIII locus on chromosome 9 and the Hprt
locus on the X chromosome. The targeting cassette was composed of a
neomycin resistance (Neo) gene driven by the herpes simplex
thymidine kinase promoter with polyoma enhancers (9) and a human
haptoglobin (Hp) gene driven by its own 1.1-kb promoter
sequence (10); haptoglobin is the major hemoglobin-binding protein in
serum, and its gene is mainly expressed in the liver. Our results
demonstrate that the specificities of the two chromosomal regions and
of the two promoters are preserved, but their interactions are not specific.
The Neo-hHp2 Transgene Cassette--
The entire
human Hp2 gene (10) is contained within a 9-kb
fragment extending from an XbaI site at approximately 1.1 kb
5' to the starting codon to an ApaI site 1.6 kb 3' to the
stop codon. This fragment with 1.1-kb DNA encoding the pMC1neopoly(A)
gene (Stratagene) attached to its 5' end in the opposite
transcriptional orientation was used as a
Neo-hHp2 transgene cassette.
Gene Targeting--
The vector to introduce the
Neo-hHp2 cassette into the Hprt locus
was a modified form of the previously described locus-specific targeting vector, pMP8 (14). The regions of homology are 4 kb of DNA 5'
to the Hprt locus and 1.6 kb of DNA extending 3' from a
HindIII site in intron 2 to an EcoRI site in
intron 3 of the Hprt gene. The Neo gene and the
hHp2 gene were inserted as shown in Fig. 1.
A 9-kb BamHI fragment of mouse genomic DNA containing the
Apoa-1 gene and a 3' part of the Apoc-3 gene (7)
was used to make the targeting construct designed to insert the
Neo-hHp2 cassette into the apoAI-CIII
region. The Neo-hHp2 cassette was
inserted so as to replace a 450-bp BglII-BstXI
fragment 3' to the Apoc-3 gene. The BstXI site is
at 30 bp downstream of the poly(A) addition signals for the
Apoc-3 transcripts. A herpes simplex thymidine kinase gene
was placed at the 3' end of the construct (Fig. 2).
Cell culture, electroporation and selection using mouse ES cells (BK4,
a subclone of E14TG2a, provided by Dr. B. Koller, University of North
Carolina) were as described (7) Microinjection of ES cells and
derivation of transgenic mice were as described (15). Mice were
maintained according to NIH/Institutional guidelines.
Southern Blot Analyses--
Southern blots were made with
HindIII digests of genomic DNA isolated from cells or from
tails of mice at weaning (3 µg). Probe 1 (Fig. 1C) is a
250-bp RsaI fragment in the intron 3 of the mouse
Hprt gene. Probe 2 (Fig. 2C) is a 600-bp
KpnI-BamHI fragment containing a part of the
Apoc-3 gene. The presence of a 7.6-kb with probe 1 or an
8-kb band with probe 2 detects targeting in the Hprt locus or
apoAI-CIII region.
LPS Treatment--
Inflammation was induced in mice (10-18
months old) by injecting 25 µg of lipopolysaccharide (LPS,
Calbiochem) intraperitoneally. Blood was collected retroorbitally
24 h after LPS injection. Tissues were collected at 12 h
after LPS injection for RNA analyses by primer extension and at 24 h for Northern blot analyses.
Detection of Plasma Haptoglobin Protein--
Sera (4-5 µl)
were mixed with 1 µl of a fresh lysate of red blood cells containing
approximately 3% hemoglobin and electrophoresed in a 5%
polyacrylamide gel at pH 8.9 (16). The gels were soaked in 0.1 M potassium acetate, pH 5.0, for 15 min before staining with 0.2% benzidine (w/v) in 0.5% acetic acid (v/v) containing 0.3%
hydrogen peroxide (w/v).
