From the
Inconsistent data have been reported on the size of the promoter
that is necessary for high levels of tissue-specific expression of the
COL1A1 gene for type I procollagen. Some of the inconsistencies may be
traced to the use of reporter gene constructs. Therefore, we prepared
transgenic mice with modifications of the intact gene engineered so
that the level of expression of the transgene could be assayed both as
mRNA and protein that were similar to the products from the endogenous
COL1A1 gene. The results with a mini-COL1A1 gene lacking 41 internal
exons and introns indicated that the first intron and 90% of the
3`-untranslated region were not essential for tissue-specific
expression. In a hybrid COL1A1/COL2A1 construct, a 1.9-kilobase
5`-fragment from the COL1A1 gene that contained only 476 of the
promoter was linked to a promoterless 29.5-kilobase fragment of the
human COL2A1 gene for type II procollagen. The hybrid COL1A1/COL2A1
construct was expressed as both mRNA and protein in tissues that
normally synthesize type I procollagen but not type II procollagen.
Apparently, 476 base pairs of the promoter are sufficient to drive
tissue-specific expression of the COL1A1 gene and totally inappropriate
expression of the COL2A1 gene.
Type I collagen is the most abundant structural protein in
vertebrates, and it is synthesized in a large number of different
tissues at different stages of development (see Refs. 1-4).
Additionally, the expression is regulated by a variety of growth
factors, hormones, and other agents
(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) .
However, there have been conflicting reports as to the size of the
promoter region and related elements of the COL1A1 gene that are
necessary both for high levels of expression and tissue-specific
expression
(2, 3, 4) .
Some of the
discrepancies have been generated by apparently similar experiments
involving transient expression of reporter gene constructs in cultured
cells
(15, 17, 18, 19, 20) .
Experiments in transgenic mice have also failed to provide a consistent
picture but most have suggested that a large promoter fragment is
required. For example, Slack et al. (21) observed that
a construct with a reporter gene of human growth hormone driven by
-2.3 kb
Inconsistent
observations have also been reported as to whether the first intron of
the COL1A1 gene contains essential regulatory elements
(2, 3, 4, 25, 26, 27) .
Some experiments have suggested that the 3`-region of the COL1A1 gene
may be important for high levels of tissue-specific expression
(28) .
Some of the discrepancies may be explained by the use
of reporter genes in experiments to define cis-regulatory elements in a
gene as complex as the COL1A1 gene. As recently observed by Moreira
et al. (29) , different reporter genes linked to the
same promoter can give very different patterns of expression when
transfected into the same cells, apparently because of differences in
the metabolic turnover of RNA transcripts and proteins from the
reporter genes. In transgenic mice, the values obtained for COL1A1
reporter gene constructs vary by as much as one order of magnitude for
expression in the same tissue from the same line of mice, even when
precautions are taken to standardize the assays per microgram of total
RNA (see and comment on page 2072 of Ref. 21). For these
reasons, we have avoided the use of reporter genes in our own
experiments
(30, 31, 32, 33, 34, 35, 36, 37, 38) .
Instead, we employed large constructs of collagen genes that are
engineered so that the level of expression can be assayed as both as
mRNA and protein that are similar in structure to mRNA and protein
synthesized from the endogenous COL1A1 gene. In addition, the
expression can be assayed over a broad range and relative to expression
of the endogenous gene in the same sample and even the same lane of an
electrophoretic gel. As a result, many of the technical problems in
assessing tissue-specific expression were reduced, including the
difficulty of isolating homogeneous samples of small tissues from
transgenic mice (see Ref. 36).
Here we report that deletion of about
90% of the 3`-untranslated sequences of a mini-COL1A1 gene construct
did not substantially change the high degree of tissue-specific
expression observed with the parent construct
(36) . In
addition, a 1.9-kb fragment from the 5`-end of the human COL1A1 gene
with only 476 bp of the promoter linked to a 30-kb promoterless
fragment of the human COL2A1 gene caused relatively high levels of
inappropriate synthesis of type II collagen in tissues that normally
synthesize type I collagen but no type II collagen.
The modified mini-COL1A1 gene lacking
90% of the sequences of the 3`-untranslated region had a deletion that
began 179 bp beyond the first polyadenylation signal (Fig. 1). It
also had a deletion of the 1.2-kb SacI/ SmaI fragment
from the first intron. To prepare the construct, a fragment of 485 bp
was amplified with seven cycles of PCR from the 3`-end of mini-COL1A1
using primers GACCAGGAATTCGGCTTCGAAGT (forward primer) spanning the
unique EcoRI site in mini-COL1A1, and primer
CATTGGATCCTGTGTCTTCTGGG (reverse primer) that was downstream from the
first polyadenylation signal. The PCR product was digested with
EcoRI and BamHI. Then it was gel-purified and ligated
with a 6.2-kb NotI- EcoRI fragment of mini-COL1A1
construct. The ligation product was digested with NotI and
BamHI and isolated by electrophoresis in a 1% agarose gel.
