©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tissue-specific Expression of the Gene for Type I Procollagen (COL1A1) in Transgenic Mice
ONLY 476 BASE PAIRS OF THE PROMOTER ARE REQUIRED IF COLLAGEN GENES ARE USED AS REPORTERS (*)

Boris P. Sokolov (§) , Leena Ala-Kokko , Rohini Dhulipala , Machiko Arita , Jaspal S. Khillan , Darwin J. Prockop (¶)

From the (1) Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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() 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.

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.


MATERIALS AND METHODS

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).

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) .

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.

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.

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.

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).

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.

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; 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).

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 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.

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 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 pro1(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 NaHPO HO, and 0.02 NaEDTA, 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 pro1(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 pro1(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 pro1(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 pro1(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) .

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.

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 10cells were grown on 60-mmdishes. The cells reached confluence in 3-4 days and were replated after splitting about 1:4.


RESULTS

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 pro1(I) chains or endogenous pro1(I) chains were seen (31) . In two additional lines (lines 73 and 85), the shortened pro1(I) chains from the mini-COL1A1 gene were detected in brain, but the values for the ratio of pro1(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 pro1(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.

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.


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).

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 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.


DISCUSSION

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 -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) .

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 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.

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.

  
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

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).


  
Table: 0p4in Absolute levels very low in brain.


FOOTNOTES

*
This work was supported in part by Grants AR-38188 and AR-39740 from the National Institutes of Health and the Lucille P. Markey Charitable Trust.

§
Present address: Dept. of Psychiatry, Box 1229, The Mount Sinai Medical Center, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029.

To whom correspondence should be addressed. Tel.: 215-955-4830.

The abbreviations used are: kb, kilobase(s); bp, base pair(s); CAT, chloramphenicol acetyltransferase; lacZ, -galactoside; RT-PCR, reverse transcriptase-polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. Larry Fisher of the National Institutes of Health for the antibodies to the pro1(I) chain of type I procollagen.


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