Tissue- and development-specific expression in transgenic mice of a type I procollagen (COL1A1) minigene construct with 2.3 kb of the promoter region and 2 kb of the 3'-flanking region. Specificity is independent of the putative regulatory sequences in the first intron

Sokolov, Boris P.; Mays, Peter K.; Khillan, Jaspal S.; Prockop, Darwin J.

doi:10.1021/bi00086a033

Cited by 33 publications

(31 citation statements)

References 54 publications

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“…The use of an ␣1(I) minigene in place of a promoter-reporter gene construct in transfection experiments (40) has averted some of these drawbacks, but even minigenes are limited in the extent to which 5Ј and 3Ј flanking sequences can be represented, and there are possible uncertainties in quantification that can result from differences in the levels of stability of the initial and mature transcripts in comparison with that of Col1A1 mRNA. The results of transgenic experiments have also been controversial (8,35,53,54), in part because of the need to compare different constructs and the possible confounding effects of copy number and site of insertion on expression of the transgene.…”

Section: Discussionmentioning

confidence: 99%

“…Since some of these elements could be placed in the first intron, numerous studies, using both transfection and transgenic approaches, have been designed to investigate transcriptional regulation by the first intron and to identify possible responsible regulatory elements, but these experiments have resulted in conflicting conclusions (reviewed in reference 8). While some studies have demonstrated a role for the intron in regulation and have identified cis-acting sequences that bind Sp1 and AP1 as important elements (6, 9, 29, 33-35, 44, 51, 52), other studies have indicated that the intron does not regulate expression of the gene (40,53,54). Thus, the role of the first intron in the regulation of the Col1A1 gene remains uncertain and controversial.…”

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confidence: 99%

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A Gene-Targeting Approach Identifies a Function for the First Intron in Expression of the α1(I) Collagen Gene

Hormuzdi

Penttinen

Jaenisch

et al. 1998

Molecular and Cellular Biology

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The role of the first intron of the Col1A1 gene in the regulation of type I collagen synthesis remains uncertain and controversial despite numerous studies that have made use of transgenic and transfection experiments. To examine the importance of the first intron in regulation of the gene, we have used the double-replacement method of gene targeting to introduce, by homologous recombination in embryonic stem (ES) cells, a mutated Col1A1 allele (Col-Int⌬). The Col-Int⌬ allele contains a 1.3-kb deletion within intron I and is also marked by the introduction of a silent mutation that created an XhoI restriction site in exon 7. Targeted mice were generated from two independently derived ES cell clones. Mice carrying two copies of the mutated gene were born in the expected Mendelian ratio, developed normally, and showed no apparent abnormalities. We used heterozygous mice to determine whether expression of the mutated allele differs from that of the normal allele. For this purpose, we developed a reverse transcription-PCR assay which takes advantage of the XhoI polymorphism in exon 7. Our results indicate that in the skin, and in cultured cells derived from the skin, the intron plays little or no role in constitutive expression of collagen I. However, in the lungs of young mice, the mutated allele was expressed at about 75% of the level of the normal allele, and in the adult lung expression was decreased to less than 50%. These results were confirmed by RNase protection assays which demonstrated a two-to threefold decrease in Col1A1 mRNA in lungs of homozygous mutant mice. Surprisingly, in cultured cells derived from the lung, the mutated allele was expressed at a level similar to that of the wild-type allele. Our results also indicated an age-dependent requirement for the intact intron in expression of the Col1A1 gene in muscle. Since the intron is spliced normally, and since the mutant allele is expressed as well as the wild-type allele in the skin, reduced mRNA stability is unlikely to contribute to the reduction in transcript levels. We conclude that the first intron of the Col1A1 gene plays a tissue-specific and developmentally regulated role in transcriptional regulation of the gene. Our experiments demonstrate the utility of gene-targeting techniques that produce subtle mutations for studies of cis-acting elements in gene regulation.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

A Gene-Targeting Approach Identifies a Function for the First Intron in Expression of the α1(I) Collagen Gene

Hormuzdi

Penttinen

Jaenisch

et al. 1998

Molecular and Cellular Biology

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“…Because the small amounts ofglycerol in the samples ignited during the first few minutes at 600C, the oven was placed in a fume hood. Preliminary experiments indicated there was no further decrease in weight if samples were incubated at 600C ( 12). As indicated in Fig.…”

Section: Methodsmentioning

confidence: 75%

“…To assay the steady-state levels of mRNA, total cellular RNA was isolated from tissues by extraction with acidic guanidinium thiocyanate-phenol-chloroform ( 1 1). The amount ofmRNA from the transgene relative to the mouse mRNA from endogenous COL 1 Al gene was measured by a quantitative reverse transcriptase (RT)-PCR method ( 12). 1-5 ug of total RNA was reverse transcribed in 20 Ml of reaction mixture using primer BS33 (5'-ATCAAG-TTTGA'3') (200 pmol) for the proal (I) chain and a preamplification system for first-strand cDNA synthesis (SuperScript<>, GIBCO BRL, Gaithersburg, MD).…”

