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

1. [1] ASPT由751个氨基酸组成，包含12个跨膜螺旋[2]ASPT具有H^+/糖转运基序、脯氨酸富区、亮氨酸拉链基序[3]在VMS中发现ASPT免疫反应神经元，高碳酸刺激使细胞数量增加我们从胚胎干细胞基因组文库中获得了含有ASPT基因外显子的克隆我们构建了一个靶向ASPT基因的载体我们获得了产生同源重组的ES细胞。髓质腹侧面（VMS）被称为中枢化学敏感神经元的位置。这些神经元感知过量的CO_2/H^+溶解在脑脊液中，使VMS超载并诱导过度通气。我们假设过度通气特异性基因在VMS神经元中的表达比在中枢神经系统（CNS）其他部分的额外VMS神经元中的表达要高得多。将差异显示技术应用于成年大鼠的大脑，我们将VMS神经元的mrna与大脑皮层神经元的mrna进行了区分我们克隆了一种新的四跨膜蛋白Rhombex-29。结构分析和系统发育树显示，大鼠Rhombex-29与主要的中枢神经系统髓磷脂蛋白PLP/DM20-M6家族同源（文献1）我们从大鼠VMS中克隆了编码核转录因子MafG的cDNA（文献2）我们得到的克隆编码了一个假定的跨膜蛋白，Rhombex-40 kDa （Rhombex-40）我们通过RT-PCR从大鼠脑中克隆了MafG-2，这是一种新的MafG剪接变体（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).