Analysis of specific gene expression of V/XI collagen genes in chondrocyte and non-chondrocyte

我们克隆了小鼠肾间质（Xi）胶原基因的5'侧翼区。如在人类基因中所见，该启动子具有几个转录起始位点。将该启动子序列与共有调控元件序列进行比较，发现该启动子序列中没有TATA盒和CCAAT盒，存在多个潜在的Sp1结合位点，并在荧光素酶上游亚克隆了两个大小为617 bp（短启动子）和5 kb（长启动子）的启动子片段。第一内含子中的3个Sma片段（3.5kb、3.0kb和7.0kb）被亚克隆到这些构建体的下游。我们将DNA转染到牛主动脉平滑肌细胞和人软骨肉瘤细胞中。两种细胞中短启动子片段的荧光素酶活性均高于长启动子片段。第一内含子的三个片段似乎抑制了启动子的活性，为了分析酸性区的选择性剪接，我们分别制作了外显子6A、6 B、7、8和6A-8缺失的小基因。我们将这些基因转染204（横纹肌肉瘤细胞）、RCS（大鼠软骨肉瘤细胞）和293（肾细胞）。在293细胞中，外显子6A-7-8是主要的剪接形式，外显子6 B不表达。在204细胞中，外显子6A-7-8是最丰富的剪接形式，而7-8也存在。在RCS单元中，存在五种拼接形式。为了分析酸性结构域在体内的功能，我们还开始工程化用于外显子6A和6 B缺失的基因靶向构建体。

We isolated the 5' flanking region of mouse alphal(XI) collagen gene. As seen in human gene, this promoter has several transcription start sites. Comparison of the sequence of the promoter with consensus regulatory elements showed that there was no TATA or CCAAT box, and that there were several potential Sp1 binding sites.Two promoter fragments , 617 bp (short promoter) and 5 kb (long promoter) in size, sharing the same 3 end were subcloned in the upstream of luciferase. And three Sma fragments, 3.5 kb, 3.0 kb and 7.0 kb in the first intron were subeloned in the downstream of the these constructs. We transfected the DNAs into bovine aorta smooth muscle cells ad human chondrosarcoma cells. The luciferase activity of short promoter fragment was higher than that of long promoter in both cells. Three fragments of first intron seemed to depress the activity of promoter.To analyze alternative splicing in the acidic region, we made mini genes for the deletion of exon 6A, 6B, 7, 8 and 6A-8, respectively. We transfected these genes in 204 (rhabdomyosarcoma cell), RCS (rat chondrosarcoma) and 293 (kidney cell). In 293 cell, the exon 6A-7-8 is main splice form, and exon 6B is not expressed. In 204 cell, exon 6A-7-8 is the abundant splice form while 7-8 is also present. In the RCS cell, five splice forms are present. In order to analyze the function of the acidic domain in vivo, we have also begun engineering the gene targeting constructs for the deletion of exon 6A and 6B.