The COL1A1/COL2A1 construct was prepared as described previously
(32) . It contained 476 bp of the promoter, the first exon, and
most of the first intron of COL1A1 gene, i.e. sequences from
-476 to +1,445 bp of the gene. It also contains 29.5 kb of
sequences of human COL2A1 gene that extended from the SphII
site in the 3`-end of the second intron of the gene to about 3.5 kb
beyond the major polyadenylation signal of the COL2A1 gene
(32) .
To prepare transgenic mice, one-cell zygotes were obtained from
mating of inbred FVB/N males and females. The DNA was injected in a
concentration of about 2 µg/ml and about 600 copies/embryo. Inbred
CD1 females were used as the pseudo-pregnant recipients. Founder mice
were identified either by Southern blot analysis of tail extracts
(31) or by PCR analysis of toe extracts (see below). For
propagation, the transgenic mice were bred into the same FVB/N strain.
To confirm the data on copy
number of the mini-COL1A1 constructs, two different PCR assays were
used. In one assay, one pair of primers was used that spanned the first
exon of the human COL1A1 gene and a second pair of primers that spanned
the first exon of the mouse COL1A1 gene. The PCR product from the human
mini-COL1A1 gene was 431 bp, and the PCR product from the mouse gene
was 400 bp. The primers were BS66-AGCGGAAGGCGCGATATAGAGTATC (human),
BS68-CTCCTCCCCCTCTCCATTCCAACT (human), BS67-CAGAACGCAATACCATAGAAGCTGT
(mouse), and BS69-CTTTCCTCCTCCCCCCTCTCGT (mouse). The conditions for
the PCR were 1 min 10 s at 94.5 °C, 2 min at 67 °C, and 25
cycles. The second PCR assay used three primers that spanned exon 1 of
both the human and mouse genes. A fragment of 255 bp was obtained from
the human sequence and a fragment of 225 bp from the mouse. The primers
were BS47-ACTCCCAAAAGTTTGGGACTTACTG (human),
BS48-ACTCCCCAGAGTTTGGAACTTACTG (mouse), and
BS49-CCAGTGTCGGAGCAGACGGGAGTTTCTCCT (human and mouse). The conditions
for PCR were the same as the first PCR assay.
To assay copy number
of the COL1A1/COL2A1 gene, one pair of primers was used that spanned
exon 50 and exon 51 of both the human and mouse COL2A1 gene. The human
gene gave a fragment of 608 bp and the mouse 562 bp. The primers were
BS39-GCTGCACCTTGGACGCCATGAA and BS40-CAGTGGTAGGTGATGTTCTGGGA. The
conditions for PCR were the same as the first PCR assay. The same PCR
assays were to screen litters for transgenic mice (see above).
To assay sequences from the 3`-end of mRNA from
the mini-COL1A1 gene relative to mRNA from the endogenous COL1A1 gene,
2 µg of total cellular RNA was reverse transcribed in a 20 µl
of reaction mixture using 2,200 pmol of a common primer for the 3`-ends
of both mRNAs (BS33-ACTAAGTTTG) and a commercial preamplification
system for first strand cDNA synthesis (SuperScript
To assay sequences from the 5`-end of mRNA from the mini-COL1A1 gene
relative to mRNA from the endogenous COL1A1 gene, reverse transcription
of 0.5 µg of total RNA from different tissues of transgenic mice
was performed with mixtures of primers: BS91-CGTCGGGGCAGA (human) and
BS92-CTTCCGGGCAGA (mouse) directed to highly homologous sequences in
exon 2 of the human and mouse COL1A1 genes. cDNA was then amplified in
three-primer PCR assay with
To assay levels of the mRNA
for type II procollagen from the COL1A1/COL2A1 hybrid construct
relative to mRNA from the endogenous COL1A1 gene, reverse transcription
of 0.5 µg of total RNA was performed with two primers:
BS94-CCTTTGTCACCAC (human COL2A1) directed to a sequence in exon 6 of
human COL2A1 gene and BS92-CTTCCGGGCAGA (mouse COL1A1) directed to a
sequence in exon 2 of mouse COL1A1 gene. cDNA was then amplified in a
three-primer PCR with a
After reaction with secondary
antibodies, blots were developed using a chemiluminescence assay based
on a phenylphosphate-substituted 1,2-dioxetane that produces light by
reaction with alkaline phosphatase (Protein Images, U. S. Biochemical
Corp.). The light emitted was detected by exposure to x-ray film, and
the film was scanned with a laser densitometer. Preliminary experiments
with varying exposure times and different amounts of tissue extracts
were used to establish the linear range of response for the assay.