Northern Blot Analyses--
Total RNA was isolated using
TRI REAGENT (Molecular Research Center, Inc.) according to
the manufacturer's protocol. RNA was electrophoresed in a 1.2%
agarose gel containing 2.2 M formaldehyde before transfer
onto a Nytran Plus membrane (Schleicher & Schuell). The blots were
sequentially hybridized to probes using conventional protocols. The
human haptoglobin Primer Extension Analyses--
Oligonucleotide primers were
labeled with [ Reverse Transcription-Polymerase Chain Reaction Analyses
(RT-PCR)--
Total RNA (1 µg) was treated with DNase I and used in
a reverse transcription-polymerase chain reaction (RT-PCR) using
a standard protocol (21). The primers specific to human
Hp2 were 5'-AGAAGGAGATGGAGTATACACC-3' and
5'-TTAAGGTGTACACTCCATCTCC-3' (10) and specific to mouse Hp
were 5'-CAGTGCCCGAGAAGAAAAACTT-3' and 5'-TACACAGAGCGACTTGAACA-3'
(18). After initial incubation at 95 °C for 2 min, PCR for 35 cycles was at 60 °C for 1 min, 72 °C for 1 min, 94 °C for
30 s, followed finally by 72 °C for 4 min.
Nuclear Run-on--
Isolation of liver nuclei and in
vitro nuclear run-on assay were carried out according to Ntambi
(22). The resulting radioactive RNA was hybridized to immobilized DNA
probes for apoC-III, apoA-I, Neo, and Gapdh
described above.
Generating Mice with Transgenes in the Hprt and ApoAI-CIII
Locus--
The two genes chosen as transgenes for the present study
are the human haptoglobin transgene (hHp2) on a
9-kb fragment of human genomic DNA that includes the
hHp2 gene with its own promoter and 3'
untranslated sequence and neomycin-resistant gene (Neo) from
the pMC1neopoly(A). The two genes are in opposite transcriptional orientations.
The overall scheme for inserting the
Neo-hHp2 transgene cassette into the
partially deleted Hprt locus on the X chromosome of BK4 ES cells is
diagrammed in Fig. 1. The targeting
vector is derived from pMP8SKB (14) used in our previous studies of targeted transgenesis at this locus. In the current vector, the promoter and exons 1 and 2 of the Hprt genes in pMP8SKB were
removed, and selection to facilitate identification of targeted cells
was with G418 instead of with hypoxanthine-aminopterin-thymidine. Approximately 1 in 10 G418-resistant colonies had a single copy of the
transgene inserted into the Hprt locus. Because the ES cell used for
targeting is karyotypically male, and thus hemizygous for the
X-chromosome-linked Hprt locus, the modified ES cell genome was
transmitted from the chimeras to their F1 female offspring. Mating of
these F1 heterozygous females with C57BL/6 males was used to generate
male progeny, and Southern blot analyses of their genomic DNA showed
either a 7.1-kb or a 7.6-kb HindIII fragment, corresponding
to the wild type or targeted Hprt locus (Fig. 1D). We
designate the resulting hemizygous males having one copy of the
hHp2 transgene on their X chromosomes as
Hprt-Hp2 mice.
The scheme for inserting the Neo-hHp2
transgene cassette into the apoAI-CIII gene cluster on chromosome 9 is
diagrammed in Fig. 2. The transgene
replaces a 450-bp BglII/BstXI fragment between the Apoa-1 and Apoc-3 genes. Approximately one in
three colonies resistant to G418 and ganciclovir had an insertion of
the transgene at the correct position as judged by the presence in
Southern blot analyses of an 8-kb HindIII fragment in
addition to a 12-kb band from the unmodified locus. The transgene was
passed from chimeric males to approximately half of their offspring. We
designate the resulting (heterozygous) mice carrying one copy of the
hHp2 transgene as Apo-Hp2 mice. Males at the F2
or F3 generation were used for further experiments.
