Geneblocks

Candidate IgVH sequences were synthesized as GeneBlocks (Integrated DNA Technologies, Inc., Coralville, IA) and cloned into the linearized vector pTR2a (Invitrogen) using two inverse PCR primers; SG101b-UB (5’-AGA GTG CAC CCT GCC GGC TGC GGC) and VH5'FP (5’-GCC AAG ACC ACC GCT CCC TCC GTT TAC CCT). Cloning was performed using the Gibson Assembly Kit (New England BioLabs, Ipswich, MA). Separately, a panel of 20 germline IgVk genes highly expressed in non-immunized mice (Table 4) (Aoki-Ota et al., 2012 ) were synthesized as GeneBlocks and cloned into pKFM, which is derived from the plasmid pCR2.0 topo (Invitrogen). Each candidate IgVH plasmid was transiently transfected (293-fectin; Invitrogen) in 96-well plates with each IgVk gene to produce mature IgG2a antibodies. Supernatants harvested 3 days later were tested for Ab and antigen binding was measured by ELISA using goat anti-mouse H+L IgG (GaM) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). For large scale production, MAbs were expressed in 1 L culture volume and purified using MabSelect™ Protein A resin (GE Healthcare). Endotoxin was removed by standard methods.

Lentiviral expression plasmids pLEX304_PHF5A-WT-STOP and pLEX304_PHF5A-Y36C-STOP were cloned using the gateway recombinase cloning strategy (Invitrogen, USA) from commercially synthesized gene blocks (IDT, USA). Lentivirus production and transduction of 22Rv1 cells was performed as described above. 22Rv1 cells stably transduced with pLEX304_PHF5A were selected with 10 μg/mL blasticidin for 7 days, at which point non-transduced control cells were completely killed.