We demonstrate that pronuclear injection of lentiviral vectors expressing small hairpin RNAs (shRNAs) that silence the expression of specific genes can be used to generate knockdown mice. A lentiviral vector capable of generating shRNA that is specific for the target gene bestrophin 1 (Best1) encodes for an anion channel that is permeable to glutamate and gamma amino butyric acid (GABA) and that also regulates intracellular calcium signaling. We confirmed that cultured cerebellar glia from these Best1 knockdown mice showed attenuation of GABA release induced by an increase in intracellular calcium. Therefore, we propose that a combined approach, the use of transgenesis together with lentiviral vectors expressing shRNAs, can successfully generate a large number of mice in which the expression of a specific gene can be downregulated gradually. We also suggest that the Best1 knockdown mouse can be a useful tool for studying Best1 gene function.