2.4. Genomic DNA Extraction and PCR

Genomic DNAs were extracted from both parental and GBA-knockout lines using InstaGene Matrix reagent (Bio-Rad Laboratories). To confirm GBA gene disruption, the targeted gene loci were PCR amplified using the primer pair: forward- CTGATGGAGTGGGCAAGAT; reverse- AAGTGATAAGCAGAGTCCCATACTC. PCR products with sizes of 425 bp (wildtype) and 1232 bp (insertion) were expected in the case of targeted gene disruption by non-homologous end joining. PCR reactions were performed using 2xTaq Master Mix (GenScript; Piscataway, NJ, USA) with a hot start at 95 °C for 2 min, followed by 35 cycles of denature at 95 °C for 30 s, annealing at 60 °C for 20 s and extension at 72 °C for 1 min. Products were electrophoresed in 1% agarose gel to confirm the size of reactions.