Expression of the Mini-COL1 Gene Minus Intron 1 and 90% of the
3`-Untranslated Region-The 3`-half of the mini-COL1 gene
construct contained the last six exons and about 3 kb of the
3`-flanking region of the COL1A1 (Fig. 1). The presence of the
3`-sequences of the gene may well explain why the mini-COL1 gene was
expressed in a tissue-specific manner in transgenic mice
(36) whereas many reporter gene constructs were not. To test
this possibility, a modification of the mini-COL1 gene was prepared in
which both the first intron and 90% of the 3`-untranslated region were
deleted. The deletion at the 3`-end of the gene removed one of the two
polyadenylation signals. Four lines of transgenic mice expressing the
construct were prepared. In addition, one founder mouse was obtained
that did not transmit the gene. All the mice prepared with the
construct had one to two copies of the exogenous gene (Fig. 2 and Table
I). To assay expression of the gene, two separate quantitative RT-PCR
assays were developed. Both assays indicated that steady-state levels
of mRNA from the exogenous gene ranged from 1 to 3% of the levels of
the mRNA from the endogenous COL1A1 gene (). Therefore, the
levels of expression in the four lines and the one founder appeared to
be somewhat less than previously seen with expression of the mini-COL1
gene or the mini-COL1 gene minus the first intron
(36) .
Data
obtained with both RT-PCR assays indicated that the expression in
different tissues of the construct lacking 90% of the 3`-untranslated
region paralleled expression of the endogenous COL1A1 gene in most
tissues (Fig. 3 and Table II). Therefore, the results were similar to
those previously obtained with the mini-COL1A1 gene and the mini-COL1A1
gene minus the first intron
(36) . However, the RT-PCR assays
indicated the levels of mRNA from the mini-COL1A1 gene lacking the
first intron and the 3`-untranslated region were higher in brain than
other tissues ( Fig. 3and ). Previously, expression
of the mini-COL1A1 gene was examined in brain only by Western blot
assays for steady-state levels of protein
(36) . In three lines
(lines J, K, and R), no detectable levels of expression in brain of
shortened pro
In further experiments, transgenic animals
from lines 73 and 85 were inbred so as to generate homozygous mice with
twice the copy number of the same transgene. As expected, the initial
levels of expression of the transgene in fibroblasts from apparently
homozygous transgenic mice were about twice as high as the level seen
with skin fibroblasts from the heterozygous mice (Fig. 4).
However, the higher levels of expression gradually fell to the level
found in the heterozygous mice as the fibroblasts were passed in
culture. The results indicated, therefore, that there appeared to be
selective pressure in culture against cells expressing high levels of
the mini-COL1A1 gene whether or not the mini-gene contained the first
intron.
To assay expression of the
COL1A1/COL2A1 hybrid gene, a RT-PCR assay was developed. In the RT
step, two primers were used, one that hybridized to exon 6 of the human
COL2A1 gene and another to exon 2 of the mouse COL1A1 gene. In the PCR
step, a three-primer assay was used in which the forward primer
complemented exon 1 of both the human and mouse COL1A1 genes, one
reverse primer complemented exon 2 of the mouse COL1A1 gene, and a
second reverse primer complemented exon 6 of the human COL2A1 gene. As
indicated in Fig. 5, specific bands from mRNA from the mouse endogenous
COL1A1 gene and the human COL1A1/COL2A1 hybrid gene were detected. Two
lines of mice expressing the hybrid gene were obtained. One had a copy
number of 1-2 and the other had a copy number of 3-4
(). RT-PCR assays indicated that the level of expression as
mRNA in one line was about 5% and in the second line about 30% of the
level of expression from the endogenous COL1A1 gene ().
RT-PCR assay of five tissues from the transgenic mice of the line
BSG2 indicated that the ratio of the levels of mRNA from the
COL1A1/COL2A1 construct and mRNA from the endogenous COL1A1 gene were
about the same (Table III). To confirm these data with an independent
assay, three tissues with high levels of the mRNAs were also assayed by
slot-blot hybridization with one cDNA probe that hybridized with mRNA
from the human COL2A1 gene but not with mRNA from the mouse COL2A1
gene, and a second cDNA probe that hybridized with mRNA from the COL1A1
gene from both species (Fig. 6). The results confirmed that the ratio
of mRNA from the COL1A1/COL2A1 construct and the endogenous COL1A1 gene
was about the same in several tissues (Table III).
