Gene expression analysis of non-cerebellar portion of Gb5-deficient mice brain

Gb5 is a divergent, evolutionarily-conserved, member of the heterotrimeric G protein b subunit family that is expressed principally in brain and neuronal tissue. Among Gb isoforms, Gb5 is unique in its ability to heterodimerize with members of the R7 subfamily of the regulator of G protein signaling (RGS) proteins that contain G protein-g like (GGL) domains. Previous studies employing Gb5 knockout mice have shown that Gb5 is an essential stabilizer of GGL domain-containing RGS proteins and regulates the deactivation of retinal phototransduction and the proper functioning of retinal bipolar cells. The purpose of this study is to better understand the functions of Gb5 in the brain outside the visual system by employing molecular biology, immunohistochemistry and confocal imaging technologies. We show here that mice lacking Gb5 have a markedly abnormal neurologic phenotype that includes neurobehavioral developmental delay, wide-based gait, motor learning and coordination deficiencies, and hyperactivity. Using immunohistochemical analysis and a green fluorescent reporter of Purkinje cell maturation we show that the phenotype of Gb5-deficient mice includes, in part, delayed development of the cerebellar cortex, an abnormality that likely contributes to the neurobehavioral phenotype. Multiple neuronally-expressed genes are dysregulated in non-cerebellar portion of Gb5 KO mice. Brain tissues(non-cerebellar portion of brain) from WT and KO with three biological replications of mice were collected, frozen in liquid nitrogen, and stored at -70 °C

Gb5是异三聚体G蛋白β亚基（heterotrimeric G protein β subunit）家族中一类分化程度较高且进化保守的成员，主要表达于大脑及神经元组织。在所有Gb亚型中，Gb5的独特之处在于其可与含有G蛋白γ样（G protein-g like, GGL）结构域的G蛋白信号调节蛋白（regulator of G protein signaling, RGS）R7亚家族成员形成异二聚体。
既往针对Gb5基因敲除小鼠的研究显示，Gb5是含GGL结构域的RGS蛋白必需的稳定因子，可调控视网膜光转导过程的失活以及视网膜双极细胞的正常功能。本研究旨在通过分子生物学、免疫组织化学（immunohistochemistry）及共聚焦成像（confocal imaging）技术，进一步阐明Gb5在视觉系统以外的大脑组织中的功能。
本研究证实，缺失Gb5的小鼠存在显著异常的神经学表型，包括神经行为发育迟缓、宽基底步态、运动学习与协调能力缺陷以及活动过度。
通过免疫组织化学分析及浦肯野细胞（Purkinje cell）成熟过程的绿色荧光报告系统，我们发现Gb5缺陷小鼠的表型部分源于小脑皮层发育延迟，该异常很可能是其神经行为表型的重要致病因素之一。
在Gb5基因敲除（knockout, KO）小鼠的非小脑脑组织中，多种神经元表达基因出现表达失调。本研究收集了野生型（wild type, WT）与Gb5基因敲除小鼠的非小脑脑组织样本，每组设置3次生物学重复；将样本置于液氮中速冻后，保存于-70 ℃环境。