Gene Editing in Antibody-Deficient Hybridomas

Genome editing experiments in B cell hybridomas were always executed in the HC9- cell line being dysfunctional in antibody expression and constitutively expressing Cas9 protein (30 (link)). Hybridoma cells were electroporated using the SF Cell Line 4D-Nucleofector X Kit L (Lonza, V4XC-2024) with the program CQ-104. The following standard conditions in 100 μl of total volume of nucleofection mix were used: 1 × 10⁶ cells, 0.156 nmol pre-complexed Alt-R duplex gRNA and 5 μg of PCR-linearized double-stranded DNA. Following transfection, cells were incubated for 5 min at RT, before adding 500 μl of pre-warmed medium to the nucleocuvette and typically transferring them to 1.5 mL of fresh growth medium in 6-well plates. The cells were usually supplemented 24 h later with 0.5–1.0 mL of fresh culturing medium.