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Genetic Methods for Detecting Astrocytes, Neurons and Neurogenesis

机译:用于检测星形胶质细胞,神经元和神经发生的遗传方法

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摘要

Two sets of reactants for modelling neurogenesis (SRMN) were developed based on the designed and tested genetic structures of lentiviral vectors. SRMN-1 contains the genetic construct LVV-GFAP-GCaMP3 and is intended for cellspecific transduction in astroglia cells. SRMN-2 contains the genetic construct LVV-PRSx8-TN-XXL and is intended for the phenotype-specific transduction in neurons. The present study examined SRMN-1 and SRMN-2 samples and assessed their efficiency in vitro and in vivo in Norvegicus rats. Specificity to particular cell types for all SRMN samples exceeded 97%. The number of induced signalling cascades was determined via activation of intracellular ingsignalling cascades in neurons and astrocytes (purinergic receptors and β-adrenoceptors). The results demonstrated dynamic recording of fluorescent signals and a two-fold increase in intensity after addition of the activator in all samples. The experimental SRMN samples revealed successful and stable transfection of catecholaminergic neurons and astrocytes, data on transfection efficiency, specificity of the developed genetic structures of SRMN, and calcium dynamics in transfected neurons and astrocytes.
机译:基于慢病毒载体的设计和测试的遗传结构,开发了两组用于建模神经发生(SRMN)的反应物。 SRMN-1含有遗传构建体LVV-GFAP-GCAMP3,用于在星形菌细胞中进行细胞特异性转导。 SRMN-2含有遗传构建体LVV-PRSX8-TN-XX1,并且旨在用于神经元的表型特异性转导。本研究检测了SRMN-1和SRMN-2样品,并在挪威大鼠体外和体内评估其效率。所有SRMN样品的特定细胞类型的特异性超过97%。通过在神经元和星形胶质细胞(嘌呤能受体和β-肾上腺素受体)中的细胞内符号级联的激活来确定感应信号级联的数量。结果表明,在所有样品中加入活化剂后,荧光信号的动态记录和强度的两倍增加。实验性SRMN样品揭示了儿茶酚胺能神经元和星形胶质细胞的成功和稳定转染,关于转染效率的数据,SRMN发育遗传结构的特异性,转染神经元和星形胶质细胞的钙动力学。

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