Expression of a Neomycin Resistance Transgene in the Apo-Hp2 and
Hprt-Hp2 Mice--
Because the Neo gene in the
Neo-hHp2 transgene cassette is driven
by a powerful herpes simplex thymidine kinase promoter and a duplicated
polyoma enhancer, the Neo transgene was expected to be
expressed in most cell types regardless of the location of insertion
(9). We, therefore, first determined the tissue distribution and
relative levels of Neo transgene expression in the Apo-Hp2
and Hprt-Hp2 mice. The Neo transcripts were detectable by
Northern blotting in the liver and small intestine of the Apo-Hp2 mice
but not in the Hprt-Hp2 mice (Fig. 3).
Comparison of the levels of Neo transcripts in different
tissues was carried out by primer extension analyses as illustrated in
Fig. 4A for the liver and
4B for the small intestine. Fig. 4C summarizes
these and also presents analyses of RNA from lung, adipose tissue, bone marrow, and spleen. The results show that expression of the
Neo transgene in the apoAI-CIII gene cluster differs
considerably in different tissues. However, in general, the relative
levels of expression in the various tissues parallel to that of the
Apoa-1 and Apoc-3 genes, which are highly
expressed in the liver and small intestine. Thus, relative to liver
expression as 100%, the amounts of Neo transcripts per µg
of total RNA in small intestine, adipose tissue, and lung were 57, 14, and 3% that of the liver. No Neo transcripts were
detectable in the spleen and bone marrow.
Expression of the Neo transgene in the Hprt-Hp2 mice was
detectable but low, at about 0.5% the expression of the
Gapdh gene in the same tissues, except that no expression
was detectable in the small intestine; the expression in the liver of
the Neo gene in the Hprt locus was about 10% that in the apoAI-CIII locus.
Expression of the Human Hp2 Transgene in the Apo-Hp2
Mice--
Liver is the major organ of haptoglobin production in humans
and in mice. Northern blot analysis of liver total RNA (Fig. 3A) using a probe (hp
To compare the expression of the human Hp2
transgene with the expression of the endogenous mouse haptoglobin gene
in tissues where the levels are too low for Northern blot analysis, we
used primer extension analyses as illustrated in Figs.
5A through 5D and
presented in a quantitative manner in Fig. 5E. The data show that the endogenous mouse Hp gene is expressed at about 15%
that of the liver level in lung, adipose tissue, and bone marrow,
respectively. Endogenous mouse Hp transcripts were also
detected (not shown) in the thymus and adrenal gland at approximately
5% that of the liver, with still lesser amounts in the intestine,
spleen, kidney, and testis.
Increased expression of the mouse gene was observed in the liver after
LPS treatment, which is known to induce synthesis of haptoglobin, an
acute-phase response protein. The amount of human Hp2 transcripts in the liver of Apo-Hp2 mice
from a single copy of the transgene was approximately 70% that of a
single copy of the endogenous mouse Hp gene. The mRNA
levels of both the endogenous mouse Hp gene and the
hHp transgene were increased equally by about 30% at
12 h after LPS treatment. Human Hp2
transcripts were also found in the small intestine, lung, adipose tissue, bone marrows, and spleen, at about 3% of the liver expression of the endogenous mouse gene. Less than 1% of liver levels of transcript were seen in the other tissues examined (not shown), including brain, thymus, heart, kidney, adrenal gland, testis, and
ovary. In general, expression of the human transgene in the Apo-Hp2
mice in these extra-hepatic tissues was approximately half or less of
the endogenous mouse gene expression, except that in the intestine
expression of the transgene, which was approximately five times that of
the mouse gene (itself very low at approximately 1.5% of the liver expression).
Expression of the hHp2 Transgene in the Hprt-Hp2
Mice--
In the Hprt-Hp2 mice, the level and tissue distribution of
transgene expression was markedly different from that in the Apo-Hp2 mice. No transcripts of the hHp2 transgene were
detectable by primer extension analyses in the liver, adipose tissue,
small intestine, and kidney in the Hprt-Hp2 mice, although they were
detectable when total RNA was subjected to RT-PCR analyses (data not
shown). Transcripts, detectable by primer extension analyses in the
lung, bone marrow, and spleen, were, respectively, at about 4, 3, and
2% levels of the endogenous mouse gene in the liver. Expression in the
brain, heart, and testis was detectable but at less than 1% of the
liver expression (data not shown). LPS treatment did not affect
expression of the transgene in any of these tissues.
