A gene-targeting approach identifies a function for the first intron in expression of the alpha1(I) collagen gene.
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-IntDelta). The Col-IntDelta 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.
Hormuzdi
S G
SG
Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
Penttinen
R
R
Jaenisch
R
R
Bornstein
P
P
D000818:Animals; D019556:COS Cells; D002874:Chromosome Mapping; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D007438:Introns / Q000502:physiology*; D008168:Lung / Q000378:metabolism; D051379:Mice; D009132:Muscles / Q000378:metabolism; D016133:Polymerase Chain Reaction
D003094:Collagen; C034510:CTCGAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific