Genetic Modification of Cell Lines

The MWCL cell line was established and characterized at Mayo Clinic^{22 (link)}. The BCWM cell line was a kind gift from Dr. Steve Treon and the HBL-1 cell line was kindly provided by Dr. Thomas Witzig. Cell line authentication is described in Supplemental Methods. All cells lines were maintained in RPMI 1640 medium with 10–20% fetal bovine serum (FBS), penicillin (50 U/ml), and streptomycin (50 µg/ml) were added. Cells were periodically checked for mycoplasma by PCR and were found to be negative. All cell lines were cultivated at 37 °C and 5% CO₂. Transcription activator-like effector nuclease (TALENs) specific for targeting exon 5 of the TNFAIP3 gene were designed by the Mayo Clinic Genetics and Model Systems Core using the FusX system as previously described^{23 (link)}. Exon 5 was targeted because it is present in all reported TNFAIP3 isoforms. Cells were transfected with 10 μg of each TALEN vector arm by using the AMAXA® nucleofection system (Amaxa, Cologne, Germany). To track successful transfection, cells were co-transfected with a 2 μg GFP expressing plasmid. After 48 h, cells that have incorporated the GFP expression plasmids were isolated by single cell sorting at the Mayo Flow Cytometry Facility. Restriction enzyme digest was used to initially identify clones that carried the frameshift mutation.