Influence of the Neo-hHp2 Transgenes on the Expression
of Nearby Apolipoprotein Genes--
To investigate whether the
insertion of the transgene 3' to the Apoc-3 gene had
influenced the expression of the nearby Apoa-1, Apoc-3, and Apoa-4 genes in the Apo-Hp2 mice, the
Northern blots used for Fig. 3 were sequentially rehybridized with
probes corresponding to each of these apolipoprotein genes. The amounts
of Apoa-1 and Apoa-4 transcripts in the Apo-Hp2
mice were not different from those in the wild type mice. However, the
amount of Apoc-3 transcripts was reduced to about 50% that
of wild type in heterozygotes, and transcripts were not detectable in
homozygous Apo-Hp2 mice. Thus the insertion of the transgene cassette
approximately 30 bp 3' to the poly(A) addition signal sequence affected
the Apoc-3 gene expression, although the transcriptional
orientations of the Neo genes and the Apoc-3
genes are opposed (Fig. 2). A preliminary nuclear run-on experiment
with the liver of a homozygous Apo-Hp2 mouse showed that initiation of
the Apoc-3 gene transcription appears to be normal (data not
shown). This suggests that the likely cause of the undetectable amounts
of Apoc-3 gene mRNA in the Apo-Hp2 homozygotes is that
insertion of the transgenes just downstream of the gene results in an
unstable message or in failure of the transcripts to be processed
properly. The levels of expression of the Apoa-1, Apoc-3,
Apoa-4, and the Neo genes were unaltered by LPS treatment.
Ready Detection of Human Haptoglobin in the Plasma of the Apo-Hp2
Mice but Not in the Hprt-Hp2 Mice--
We compared the levels of the
endogenous and human haptoglobin proteins in the circulation of the
transgenic mice by nondenaturing polyacrylamide gel electrophoresis of
plasma proteins at pH 8.9 (Fig. 6). The
amount of endogenous mouse haptoglobin differs in the plasma of
different individuals, with no protein being detected in some mice.
Plasma haptoglobin levels are much more variable than the corresponding
mRNA levels of the animals. Reliable detection of haptoglobin in
the plasma of all mice was, however, achieved by treating them with
LPS, which induces the synthesis of haptoglobin. Mouse haptoglobin,
like the monomeric form of human haptoglobin, hp1, contains one free
sulfydryl group in its
In marked contrast to these observations with the Apo-Hp2 mice, no
human haptoglobin was detectable in the plasma of Hprt-Hp2 mice, even
after LPS-treatment, although the endogenous mouse haptoglobin was
normally present.
Our results show that the chromosomal location of a transgene
markedly and with a considerable degree of predictability affects the
levels of its expression. Thus the Neo gene in the Hprt
locus is expressed in many tissues of the Hprt-Hp2 mice at a uniform level, reflecting the ubiquitous pattern of the Hprt gene
expression. However, although the Neo gene is driven by a
strong viral promoter, its expression in the Hprt locus is relatively
low. In contrast, the tissue expression of the same Neo gene
in the apoAI-CIII region of the Apo-Hp2 mice takes on the same strong
expression pattern as the apolipoprotein locus, which is highly
expressed in the liver and intestine. Expression in the liver of the
Neo gene in the apoAI-CIII locus is at least 9-fold that of
the same gene in the Hprt locus.
