Genetic Methods for Detecting Astrocytes, Neurons and Neurogenesis

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.These results confirm the crucial role of astrocytes in ensuring neurogenesis. The results in pure cell culture (in vitro) were identical to the in vivo results in animals.