Functional Validation of Genetic Variants in Cancer Cells

pcDNA3.1-FLAG- BCHE, DARS, NGDN, or UNC5B wild-type (WT) was purchased from Origene (Rockville, MD, USA), and each mutant plasmid of target genes was generated with Site-Directed Mutagenesis Kits according to the manufacturer’s protocol (Thermo Fisher Scientific). To determine the effects of each variant on cancer cell properties, GH3 cells were transfected with pcDNA3.1 control vector, pcDNA3.1-FLAG-WT of target genes, or pcDNA3.1-FLAG-mutant of target genes using Polyjet reagent (Invitrogen, Carlsbad, CA, USA). The cells were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, MTS assay (Promega, #G3582, Madison, WI, USA), and ELISA for GH (Merck, Kenilworth, NJ, USA) at 48 h after transfection^{42 (link)}.