In contrast to the Neo gene with its viral promoter, the
liver expression in the Hprt-Hp2 mice of the
hHp2 transgene driven by its own promoter
sequence was only detectable by RT-PCR. This agrees with earlier work
in which a sequence that included the 1.1-kilobase pair promoter of
hHp2 gene was not able to support high levels of
liver expression of reporter genes randomly inserted into the genome as
transgenes (12, 13), despite its being sufficient for liver-specific transient expression of the gene in hepatic cells in tissue culture (11). Thus, high levels of liver expression of the haptoglobin gene
require not only a liver-competent promoter but also enhancer element(s) outside of the 9-kb DNA containing the
hHp2 gene. The apoAI-CIII region, but not the
Hprt locus, can supply the enhancer element(s), and the
hHp2 transgene inserted close to the
Apoa-1 gene is expressed in the liver at approximately 70%
that of the level of the endogenous mouse Hp gene. The
apoAI-CIII region is also known to contain enhancers that functions in
the intestine, and the expression of the hHp2
transgene in this tissue in the apoAI-CIII locus was five times that of
the endogenous mouse Hp gene in the same tissue. These results demonstrate that tissue-specific enhancer elements present in
the apoAI-CIII region can interact with the hHp2
promoter as well as with the viral thymidine kinase promoter and that
high levels of expression of the transgene can be obtained in the liver
by interactions between either of these quite different promoters when
they are inserted into the genome within the range of the
tissue-specific enhancers.
Studies using transgenic mice have shown that a 256-bp sequence from
the human apoA-1 gene (24) or a 780-bp sequence from the human apoC-III
gene (25) are sufficient for driving their respective gene expression
at high levels in the liver. The cis-elements within these regions that
are necessary for the liver-specific expression have been well
characterized (26). The same studies also showed that neither element
is able to support the expression of the transgenes in the intestine,
but that high levels of intestinal expression of the transgenes can be
achieved by the simultaneous presence of a DNA segment 5' to the
apoC-III gene ( Unusual aberrant expression of a foreign gene in a particular
transgenic mouse line is frequently attributed to chromosomal position
effect, although proving this effect is often difficult. In one such
case of an aberrant tissue expression of a transgene, Al-Shawi et
al. (27) demonstrated that the expression patterns of the
transgene changed by recovering and reintroducing it into the secondary
transgenic mice. Position independence of transgene expression can be
obtained when relatively large genomic segments are introduced or when
the transgene is attached to a sequence for matrix attachment sites (or
scaffold attachment sites) (28) or to very strong enhancers such as the
locus-controlling region of the The predictable expression of the Neo and
hHp2 transgene in the liver of the Apo-Hp2 mice
demonstrates the usefulness of the apoAI-CIII region for introducing a
targeted transgene when expression in the liver is desirable. The
transcriptional properties of the foreign promoters appear to be
maintained, but their promoter activities in the liver and intestine
(and potentially in the adipose tissue) are greatly enhanced. Thus the
acute phase response of the Hp promoter was retained, as
shown by the increase in the hHp2 gene
expression, under LPS stimulation to approximately the same extent as
the increase in endogenous gene expression. Inactivation of the
Apoc-3 gene by an insertion in opposite transcriptional orientation of the Neo gene at approximately 30 bp 3' to the
poly(A) addition signal sequence was a slight complication in the
present experiments, and adjustment of the position of the insertion
may be desirable to eliminate or reduce the influence of the transgene on expression of the Apoc-3 gene. However, the reduction of
apoCIII expression itself is unlikely to influence the phenotype
ascribable to the transgene, because mice completely lacking apoCIII
are healthy and exhibit only a mild hypotriglyceridemia (30).