To examine
expression of the COL1A1/COL2A1 construct as protein, Western blot
assays were carried out with polyclonal antibodies that were specific
for the C-terminal telopeptide of human type II collagen and that did
not cross-react with either mouse type I collagen or mouse type II
collagen
(32, 33, 35) . The results (Fig. 7)
indicated that the COL1A1/COL2A1 construct was expressed as human type
II collagen in bone, skin, tail, muscle, and aorta. There was no
evidence of expression in xiphoid cartilage (not shown). Of special
interest was that the protein synthesized from the construct was
processed to
Defining the cis-regulatory elements required for high levels
of tissue-specific expression of many genes has proven to be difficult.
In the classic example of the cluster of
In the case of the COL1A1
gene, inconsistent data have been reported as to the cis-regulatory
sequences required for high levels of tissue specific expression. Some
initial observations suggested that only about -350 bp of the
promoter were necessary
(17, 18) . More recent data
indicate the -3.5 kb or more of the promoter are required
(15, 20, 21, 22, 23, 24) .
Other data suggest that the first intron is of critical importance
(2, 3, 4, 25, 26, 27) ,
and still other data suggest 3`-flanking sequences are important
(28) . Interpretation of data on cis-regulatory sequences is
simplified if the level of expression of test constructs can be related
to the level of expression of the endogenous gene. Interpretation of
data is further simplified if expression can be assayed as both mRNA
and protein that are similar in structure to mRNA and protein from the
endogenous gene, since the only practical assays for expression in
tissues from transgenic animals measure steady-state levels of mRNA or
intermediates in biosynthesis such pro
One of the questions addressed in the experiments here was
whether the 3`-untranslated region of the mini-COL1 gene contains
critical sequences for regulation of expression. A construct lacking
90% of the 3`-untranslated region appeared to be expressed at somewhat
lower levels than the mini-COL1 gene in several transgenic lines, but
there were no critical differences in tissue specificity of expression.
Since the construct lacking the 3`-untranslated regions also lacked
most of the first intron, the results indicated that 90% of the
sequences in the 3`-untranslated region and most of the sequences in
the first intron were not essential for a high degree of tissue
specificity of expression.
Another of the questions addressed here
was whether, as suggested by Liska et al. (27) , the
first intron of the COL1A1 gene was important to maintain expression of
the gene in cultured fibroblasts. The results did not reveal any
important role for the first intron in maintaining expression of the
mini-COL1A1 gene in cultured fibroblasts from transgenic mice. Instead,
a decrease in expression with passage number was seen only with
fibroblasts that expressed unusually high levels of mini-COL1A1 gene
constructs with or without the first intron. The results suggested,
therefore, that there was a growth disadvantage in culture against
fibroblasts expressing high levels of the mini-COL1A1 gene. The results
were analogous to the growth disadvantage in culture for fibroblasts
expressing mutated collagen genes that were seen previously with skin
fibroblasts from individuals who had somatic cell mosaicism for
mutations in the COL1A1 gene (see Ref. 45).
The further results
obtained with the COL1A1/COL2A1 hybrid gene were unexpected and highly
informative. Type I collagen and type II collagen arose as distinct
fibrillar collagens with different tissue distributions over 500
million years ago (see Refs. 46, 47). Therefore, it seemed reasonable
to assume that multiple inhibitory mechanisms were present that
prevented expression of the COL2A1 gene in cells and tissues expressing
the COL1A1 gene. Instead, the results here demonstrated that the
construct containing 1.9 kb from the 5`-end of the COL1A1 gene linked
to a promoterless 30-kb fragment of the human COL2A1 gene was expressed
in cells and tissues that normally synthesize type I procollagen. The
COL1A1/COL2A1 construct was not only expressed as mRNA but also as type
II procollagen that was processed to type II collagen and then was
cross-linked. Therefore, the protein was probably incorporated into
extracellular fibrils. These results clearly demonstrate that the
1.9-kb fragment from the 5`-end of the COL1A1 gene consisting only of
476 bp of the promoter, the 222 bp of exon 1, and 1,223 bp of intron 1
contains all the elements necessary to drive inappropriate expression
of the COL2A1 gene in tissues that normally synthesize type I collagen
but not type II collagen. They also demonstrate that the 30-kb
promoterless fragment of the COL2A1 gene extending from the second
intron to 3.5 kb beyond the site for termination of translation does
not contain any elements that prevent expression of the gene as mRNA or
protein in tissues that normally synthesize type I collagen but not
type II collagen. Since the results with variants of the mini-COL1A1
gene indicated that the first intron does not contain essential
tissue-specific elements, the data as a whole suggest that most of the
critical elements are in 476 bp of the promoter.
