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Gene targeting approach to study functions on respiratory and circulatory regulation of novel H^+ Channel

基因打靶方法研究新型H^通道对呼吸和循环调节的功能

基本信息

批准号：
11680779
负责人：
SHIMOKAWA Noriaki
金额：
$ 2.11万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680779/
关键词：
Differential display Gene targeting Ventral medullary surface H^+-sensitivity Hypercapnia Rhombex-29 40 MafG CO_2 H^+sensitivity hypercapnia Transmembrane Protein H^+ sensitivity Maf protein proteolipid protein Mafg Naclear

项目摘要

1. Characterization and gene targeting study of ASPT[1] ASPT comprises 751 amino acids containing 12 membrane-spanning helices[2] ASPT has H^+/Sugar transport motif, Proline-rich region, leucine zipper motif.[3] ASPT immunoreactive neurons were found in the VMS and the number of cells was increased by hypercapnic stimulation.[4] We got clones containing exons of ASPT gene from genomic library of ES cells.[5] We constructed a vector for targeting of ASPT gene.[6] We obtained ES cells that generated homologous recombination.2. Detection of novel genes involved in H^+-sensitivity in the VMSThe ventral medullary surface (VMS) in known as the site of the central chemosensitive neurons. These neurons sense excess CO_2/H^+ dissolved in the cerebrospinal fluid that superfuses the VMS and induce hyperventilation. We hypothesized that genes specific for hyperventilation are expressed much more highly in VMS neurons than in extra-VMS neurons in other parts of the central nervous system (CNS). Applying the differential display technique to the brain of adult rats, we differentiated the mRNAs of the VMS neurons from those of cerebral cortex neurons.[1] We cloned a novel four-transmembrane protein, Rhombex-29. Structural analysis and the phylogenic tree showed that rat Rhombex-29 is homologous to the major CNS myelin protein PLP/DM20-M6 family (REFERENCES 1).[2] We cloned cDNA encoding nuclear transcription factor MafG from rat VMS (REFERENCES 2).[3] We got clones encoded a putative transmembrane protein, rhombencephalic expression protein-40 kDa (Rhombex-40) (REFERENCES 3).[4] We cloned MafG-2, a novel splice variant of MafG, from rat brain by RT-PCR (REFERENCES 4).

1. ASPT的表征和基因打靶研究[1] ASPT由751个氨基酸组成，含有12个跨膜螺旋[2] ASPT具有H^+/糖转运基序、富含脯氨酸区、亮氨酸拉链基序。[3]在VMS内发现ASPT免疫反应阳性神经元，高碳酸刺激可使ASPT免疫反应阳性神经元增多。[4]从ES细胞基因组文库中获得了含有ASPT基因外显子的克隆。[5]我们构建了ASPT基因的靶向载体。[6]获得了能产生同源重组的ES细胞.延髓腹侧表面（VMS）是化学敏感性神经元的中枢部位。这些神经元感知到溶解在脑脊液中的过量CO_2/H^+，这些CO_2/H^+使VMS过度通气。我们假设，过度通气的基因表达更高的VMS神经元比在其他部分的中枢神经系统（CNS）的VMS神经元。将差异显示技术应用于成年大鼠脑，我们区分了VMS神经元和大脑皮层神经元的mRNA。[1]我们克隆了一个新的四跨膜蛋白，Rhombex-29。结构分析和基因树显示大鼠Rhombex-29与主要CNS髓鞘蛋白PLP/DM 20-M6家族同源（参考文献1）。[2]我们从大鼠VMS中克隆了编码核转录因子MafG的cDNA（参考文献2）。[3]我们得到了编码推定的跨膜蛋白、菱形表达蛋白-40 kDa（Rhombex-40）的克隆（参考文献3）。[4]我们通过RT-PCR从大鼠脑中克隆了MafG-2，一种新的MafG剪接变体（参考文献4）。