In conclusion, the ability to control the levels of tissue-specific
expression of a transgene in vivo by choosing both the promoter driving the gene and the site of chromosomal integration with
its nearby enhancers makes targeted transgenesis a very versatile approach for achieving a predictable in vivo function of the
transgene. Furthermore, by using repeated insertion of variant
transgenes into the same locus, targeted transgenesis allows the
systematic dissection of in vivo promoter functions or
effects of subtle structural changes in the products of the transgenes.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
-actin promoter or by a human
actin
promoter and demonstrated that expression of the two transgenes differed in a predictable fashion. The Hprt gene is a
housekeeping gene, transcriptionally active in all tissues, and
modification of the locus does not affect the overall development and
health of the animals. Furthermore, the availability of
Hprt
ES cells, such as E14TG2a (5) and HM-1
(6) and of vectors capable of mediating highly efficient and directly
selectable homologous recombination at the Hprt locus makes this system
simple to use. The previous work, however, also showed that despite
expression of the Hprt locus in all tissues, expression of the Bcl-2
transgene driven by the human or the chicken
actin promoter was
very low or undetectable in the liver and kidney; this has prompted us to search for a locus in which liver-specific expression of a targeted
transgene can be readily studied.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
probe was a 650-bp
BamHI-HindIII fragment corresponding to exon 7 of
the Hp2 gene. The mouse apoA-I probe was a
180-bp EcoRI-NheI fragment corresponding to exon
4. The mouse apoC-III probe was a 410-bp SauIIIA fragment
corresponding to exon 4. The mouse apoA-IV probe was a 321-bp
PvuII fragment corresponding to exon 3. The Neo
probe was a 380-bp PstI-NcoI fragment in
pMC1neopoly(A). The Gapdh probe was a 315-bp
SacI-BamHI fragment corresponding to exons 5 through 8 (pTRI-GAPDH-mouse, Ambion).
-32P]ATP (1.3 × 107
cpm/pmol) and incubated for 10 min at 68 °C with 20 µg of total RNA in 30 µl of 0.05 M PIPES buffer, pH 6.4, containing
0.2 M NaCl. The reaction mixture was then incubated
overnight at 42 °C before treatment with reverse transcriptase, as
described (17). The reaction products were resolved in a 20%
polyacrylamide gel containing 8.3 M urea. Gels were then
dried and exposed to Kodak XAR films for at least two different times.
Densitometoric analyses were performed using NIH Image, and the
relative amount of mouse Hp, human
Hp2, or Neo transcripts were
estimated in each sample by the intensities of corresponding bands
relative to that of Gapdh. The following primers were
used: 5'-GCCACAGGCAGCATGACATACTT-3' for haptoglobins (nucleotides
901-923 of human Hp2 (10), 761-783 of mouse,
(18)); 5'-ACCCATCACAAACATGGGGGCAT-3' for mouse Gapdh
(nucleotides 420-442(19)); and
5'-CCGATCCCCTCAGAAGAACTCTCGA-3'for Neo (nucleotides
1522-1544). The validity of the assay for quantitating amounts of
transcript was established by the linearity of the signal for the
Hp and the Gapdh genes over a range of total
liver RNA from 0 to 40 µg and with primer amount varying from 0 to
105 cpm. Because the haptoglobin primer hybridizes to both
human and mouse transcripts equally well, the relative amounts of
transcripts within a sample can be readily compared. For comparisons
between samples or between different experiments, the expression levels were adjusted against that of Gapdh with the caution that
the normalized values in different tissues may not be completely
comparable, because expression of Gapdh itself is somewhat
variable in different tissues (20).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Insertion of the
Neo-hHp2 transgenes into the mouse
Hprt gene locus on the X chromosome. A, the
partially deleted Hprt gene ( Hprt) of BK4 ES
cells. The numbered black rectangles denote exons. The
double arrow indicates the 7.1-kb HindIII
fragment characteristic of the endogenous partially deleted
Hprt gene. The open arrow indicates the direction
of the gene, and the vertical zig-zag line represents the
deletion. B, H, R, and S
indicate BamHI, HindIII, EcoRI, and
SalI sites, respectively. B, the targeting
construct. Neo indicates the neomycin resistance gene
oriented as indicated by the arrow. The wavy
lines indicate plasmid sequences (not drawn to scale). The
two shaded areas represent the homologous regions in which
recombination occurs. C, the inserted
Neo-hHp2 in the Hprt gene
locus. Probe 1 is used to demonstrate the 7.6-kb HindIII
fragment characteristic of the inserted hHp2
gene. D, autoradiogram with probe 1 of a Southern blot of F2
mouse genomic DNA digested with HindIII, showing the
expected fragments for the wild type (7.1 kb) and targeted alleles (7.6 kb). X/Y and X/X are DNA from wild type animals;
T/Y and T/X are from animals carrying the
targeted X chromosome.
