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

我们克隆了小鼠α(XI)胶原基因的5‘侧翼区。正如在人类基因中看到的，该启动子有几个转录起始点。将该启动子序列与已知的启动子序列进行比对，发现没有TATA和CCAAT盒，存在多个潜在的Sp1结合位点。在荧光素酶的上游亚克隆了两个启动子片段，大小分别为617bp(短启动子)和5kb(长启动子)，具有相同的3端。第一内含子的3.5kb、3.0kb和7.0kb三个Sma片段位于这些构建体下游。我们将DNA导入牛主动脉平滑肌细胞和人软骨肉瘤细胞。在两种细胞中，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，为了分析酸性区域的选择性剪接，我们分别构建了缺失外显子6A、6B、7、8和6A-8的微型基因。我们将这些基因分别导入204(横纹肌肉瘤细胞)、RCS(大鼠软骨肉瘤)和293(肾脏细胞)。在293细胞中，外显子6A-7-8是主要的剪接形式，外显子6B不表达。在204细胞中，外显子6A-7-8是丰富的剪接形式，同时也存在7-8。在RCS细胞中，存在五种剪接形式。为了分析酸性结构域在体内的功能，我们还开始了针对外显子6A和6B缺失的基因靶向构建。

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.