Separate cis-acting DNA elements of the mouse pro-alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice.
Separate cis-acting DNA elements of the mouse pro-alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice.
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DOI：
10.1083/jcb.129.5.1421
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发表时间：
1995-06
期刊：
影响因子：
--
通讯作者：
de Crombrugghe B
中科院分区：
文献类型：
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作者：
Rossert J;Eberspaecher H;de Crombrugghe B
编码两种 I 型胶原链的基因在成骨细胞、成牙本质细胞、成纤维细胞和一些间充质细胞中选择性活跃，构成了研究在少数离散细胞类型中表达的基因的细胞特异性活性机制的良好模型。为了测试单独的遗传元件是否可以将小鼠 pro-alpha 1(I) 胶原蛋白基因的活性引导至其表达的不同细胞类型，产生了转基因小鼠，其含有克隆到大肠杆菌 β-半乳糖苷酶基因上游的该基因的近端启动子的各种片段。在胚胎发育过程中，X-gal 染色可以精确识别 β-半乳糖苷酶基因处于活跃状态的不同细胞类型。含有 900 bp 的 pro-alpha 1(I) 近端启动子的转基因小鼠几乎只在皮肤中以相对较低的水平表达转基因。在含有 2.3 kb 近端启动子的小鼠中，转基因也在成骨细胞和成牙本质细胞中高水平表达，但在其他 I 型胶原蛋白生成细胞中不表达。含有 3.2 kb 近端启动子的转基因小鼠在肌腱和筋膜成纤维细胞中显示出额外的高水平转基因表达。通过使用萤火虫荧光素酶基因作为报告基因，证实了由 0.9-和 2.3-kb pro-alpha 1(I) 近端启动子指导的 lacZ 转基因的表达模式。该转基因的表达模式与 β-半乳糖苷酶基因的表达模式相似，即使它的活性水平非常低，也可以被检测到。这些数据强烈表明，单独的细胞特异性顺式作用元件的模块化排列可以激活不同 I 型胶原蛋白生成细胞中的小鼠 pro-alpha(I) 胶原蛋白基因。至少三种不同类型的细胞特异性元件将位于启动子的前3.2kb中：(a)在真皮成纤维细胞中赋予低水平表达的元件； (b) 第二种介导成骨细胞和成牙本质细胞的高水平表达； (c) 一种负责在肌腱和筋膜成纤维细胞中高水平表达的基因。我们的数据还表明，将基因活性引导至其他 I 型胶原蛋白生成细胞的其他顺式作用细胞特异性元件仍有待鉴定。
The genes coding for the two type I collagen chains, which are active selectively in osteoblasts, odontoblasts, fibroblasts, and some mesenchymal cells, constitute good models for studying the mechanisms responsible for the cell-specific activity of genes which are expressed in a small number of discrete cell types. To test whether separate genetic elements could direct the activity of the mouse pro-alpha 1(I) collagen gene to different cell types in which it is expressed, transgenic mice were generated harboring various fragments of the proximal promoter of this gene cloned upstream of the Escherichia coli beta-galactosidase gene. During embryonic development, X-gal staining allows for the precise identification of the different cell types in which the beta-galactosidase gene is active. Transgenic mice harboring 900 bp of the pro-alpha 1(I) proximal promoter expressed the transgene at relatively low levels almost exclusively in skin. In mice containing 2.3 kb of this proximal promoter, the transgene was also expressed at high levels in osteoblasts and odontoblasts, but not in other type I collagen-producing cells. Transgenic mice harboring 3.2 kb of the proximal promoter showed an additional high level expression of the transgene in tendon and fascia fibroblasts. The pattern of expression of the lacZ transgene directed by the 0.9- and 2.3-kb pro-alpha 1(I) proximal promoters was confirmed by using the firefly luciferase gene as a reporter gene. The pattern of expression of this transgene, which can be detected even when it is active at very low levels, paralleled that of the beta-galactosidase gene. These data strongly suggest a modular arrangement of separate cell-specific cis-acting elements that can activate the mouse pro-alpha(I) collagen gene in different type I collagen-producing cells. At least three different types of cell- specific elements would be located in the first 3.2 kb of the promoter: (a) an element that confers low level expression in dermal fibroblasts; (b) a second that mediates high level expression in osteoblasts and odontoblasts; and (c) one responsible for high level expression in tendon and fascia fibroblasts. Our data also imply that other cis- acting cell-specific elements which direct activity of the gene to still other type I collagen-producing cells remain to be identified.