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Fig. 2.
Insertion of the
Neo-hHp2 transgenes into the mouse
apoAI-CIII gene locus on chromosome 9. A, the endogenous
apoAI-CIII locus. B, the targeting construct. C,
the inserted Neo-hHp2 transgenes in
the apoAI-CIII gene locus. The symbols are as in Fig. 1,
with additional restriction sites for BglII,
BstI, and NheI labeled Bgl, Bst, and Nh. TK
indicates the herpes thymidine kinase gene. The double
headed arrow indicates the 8-kb HindIII
fragment characteristic of the inserted hHp2
gene. D, Southern blot analysis of tail DNA. DNA samples
were isolated from the tails of 3-week-old animals resulting from
heterozygote matings. Probe 2 was used to identify the 8.0-kb
HindIII band diagnostic of the modified Apoc-3
gene. Animal 1 is wild type (+/+); animals 2, 3, and 5 are
heterozygous (T/+) for the insertion; animals 4 and 5 are
homozygous (T/T) for the insertion.
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Fig. 3.
Northern blot analysis of the mRNA from
liver (A) and small intestine (B). RNAs
blots were sequentially hybridized to the six probes indicated. The
haptoglobin probe was used first; it is a fragment corresponding to the
subunit of hHp2. Subsequent probes were
specific for the genes coding for Apoa-1, Apoc-3,
Apoa-4, Neo, and Gapdh. RNAs were
collected without treatment (LPS
) on 24 h after LPS
injection (LPS +). Each lane contained 15 µg of
total RNA. WT, wild type.
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Fig. 4.
Primer extension analysis of expression of
the Neo transgene. mRNAs (20 µg) from the liver
(A) and from the small intestine (B) of the
indicated animals were subjected to primer extension analyses with Neo
primer (105 cpm) and with GAPDH primer (105
cpm). LPS+ or LPS indicate RNA from 12 h
after LPS injection or from untreated mice. C, densitometric
analyses of the Neo gene expression in various tissues from
the Apo-Hp2 and the Hprt-Hp2 mice. The values are means of three mice
S.E. and are expressed relative to the signals of Gapdh in
the liver as 100. LI, LU, AD,
BM, SP, and SI are liver, lung,
adipose tissue, bone marrow, spleen, and small intestine.
WT, wild type.
) derived from the
region of the
human Hp2 gene showed that human haptoglobin
transcripts were readily detectable in the Apo-Hp2 mice. The level of
expression in the transgene homozygotes with two copies of the
transgene was approximately twice as much as that in the single copy
heterozygotes. Human Hp2 transcripts were also
detectable by Northern blot in the intestine of the Apo-Hp2 mice, but
in considerably less amounts (Fig. 3B). Expression of the
endogenous mouse Hp gene (detectable by a low level
cross-reaction of its mRNA with the hp
probe) was stimulated by
LPS in the liver but not in the intestine.
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Fig. 5.
Primer extension analysis of expression of
the hHp2 transgene. mRNAs (20 µg)
from the liver (A), lung (B), small intestine
(C), and spleen (D) were analyzed as in Fig. 4
with primers for haptoglobin (105 for the liver and
106 cpm for other tissues) and GAPDH (105 cpm).