How can the
observations here be reconciled with previously indications that
-3.6 kb or more of the promoter are required for tissue-specific
expression of the COL1A1 gene
(15, 20, 21, 22, 23, 24) ?
One simple explanation is that the COL1A1 gene may be associated with
several cis-regulatory elements that have redundant activities but that
are of varying potency. Therefore, assays with sensitive reporter genes
may detect sequences between -0.48 and -3.6 kb of the
promoter that direct tissue-specific expression if placed in some
sequence contexts. However, the results here demonstrate that these
upstream elements are not necessary for tissue-specific expression if
just 0.48 kb of the promoter is placed in the natural sequence context
of the COL1A1 or COL2A1 gene. Moreover, the results demonstrated that
the sequences between -0.48 and +1.4 kb are highly potent in
their effects, since they drive totally inappropriate expression of the
COL2A1 gene. By deleting and mutation sequences in the 1.9-kb fragment
of the COL1A1 and COL2A1 construct, it should be possible to define the
specific sequences that have major effects on the tissue-specific
expression of the COL1A1 gene.
Values are mRNA from mini-COL1A1 gene as
percent of mRNA from endogenous COL1A1 gene. Values are either mean
± standard deviation ( n = 5-10) or mean
( n = 2-4). Lines 73 and 85 were assayed with the
two-primer assay for the 3`-ends of the human and mouse COL1A1 genes
(see ``Materials and Methods''). Similar values were obtained
when the three-primer assays for the 5`-ends of the genes were used to
reassay tail and brain from line 73, and tail and skin from line 85.
Lines BS2 and BS4 were assayed with the three-primer assay for the
human COL1A1/COL2A1 hybrid gene (Fig. 5).
We thank Dr. Larry Fisher of the National Institutes
of Health for the antibodies to the pro
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
of the human COL1A1 gene was
expressed in a largely tissue-specific manner in most tissues but at an
anomalously high level in lung and at an anomalously low level in
muscle. In similar experiments, Bedalov et al. (22) observed that a CAT construct driven by -3.5 kb of
the rat COL1A1 promoter was expressed in a manner that paralleled
expression of the endogenous gene except for anomalously low levels in
vascular smooth muscle cells. Bogdanovic et al. (23) reported that sequences between -2.3 and -1.7
kb are required for COL1A1 promoter expression in bones and teeth, and
sequences between -3.5 and -1.7 kb controlled expression in
tendon. More recently, Rossert et al. (24) reported
experiments with lacZ reporter gene that were consistent with
a modular arrangement of cis-regulatory element in which sequences
between the start site of transcription and -2.3 kb activated the
gene in osteoblasts and odontoblasts, sequences between -2.3 and
-3.2 kb contained an enhancer for tendon and skin fibroblasts,
and elements further upstream than -3.2 kb might activate
expression in still other subpopulations of cells.
The Gene Constructs
The mini-COL1A1 gene,
previously designated as pMG155
(30) , consisted of 11 kb of the
human COL1A1 gene that contained the first five exons and introns
joined to the last six exons and introns. The construct (Fig. 1)
also contained -2.3 kb of 5`-flanking sequence and about 2 kb of
3`-flanking sequence beyond the second polyadenylation site. The
junction between the 5`- and 3`-halves was made in an intron so as to
preserve all the known consensus sequences required for correct RNA
splicing. The first half of the construct encoded 23 amino acids of the
signal peptide, 85 amino acids of the non-helical domain of the
N-propeptide, and 48 amino acids of the Gly-Xaa-Yaa sequence
from the triple-helical domain of the N-propeptide followed by
a single glycine. The second half encoded the last 69 amino acids of
the Gly-Xaa-Yaa sequence of the triple-helical domain, the
C-telopeptide, and the complete C-propeptide.
Figure 1:
Schematic of gene
constructs and summary of data on number of lines, levels of
expression, and tissue specificity of expression. Symbols for
3`-untranslated region: , CPE (ACE) sequence; +, NFI motif;
▾, AP2 motif;
, glucocorticoid-responsive element-like
sequence;
, SP1 motif;
, viral enhancer-like sequence; *,
adenovirus EA1 enhancer-like sequence.
The modified
mini-COL1A1 gene lacking the first intron was prepared by deleting a
1.2-kb SacI/ SmaI fragment
(30) . The deletion
removed the sequences between +380 and +1,610 and eliminated
most of the putative regulatory sequences in the first intron
(+222 to +1,675).
