The presence of both negative and positive elements in the 5'-flanking sequence of the rat Na,K-ATPase alpha 3 subunit gene are required for brain expression in transgenic mice.
The presence of both negative and positive elements in the 5'-flanking sequence of the rat Na,K-ATPase alpha 3 subunit gene are required for brain expression in transgenic mice.
复制标题
转基因小鼠的脑表达需要大鼠 Na,K-ATPase α 3 亚基基因的 5 侧翼序列中同时存在阴性和阳性元件。
DOI：
10.1093/nar/22.22.4748
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发表时间：
1994
影响因子：
14.9
通讯作者：
Lingrel,JB
中科院分区：
文献类型：
--
作者：
Pathak,BG;Neumann,JC;Croyle,ML;Lingrel,JB
Na，K-ATP酶是一种完整的质膜蛋白，由α和β亚基组成，每个亚基都有以组织特异性方式表达的离散异构体。在三种功能性α亚型基因中，编码α3亚型的基因在其表达中是最受组织限制的，主要在脑中发现。为了鉴定α3亚型基因中参与指导脑中表达的区域，将1.6 kb的5′侧翼序列连接到报告基因氯霉素乙酰转移酶（CAT）上。将α3 - CAT嵌合基因构建体显微注射入小鼠受精卵中，获得转基因小鼠。对来自不同品系的成年转基因小鼠的分析表明，转基因主要在大脑中表达。为了进一步描绘在该组织中赋予表达所需的区域，再次使用转基因小鼠对α3-CAT融合构建体的5′-侧翼序列进行系统性缺失并进行分析。这些分析的结果表明，介导α3亚型基因脑特异性表达所需的DNA序列存在于转录起始位点上游210 bp内。还在转基因小鼠中测定了在该区域含有扫描突变的α3-CAT启动子构建体。这些研究已经确定了一个功能性的神经限制性沉默元件以及一个积极作用的顺式元件。
The Na, K-ATPase is an integral plasma membrane protein consisting of α and β subunits, each of which has discrete isoforms expressed in a tissue-specific manner. Of the three functional α isoform genes, the one encoding the α3 isoform is the most tissuerestricted in its expression, being found primarily in the brain. To identify regions of the α3 isoform gene that are involved in directing expression in the brain, a 1.6 kb 5′-flanking sequence was attached to a reporter gene, chloramphenicol acetyltransferase (CAT). The α3 – CAT chimeric gene construct was microinjected into fertilized mouse eggs, and transgenic mice were produced. Analysis of adult transgenic mice from different lines revealed that the transgene is expressed primarily in the brain. To further delineate regions that are needed for conferring expression in this tissue, systematic deletions of the 5′-f lanking sequence of the α3–CAT fusion constructs were made and analyzed, again using transgenic mice. The results from these analyses indicate that DNA sequences required for mediating brain-specific expression of the α3 isoform gene are present within 210 bp upstream of the transcription initiation site. α3-CAT promoter constructs containing scanning mutations in this region were also assayed in transgenic mice. These studies have identified both a functional neural-restrictive silencer element as well as a positively actingciselement.