Nucleofector 2b

HEK293 cells (ATCC #CRL-1573) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies) without glucose (Catalog # 11966–025) and supplemented with 10 mM galactose, 6 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% FBS. Cells were grown at 37°C in a humidified atmosphere with 10% CO₂ in air. Cells were routinely passaged by washing with PBS (pH 7.4), incubating with 0.25% trypsin, 1 mM EDTA (Life Technologies) for 2 min and then neutralized with media. Cells were next plated at 1:10 and 1:20 dilutions to maintain cells in the replicative growth phase. Transfection was performed using the Lonza Nucleofector^TM 2b with the corresponding kit (Amaxa^TM cell line kit V) according to the manufacturers protocol. Cells were transfected with the previously described pJ603-NGFP-POLG2 plasmid with and without the R182W mutation. Cells were transfected using the Lonza Nucleofector^TM 2b and then grown in the presence of 800 μg/mL G418 for selection. After 4–5 days, selection was maintained with 50 μg/mL G418. Vials of frozen cells were prepared in freezing medium containing 40% growth DMEM, 50% FBS, 10% DMSO and stored in the gas-phase of a nitrogen freezer.

The knock-in vectors were transfected by GenePulser (Bio-Rad Laboratories) as previously described.²⁴ Vectors were linearized by NotI, except that SalI and ScaI were used for HC constant region knock-in and the hV_H knock-in vector, respectively. RMCE donor vectors were transfected using Nucleofector 2b (Lonza) and Cell Line Nucleofector Kit T (Lonza). A total of 1 × 10⁷ cells were collected and transfected with 8 μg of DNA mixture (7 μg of RMCE construct and 1 μg of Cre recombinase expression vector) with the Nucleofector 2b (Lonza) optimized transfection program B-023.

Strep-tagged ADAP1 or GFP were expressed using the Jurkat T-REx system. Briefly, the Jurkat T-REx cells (Thermo Fisher) were electroporated with 1 μg of Ssp1-linearized pcDNA4/TO Strep-tagged ADAP1 or GFP vector using Nucleofector2b (Lonza). Positive clones were selected with 10 μg/ml Blasticidin and 100 μg/ml Zeocin. To induce protein expression, the stable cell lines were treated with 1 μg/ml Doxycycline Hydrochloride (Fisher Scientific, BP2653-1) for 48 h. Next, for each transcription reporter assay reaction, 1 × 10⁶ Jurkat cells expressing Strep-tagged ADAP1 or GFP were resuspended in 0.1 ml Mirusbio ingenio (Mirus, MIR50115) solution containing 2 μg of TF-responsive reporters driving Firefly luciferase (pGL3-3xAP1-luciferase (Addgene, 40342), pGL3-NF-κB-luciferase (Promega), pGL3-NFAT-luciferase (Addgene, 17870)) and 0.2 μg Renilla plasmid (pRL-CMV, Promega, E2261) at room temperature. Triplicate reactions were set up for each reporter plasmid. Cells were transferred into cuvettes (2 mm gap, Mirus, MIR50121) and subjected for electroporation using Nucleofector2b (Lonza). Cells were transferred to pre-warmed plate containing 1 ml RPMI/10% FBS and cultured for 24 h at 37 °C. Cells were harvested and analyzed using Dual Luciferase Kit (Promega, E1910). Firefly luciferase signal was normalized to the internal Renilla luciferase control.