Preparation of Transgenic Mice
To prepare the
mini-COL1A1 constructs for microinjection, one of two protocols was
used. One was to cleave plasmids with the appropriate restriction
endonuclease and isolate the inserts by sucrose gradient centrifugation
and dialysis against 10 m
M Tris-HCl buffer, pH 7.5, and 1
m
M EDTA. The second procedure was to isolate plasmid inserts
by electrophoresis on agarose gels and cutting out the appropriate gel
slices. The DNA was extracted from the gel slices with an equal volume
of isoamyl alcohol and concentrated on an ion-exchange column (ELUTIP,
Schleicher and Schuell) followed by ethanol precipitation.
Alternatively, the DNA was isolated from gel slices using a commercial
DNA extraction kit (QIAquik; Qiagen Inc.). In the case of the
COL1A1/COL2A1 hybrid construct, the cosmid containing the construct was
isolated by CsCl equilibrium centrifugation and digested by
SalI. The mixture of insert and vector was used for injection.
Assays of Gene Copy Number
For assay of copy
number in the lines expressing the mini-COL1A1 constructs, Southern
blot analysis and two different PCR assays were used. For the Southern
blot assays, genomic DNA was digested with EcoRI, and filters
were probed with the intact mini-COL1A1 construct labeled with
[P]dCTP by random primer extension with a
commercial kit (Amersham Corp.). The mini-COL1A1 constructs contained a
single EcoRI site. The probe hybridized to both EcoRI
fragments of the human mini-COL1A1 gene but none from the endogenous
mouse COL1A1 gene. Therefore, two EcoRI fragments from the
construct plus flanking sequences were detected in line 85 that had a
single copy of the mini-COL1A1 gene lacking the first intron, three
bands of about equal intensity in line 75 that had 2-4 copies of
the mini-COL1A1 gene, and a very intense band in line 73 that had
multiple copies of the mini-COL1A1 gene.
mRNA Assays
Total cellular RNA was isolated from
tissues by extraction with guanidine thiocyanate, extraction with
acidic phenol-chloroform, and precipitation with isopropyl alcohol
(39) . The levels of mRNA from the transgenes relative to the
mRNA from the endogenous COL1A1 gene were assayed with three different
RT-PCR protocols.
; Life
Technologies, Inc.). After RNaseH treatment, the cDNA was amplified by
PCR (GeneAmpPCR reagent kit; Perkin-Elmer Cetus) with two primers
for the 3`-untranslated region. The primers (BS31-TTGGCCCTGTCTGCTT and
BS32-TGAATGCAAAGGAAAAAAAT) were directed to sequences in the
3`-untranslated regions of both the human and mouse cDNA. The primers
were used at concentrations of 4 pmol/100 µl of reaction mixture.
PCR conditions were 1 min 20 s at 94 °C, 1 min at 47 °C, and 20
s at 72 °C for 15 cycles. One of the primers used in PCR was
labeled with
P using a 5`-DNA terminus labeling system
(Life Technologies, Inc.). After the PCR, 10 µl of reaction mixture
was treated by 2 units of BstNI for 1 h at 60 °C. Three
µl of the product was heat denatured and separated in 15% PAGE
containing 6
M urea. The gel was fixed, dried, and exposed to
x-ray film. After cleavage with BstNI, the human PCR product
gave a band of 135 bp and the mouse 100 bp. The relative intensities of
the bands were measured with a laser densitometer (Ultroscan XL; KLB).
P-labeled primer directed to
identical sequences in exon 1 of human and mouse COL1A1 gene
(BS84-CTCCGGCTCCTGCTCCTCTTA) and two reverse primers directed to highly
homologous sequences in exon 2 of the mouse COL1A1 gene
(BS81-GCACAGCACTCGCCCTCCC) and the human COL1A1 gene
(BS82-GGACAGCACTCGCCCTCGG) upstream to the primers used for reverse
transcription. The product from the human gene was 260 bp and from the
mouse gene was 232 bp. Conditions for PCR were the same as in the first
RT-PCR assay. PCR products were electrophoresed in 10% PAGE without
urea or 8% PAGE containing 2
M UREA.
P-labeled primer directed to
identical sequences in exon 1 of human COL1A1 and mouse COL1A1 genes
(BS84-CTCCGGCTCCTGCTCCTCTTA), a primer directed to sequences in exon 2
of mouse COL1A1 gene (BS81-GCACGCACTCGCCCTCCC), and a primer directed
to sequences in exon 6 of human COL2A1 gene upstream to the primers
used for reverse transcription (BS93-TCCTTGTTCCCCTGCAGGTCC). The
product from the human gene was 189 bp, and the product from the mouse
gene was 232 bp. Conditions for PCR were as in the first RT-PCR assay.
The PCR products were then electrophoresed in 8% PAGE containing 2
M UREA.