E, densitometric analyses of the endogenous mouse
Hp gene in wild type (WT) mice (hatched
bars) and the hHp2 transgene (shaded
bars) in Apo-Hp2 and Hprt-Hp2 mice. The values are means of three
mice with S.E. and are expressed as haptoglobin signal/gene copy/µg
of RNA relative to the signal for Gapdh in the liver as 100. LI, LU, AD, BM,
SP, and SI are liver, lung, adipose tissue, bone
marrow, spleen, and small intestine.
subunit and migrates as a single tetrameric
(
)2 band, like human haptoglobin type 1-1 individuals. The human hp2 protein
subunit, in contrast, contains
two free sulfydryl groups and forms a series of heteromultimers in the
presence of hp1 proteins. Thus, in human type 2-1 heterozygotes, haptoglobin migrates as a ladder during gel electrophoresis. A similar
ladder of haptoglobin heteromultimers, detectable in the Apo-Hp2 mice
after LPS treatment, demonstrates that the haptoglobin hp2 protein is
secreted in combination with the type 1-like mouse haptoglobin. Human
haptoglobin was readily detected in the plasma of (homozygous) Apo-Hp2
mice having two copies of the transgene, even without LPS treatment.
Plasma from human type 2-1 heterozygous individuals yields a ladder
with up to 9 identifiable bands. But only three bands are detected in
the plasma of Apo-Hp2 mice, with the first being the heaviest. This
pattern is very similar to that seen in individuals having the
haptoglobin 2-1-modified phenotype (23), which is caused by the
production of less hp2 protein than hp1 (16). Polyacrylamide gel
electrophoresis analysis of plasma from the Apo-Hp2 mice, therefore,
demonstrates that the human haptoglobin is made and secreted in these
mice, but the amount of the human hp2 protein is less than the mouse
protein, consistent with the mRNA data presented above, showing
that the expression of the Hp2 transgene is
somewhat lower than that of the endogenous mouse gene.
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Fig. 6.
Polyacrylamide gel electrophoresis of plasma
isolated from mice treated (+) or untreated ( ) with LPS.
LPS + indicates the mice for 24 h after LPS injection.
Free hemoglobin (Free Hb) and the hemoglobin-haptoglobin
complexes (Hp 1-1 or Hp 2-1) are detected by
benzidine staining. Human plasma haptoglobins from individuals with
haptoglobin types 2-1 and 1-1 are shown for comparison. WT,
wild type.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
500 to
900) that functions as a classical enhancer.
This distal DNA segment, which contains multiple regulatory elements,
also enhances expression from the apoA-I and apoC-III promoters in the
liver 5- to 10-fold in the tissue culture system (24). The enhancement
of expression in both the liver and the intestine of the
Neo-hHp2 transgenes when inserted into the mouse
apoAI-CIII region is most likely the consequence of similar interactions.
-globin genes (29). Our work clearly
demonstrates how expression levels in various tissues is affected by
interaction between the promoter elements of the introduced transgene
and enhancers that reside near the location of the transgene. It will be of interest to test how the introduction of matrix attachment sites
or a locus-controlling region will influence the expression patterns of
endogenous genes near a chosen chromosomal insertion site.
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ACKNOWLEDGEMENTS |
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We thank J. Cowhig, K. Kluckman, S. Hiller, and A. Staton for their technical help and Dr. K. Caron, J. Knowles, Dr. N. Takahashi, and Dr. R. Thresher for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM37567 and GM20069.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Dept. of Microbiology, University of Texas at
Austin, ESB 226, Austin, TX 78712-1095.
§ To whom correspondence and reprint requests should be addressed: Dept. of Pathology and Laboratory Medicine, University of North Carolina, CB 7525, Brinkhous-Bullitt Bldg., Chapel Hill, NC 27599-7525. Tel.: 919-966-6913; Fax: 919-966-6718; E-mail address: nobuyo{at}med.unc.edu.
The abbreviations used are: ES cells, embryonic stem cells; apo, apolipoprotein; Gapdh, glyceraldehyde-3-phosphate dehydrogenase gene; Hp, human haptoglobin gene; Hp2, Hp 2 allele; hHp2, Hp 2 transgene; mHp, mouse haptoglobin gene; Hprt, hypoxanthine phophoribosyl transferase gene; LPS, lipopolysaccharide; Neo, neomycin resistance gene; kb, kilobase(s); bp, base pair(s); PIPES, 1,4-piperazinediethanesulfonic acid; RT-PCR, reverse transcription-polymerase chain reaction.
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REFERENCES |
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