Slack J L, Liska D J, Bornstein P
Department of Biochemistry, University of Washington, Seattle 98195.
Mol Cell Biol. 1991 Apr;11(4):2066-74. doi: 10.1128/mcb.11.4.2066-2074.1991.
Studies in vitro have not adequately resolved the role of intronic and upstream elements in regulating expression of the alpha 1(I) collagen gene. To address this issue, we generated 12 separate lines of transgenic mice with alpha 1(I) collagen-human growth hormone (hGH) constructs containing different amounts of 5'-flanking sequence, with or without most of the first intron. Transgenes driven by 2.3 kb of alpha 1(I) 5'-flanking sequence, whether or not they contained the first intron, were expressed at a high level and in a tissue-specific manner in seven out of seven independent lines of transgenic mice. In most tissues, the transgene was expressed at levels approaching that of the endogenous alpha 1(I) gene and was regulated identically with the endogenous gene as animals aged. However, in lung, expression of the transgene was anomalously high, and in muscle, expression was lower than that of the endogenous gene, suggesting that in these tissues other regions of the gene may participate in directing appropriate expression. Five lines of mice were generated containing transgenes driven by 0.44 kb of alpha 1(I) 5'-flanking sequence (with or without the first intron), and expression was detected in four out of five of these lines. The level of expression of the 0.44-kb constructs in the major collagen-producing tissues was 15- to 500-fold lower than that observed with the longer 2.3-kb promoter. While transgenes containing the 0.44-kb promoter and the first intron retained a modest degree of tissue-specific expression, those without the first intron lacked tissue specificity and were poorly expressed in all tissues except lung. These results contribute to our understanding of the role of the first intron in regulating alpha1(I) gene expression and identify a region, upstream of the basal alpha1(I) promotor, which is necessary for full tissue-specific, developmentally regulated expression of the alpha1(I) collagen gene.
体外研究尚未充分阐明内含子和上游元件在调节α1（I）型胶原基因表达中的作用。为解决这一问题，我们构建了12种不同的转基因小鼠品系，其α1（I）型胶原-人生长激素（hGH）构建体包含不同长度的5'侧翼序列，有的含有大部分第一个内含子，有的则没有。由2.3 kb的α1（I）型胶原5'侧翼序列驱动的转基因，无论是否包含第一个内含子，在7个独立的转基因小鼠品系中均以高水平且组织特异性的方式表达。在大多数组织中，转基因的表达水平接近内源性α1（I）基因，并且随着动物年龄增长，其表达调控方式与内源性基因相同。然而，在肺中，转基因的表达异常高，而在肌肉中，表达低于内源性基因，这表明在这些组织中，基因的其他区域可能参与指导适当的表达。我们构建了5种含有由0.44 kb的α1（I）型胶原5'侧翼序列驱动的转基因的小鼠品系（有的含有第一个内含子，有的没有），其中4个品系检测到了表达。在主要的胶原产生组织中，0.44 kb构建体的表达水平比使用更长的2.3 kb启动子观察到的水平低15至500倍。虽然含有0.44 kb启动子和第一个内含子的转基因保留了一定程度的组织特异性表达，但那些没有第一个内含子的转基因缺乏组织特异性，并且除肺以外在所有组织中的表达都很低。这些结果有助于我们理解第一个内含子在调节α1（I）基因表达中的作用，并确定了一个位于基础α1（I）启动子上游的区域，该区域对于α1（I）型胶原基因的完全组织特异性、发育调控表达是必需的。
