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

1.ASPT的特性及基因打靶研究[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Pro区、亮氨酸拉链基序。[3]ASPT免疫反应阳性神经元存在于VMS中，细胞数在高二氧化碳刺激下增加。[4]我们从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]我们获得了产生同源重组的ES细胞。在VMST腹侧延髓表面(VMS)检测到与H~(++)敏感有关的新基因，该区域被称为中枢化疗敏感神经元的位置。这些神经元感觉到溶解在脑脊液中的过量CO2/H^+，脑脊液对VMS进行灌流，导致过度换气。我们假设，过度换气的特异性基因在VMS神经元中的表达比在中枢神经系统(CNS)其他部分的VMS外神经元中高得多。将差异显示技术应用于成年大鼠的大脑，我们区分了VMS神经元和大脑皮层神经元的mRNAs。[1]我们克隆了一个新的四跨膜蛋白Rhombex-29。结构分析和系统发育树表明，大鼠Rhombex-29与主要的中枢神经系统髓鞘蛋白PLP/DM20-M6家族同源(参考文献1)。[2]我们从大鼠VMS中克隆了核转录因子MafG的编码基因(参考文献2)。[3]我们获得了编码一个可能的跨膜蛋白--菱形脑表达蛋白-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的一个新的剪接变体MafG-2(参考文献4)。

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).