Slot-blot Assays for mRNAs
Assays were performed
by blotting denatured total RNA on nylon filters in a commercial
apparatus (Schleicher and Schuell). The filters were probed either with
a 1.5-kb EcoRI/ XhoI fragment from the cDNA for human
pro1(II) chains of type II procollagen
(40) or a complete
cDNA of 4.8 kb for the human pro
1(I) chain of type I procollagen
(41) . The probes were labeled with
P by nick
translation. Filters were hybridized at 42 °C for 24 h in 5
SSPE (3
M NaCl, 0.2
M NaH
PO
H
O, and 0.02 Na
EDTA, pH 7.4), 10
Denhardt's solution, 100 µg/ml denatured and sheared
salmon sperm DNA, 50% formamide, and 2% SDS. Filters hybridized with
the probe for the human pro
1(I) chain were washed twice at 58
°C for 20 min in 0.2
SSC and 0.1% SDS. Filters hybridized
with the probe for human pro
1(II) chains were washed twice at 65
°C for 20 min in 0.1
SSC and 0.1% SDS. After washing, the
filters were exposed to x-ray film.
Protein Assays
For assays of expression of the
mini-COL1A1 gene as protein, 50-200 mg of tissue was crushed to a
powder after cooling in liquid nitrogen, and 10-40 mg of the
powder was homogenized in 0.5 ml of buffer that contained 50 m
M Tris-HCl, pH 6.8, 2% SDS, 6
M urea, 0.0015% bromphenol
blue, 5% 2-mercaptoethanol, 25 m
M EDTA, 10 m
M ethylmaleimide, 1 m
M phenylmethanesulfonyl fluoride, and
0.01% NaN. The homogenate was shaken at 4 °C for 2 h,
heated at 100 °C for 5 min, and centrifuged for 5 min at 12,000
g. Two to 15 µl of the supernatant (0.03-0.4
µg of protein) was electrophoresed in a 4-15% polyacrylamide
gel in a mini-gel apparatus (Protein II, BioRad). The protein was
electroeluted onto a filter (polyvinyldifluoride membrane; product
number 71925; United States Biochemical Corp., Cleveland OH). The
filter was then reacted with polyclonal antibodies that reacted with
the C-propeptide of the pro
1(I) chain from both human and mouse
type I procollagen
(30) . The polyclonal antibodies were kindly
provided by Dr. Larry Fisher, NIDR, National Institutes of Health,
Bethesda, MD. The secondary antibodies were anti-rabbit IgG coupled to
alkaline phosphatase (Promega Biotek). For assays of expression of the
COL1A1/COL2A1 hybrid construct as human pro
1(II) chains,
polyclonal antibodies prepared with a synthetic peptide containing
sequences from the C-telopeptide of human type II collagen were used
with the same procedures
(32) .
Culture of Skin Fibroblasts from Transgenic
Mice
To prepare skin fibroblasts, embryos were removed from
pregnant females at 15-16 days post-coitum. The heads and
internal soft tissues were removed. The remaining carcasses were minced
with scissors, and the minced tissues were incubated in 5-10 ml
of 250 µg/ml of trypsin in 1.0 m
M EDTA for 5-10 min.
An equal volume of Dulbecco's modified Eagle's medium
containing 10% fetal calf serum was added, and the suspension was
allowed to settle in a 50-ml centrifuge tube for 2-3 min. The
supernatant was transferred to 100-mmplastic dish, and the
cells were allowed to attach by incubation at 37 °C overnight. The
attached cells were washed with Dulbecco's modified Eagle's
medium containing 10% fetal calf serum, and the cells were grown in the
same medium. The cells reached confluence in 8-10 days, after
which they were recovered by trypsinization and replated for continuous
culture or frozen in liquid nitrogen. To obtain cells with increasing
passage number, about 10
cells were grown on 60-mm
dishes. The cells reached confluence in 3-4 days and were
replated after splitting about 1:4.
1(I) chains or endogenous pro
1(I) chains were
seen
(31) . In two additional lines (lines 73 and 85), the
shortened pro
1(I) chains from the mini-COL1A1 gene were detected
in brain, but the values for the ratio of pro
1(I) chains from the
mini-COL1A1 gene and the endogenous COL1A1 gene were about the same as
in other tissues
(36) . Therefore, the results were explainable
by contamination of the samples of brain by meninges and related
structures that contain type I collagen. Expression of the mini-COL1A1
gene as mRNA was not assayed in those experiments.
Figure 3:
RT-PCR
assay for expression of endogenous COL1A1 gene and the mini-COL1A1 gene
minus intron 1 and 90% of the 3`-untranslated sequences. About 1 µg
of total RNA from each tissue was used for the RT-PCR assay. Equal
aliquots were loaded in each well of an electrophoretic gel (see Fig.
2), and the gel was analyzed with a laser densitometer. Values are
expressed in arbitrary units of absorbance/milligram of total
RNA.
