Xiong W, Wang J, Chao L, Chao J
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA.
Biochem J. 1997 Jul 1;325 ( Pt 1)(Pt 1):111-6. doi: 10.1042/bj3250111.
The expression of the tissue kallikrein gene is tissue-specific and exhibits a complex pattern of transcriptional and post-translational regulation. Information concerning the mechanism of its tissue-specific expression has been limited owing to the lack of suitable cell lines for the expression study. We approached this problem by introducing human tissue kallikrein gene constructs into mouse embryos, creating transgenic lines carrying its coding sequence with varying lengths of the promoter region. One construct (PHK) contained 801 bp in the 5'-flanking region and two deletion constructs contained either 302 bp (D300) or 202 bp (D200) of the promoter region. The expression of human tissue kallikrein in these transgenic mice was monitored by Northern blot, reverse transcriptase-PCR followed by Southern blot, and radioimmunoassay. In all three lines, human tissue kallikrein was expressed predominantly in the pancreas and at lower levels in other tissues, including salivary gland, kidney and spleen. This pattern was similar to that of tissue kallikrein expression in human tissues. The D300 line has higher levels of transgene expression than the D200 and PHK lines. The results indicate that the 202 bp segment immediately upstream of the translation starting site is sufficient to direct a tissue-specific expression pattern of the human tissue kallikrein gene, and that regulatory elements might exist between -801 and -202.
组织激肽释放酶基因的表达具有组织特异性,并呈现出复杂的转录和翻译后调控模式。由于缺乏适合进行表达研究的细胞系,关于其组织特异性表达机制的信息一直有限。我们通过将人组织激肽释放酶基因构建体导入小鼠胚胎来解决这个问题,创建了携带其编码序列且启动子区域长度不同的转基因品系。一个构建体(PHK)在5'侧翼区域包含801 bp,两个缺失构建体分别包含启动子区域的302 bp(D300)或202 bp(D200)。通过Northern印迹、逆转录PCR随后进行Southern印迹以及放射免疫测定来监测这些转基因小鼠中人组织激肽释放酶的表达。在所有三个品系中,人组织激肽释放酶主要在胰腺中表达,在其他组织(包括唾液腺、肾脏和脾脏)中表达水平较低。这种模式与人类组织中组织激肽释放酶的表达模式相似。D300品系的转基因表达水平高于D200和PHK品系。结果表明,翻译起始位点上游紧邻的202 bp片段足以指导人组织激肽释放酶基因的组织特异性表达模式,并且在-801和-202之间可能存在调控元件。