人干细胞因子基因5′旁侧序列的克隆和功能研究

Cloning and Function on 5′ Flanking Sequence of Human Stem Cell Factor Gene

人干细胞因子 ( human stem cell factor,h SCF)在多种哺乳动物干细胞的增殖、分化和移居中发挥重要作用 .利用改进的 PCR体系 ,从人类基因组 DNA中扩增出自 h SCF基因编码起始位点( + 1 81 )至 5′旁侧 - 1 1 90位点共 1 372 bp的片段 ,将其克隆至 p GEM- T载体中 ,经序列分析证明无误 .为探寻此片段中对转录调控起作用的具体区域 ,用基因缺失技术从 - 1 62 ( Bst X )位点向上游分别延伸至 - 2 73( Pst )、- 339( Sma )、- 381 ( Sac )、- 44 0 ( Eco R )、- 570 ( H inc )、-853( Bgl )及 - 1 1 90 ( Xho )等位点 ,共获 7个不同长度的 h SCF基因 5′旁侧序列片段 ,随后将这7个片段分别克隆入荧光素酶 ( luc)报告基因载体 p GL2 - Promoter.经阳离子脂质体包埋技术介导转染 He La细胞 ,培养 48h后检测 Luc活性 .结果表明 :h SCF基因 5′旁侧 - 1 1 90～ - 853区域对luc表达有明显的增强作用 ,而 - 339～ - 2 73区域则显示有抑制作用 .分别以包含这两个区域的片段为探针进行竞争性凝胶阻滞实验 ,结果均出现一个或多个特异性滞留区带 ,说明这两个区域存在转录因子的结合位点 .实验结果表明 ,h SCF基因 5′旁侧 - 1 1 90～ - 853区域富含 AT,是一典型基质结合区 ,而 - 339～ - 2 73区域为一新的沉默子 。

Human stem cell factor (hSCF) plays an important role in the proliferation,differentiation and migration of certain mammalian stem cells.Up to now the definite regulatory mechanism of hSCF gene is unclear except it has been shown that its 5′ flanking sequence contains some essential elements for transcription regulation.The investigation has focused on cloning the 5′ flanking sequence and localizing cis acting elements responsible for the regulation of the hSCF gene.Through accurate PCR,a 1 372 bp fragment was amplified,extending from the coding origin site (+181) to -1 190 of hSCF gene 5′ flanking sequence.Then it was cloned into pGEM T vector and confirmed by DNA sequencing.A series of 5′ DNA deleted fragments extending from -162 ( Bst XⅠ) to -273( Pst Ⅰ),-339( Sma Ⅰ),-381( Sac Ⅰ),-440( Eco RⅠ),-570( Hinc Ⅱ),-853( Bgl Ⅱ) or -1190( Xho Ⅰ) were subcloned respectively into the pGL2 promoter vector for identification of the specific functional regions.Then these recombinant reporter genes were transfected into HeLa cells via cationic liposome mediated transduction and luc gene activities were subsequently detected by Luminometer.The result indicates that the distal rigion of -1 190 ～-853 of the hSCF 5′ flanking sequence significantly enhances luc expression gene,while the proximal fragment -339～-273 inhibits the expression.Competitive gel retardation assay was performed to further the functional research.One or three retarded bands bound by the probe A(-1190～-1086),B(-1094～-966),C(-965～-846) and probe 67 bp(-339～-273) with HeLa nuclear extract proteins were observed in PAGE lanes,and the specific DNA protein complexes forming in some retarded bands were demonstrated by competitive GRA.Therefore,the region -1 190～-853 is a AT rich sequence and may be a matrix attachment rigion;-339～-273 fragments of the hSCF 5′ flanking sequence is a novel silencer. They both play certain roles in the regulation of hSCF gene expression.