Because of the
higher ratio of mRNA in brain seen with the mini-COL1A1 gene minus
intron 1 and the 3`-untranslated region ( Fig. 3and Table II),
expression of the mini-COL1 gene in brain was re-examined in one line
(line 73). As indicated in , the ratio of mRNA from the
mini-COL1A1 gene relative to mRNA from the exogenous gene were six to
eight times higher in brain than other tissues. Therefore, high values
for the relative levels of mRNA in brain were common to expression of
both the mini-COL1 gene and the mini-COL1 gene minus intron 1 and the
3`-untranslated region. However, since the absolute levels of COL1A1
mRNA in brain were very low, the absolute levels of mRNA from the
exogenous genes were also low.
Expression of the Mini-COL1 Gene Constructs in Cultured
Fibroblasts from Transgenic Mice
Liska et al. (27) reported that the first intron of the COL1A1 gene was
important to maintain high levels of expression of a reporter gene
construct when fibroblasts from transgenic animals were cultured ex
vivo. To test this hypothesis, we prepared cultured skin
fibroblasts from two lines of transgenic mice expressing a mini-COL1A1
gene and one line of transgenic mice expressing the mini-COL1A1 gene
lacking the first intron. As indicated in Fig. 4, the initial values
for expression of the transgenes as shortened pro1(I) chains were
about the same as previously observed in skin and other tissues taken
from the transgenic mice (see in Ref. 36). As the cells
were passed in culture, however, the relative levels of expression of
the transgene from one line that had a high initial level (line R) fell
to about one-third of the initial level. In the other two lines that
began with lower initial levels, the level of expression of shortened
pro
1(I) chains remained constant. Since one of the lines had an
intact first intron (lines 73), whereas the other had a deleted first
intron (line 85), there was no apparent effect from the presence or
absence of the intron.
Figure 4:
Effect
of passage number on expression of mini-COL1A1 gene constructs in
cultured skin fibroblasts from transgenic mice. Expression was assayed
by Western blot (35). Symbols: , skin fibroblasts
isolated from each line;
, skin fibroblasts from transgenic mice
that were from inbreeding of the same line and, therefore, apparently
homozygous for the transgene.
Expression of a COL1A1/COL2A1 Hybrid Gene
If the
promoter region and first intron of the COL1A1 gene contain the
critical sequences for tissue-specific expression, joining a
5`-fragment from the COL1A1 gene containing these sequences to the gene
for a second collagen normally expressed in different tissues should
cause inappropriate expression of the second collagen. To test this
hypothesis, a 1.9-kb 5`-fragment from the COL1A1 gene linked to a
promoterless gene for COL2A1
(32) was used to prepare
transgenic mice (Fig. 1).
1(II) chains of type II collagen, and some of the
1(II) chains were cross-linked. Surprisingly, the transgenic mice
expressing the COL1A1/COL2A1 construct had no apparent phenotype. They
were about 25% smaller by body weight than littermates but readily
reproduced.
-globin genes, the locus
control region that exerts a powerful enhancer effect is located about
20 kb upstream of the
-globin gene (see Ref. 43). The locus
control region linked to the
-globin gene can generate copy
number-proportional and position-independent expression of the gene in
transgenic mice, but the cis-regulatory elements that are critical for
the time-dependent expression of embryonic and adult genes during
development have not been defined. The data on several other genes
suggest a complex interplay among cis-regulatory elements. For example,
the keratin 18 gene requires the presence of either 0.8 kb of
5`-flanking promoter sequence or 3.5 kb of 3`-flanking sequence but not
both for integration site-independent expression of tandemly duplicated
genes in transgenic mice
(44) .
chains of procollagen. Our
previous observations indicated that the mini-COL1A1 gene containing
-2.3 kb of the promoter was expressed as both mRNA and protein in
a highly tissue-specific and developmental-specific manner
(36) . The levels of expression were not proportional to copy
number
(31) , but the expression relative to expression of the
endogenous gene was highly consistent in many tissues within a given
line
(36) . As indicated here, expression of the mini-COL1A1
gene as mRNA was high in brain when assayed relative to mRNA from the
endogenous COL1A1 gene. However, the absolute levels of expression of
both the mini-COL1A1 gene and the endogenous COL1A1 gene were very low
in brain.
Table: Expression of constructs in tails of
transgenic mice
Table: RT-PCR assays
for tissue specificity of expression as mRNA of constructs in
transgenic mice
Table: 0p4in
Absolute levels very low in brain.
-galactoside; RT-PCR, reverse
transcriptase-polymerase chain reaction; PAGE, polyacrylamide gel
electrophoresis.
1(I) chain of type I
procollagen.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.