Section: Methodsmentioning

confidence: 99%

“…1-5 ug of total RNA was reverse transcribed in 20 Ml of reaction mixture using primer BS33 (5'-ATCAAG-TTTGA'3') (200 pmol) for the proal (I) chain and a preamplification system for first-strand cDNA synthesis (SuperScript<>, GIBCO BRL, Gaithersburg, MD). The sample was treated with RNase H and the cDNA was amplified directly using a PCR reagent kit (GeneAmpo, Perkin-Elmer Cetus, Cochranville, PA), and primers BS31 (5'-TTG-GCCCTGTCTGCTT-3') and BS32 (5'-TGAATGCAAAGGAAA-AAAAT-3') using 4 pmol of each in a 100-Al reaction mixture (12).…”

Section: Methodsmentioning

confidence: 99%

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Phenotypic variability and incomplete penetrance of spontaneous fractures in an inbred strain of transgenic mice expressing a mutated collagen gene (COL1A1).

Pereira¹,

Halford²,

Sokolov³

et al. 1994

J. Clin. Invest.

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Phenotype variability and incomplete penetrance are frequently observed in human monogenic diseases such as osteogenesis imperfecta. Here an inbred strain of transgenic mice expressing an internally deleted gene for the proal (I) chain of type I procollagen (COLIAl) was bred to wild type mice of the same strain so that the inheritance of a fracture phenotype could be examined in a homogeneous genetic background. To minimize the effects of environmental factors, the phenotype was evaluated in embryos that were removed from impregnated females 1 d before term. Examination of stained skeletons from 51 transgenic embryos from 11 separate litters demonstrated that -22% had a severe phenotype with extensive fractures of both long bones and ribs, -51% had a mild phenotype with fractures of ribs only, and -27% had no fractures. The ratio of steady-state levels of the mRNA from the transgene to the level of mRNA from the endogenous gene was the same in all transgenic embryos. The results demonstrated that the phenotypic variability and incomplete penetrance were not explained by variations in genetic background or levels in gene expression. Instead, they suggested that phenotypic variation is an inherent feature of expression of a mutated collagen gene. (J. Clin. Invest. 1994Invest. . 93:1765Invest. -1769

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Transgenic mice with a mutated collagen promoter display normal response during bleomycin-induced fibrosis and possess neurological abnormalities

et al. 2000

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We have previously identified a potential TGF-beta activation element (TAE) in the rat collagen alpha1(I) promoter at -1624 upstream of the transcriptional start site [Ritzenthaler et al., 1991, 1993]. To determine the importance of the TAE in vivo, we produced transgenic mice carrying 3.6 kb of the rat collagen alpha1(I) promoter linked to the reporter gene chloramphenicol acetyl transferase with and without site-directed mutations that eliminate DNA-protein binding at the TAE site. Tissue-specific expression of the reporter gene in transgenic mice with the mutated collagen promoter was similar to that of transgenic mice with the normal promoter in two genetic backgrounds as judged by in situ hybridization, reporter assays, and immunochemistry. Endotracheal instillation of bleomycin induces lung fibrosis, mediated in part by TGF-beta. Earlier studies indicated that expression of wild-type collagen-reporter gene was upregulated in transgenic mice lungs in response to endotracheal instillation of bleomycin. A similar level of reporter gene upregulation was observed in transgenic mice carrying the mutation in the TAE. Two lines of transgenic mice carrying the mutated promoter construct displayed unexpected neurological abnormalities. In the FVB genetic background, there was a higher than normal incidence of mortality, spontaneous seizures, and an inability to nurture offspring. Histological evidence demonstrated clear abnormalities, including disorderly arrangement of neurons in the hippocampus and significant laminar cortical necrosis in the cerebrum in animals after seizures. In the C57Bl/6 background, there was a high incidence of severe communicating hydrocephalus, early runting, and increased mortality similar to that in transgenic animals with astroglial overexpression of TGF-beta. These animals provide an interesting model system to investigate molecular mechanisms responsible for seizures and hydrocephalus.

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Tissue- and development-specific expression in transgenic mice of a type I procollagen (COL1A1) minigene construct with 2.3 kb of the promoter region and 2 kb of the 3'-flanking region. Specificity is independent of the putative regulatory sequences in the first intron

Cited by 33 publications

References 54 publications

A Gene-Targeting Approach Identifies a Function for the First Intron in Expression of the α1(I) Collagen Gene

A Gene-Targeting Approach Identifies a Function for the First Intron in Expression of the α1(I) Collagen Gene

Phenotypic variability and incomplete penetrance of spontaneous fractures in an inbred strain of transgenic mice expressing a mutated collagen gene (COL1A1).

Transgenic mice with a mutated collagen promoter display normal response during bleomycin-induced fibrosis and possess neurological abnormalities

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