Amaxatm 4d nucleofectortm

HEK293T, PC12, Vero, and SK-N-AS cells at 80% confluence were transfected with the desired expression plasmids using Lipofectamine 2000® (Thermo Fisher Scientific^TM) in Opti-MEM (Gibco), following the manufacturer’s instructions.
SH-SY5Y cells were transfected using the Amaxa^TM 4D-Nucleofector^TM (Lonza, Basilea, Switzerland) system. Cell lines were transfected with FAIM isoforms subcloned into pcDNA3containing 3x-FLAG or GFP.

The pBABE-neo-hTERT plasmid was a gift from Dr. Bob Weinberg (Addgene plasmid # 1774) and was the source of the hTERT cDNA. The pTT-PB-SOKMLN-puro as well as pcA3-PBase-Tomato (for transient expression of piggyBac transposase) plasmid were described previously [37 (link)]. The pTT-PB-SOKMLN-puro was used as the backbone for the piggyBac transposon after excision of the SOKMLN cassette with EcoRI and SalI. All restriction enzymes and DNA ligase were purchased from New England Biolabs (NEB). The pBABE-neo-hTERT was digested with EcoRI and SalI and the DNA fragment containing the hTERT cDNA was ligated into the pTT-PB-puro (without SOKMLN). The resulting plasmid pTT-PB-hTERT-puro was transformed into NEB10-beta competent E.coli cells. Afterwards, plasmid DNA was isolated using Maxiprep kit (Qiagen) according to the manufacturer’s instructions.
Common marmoset primary fibroblasts were nucleofected with pTT-PB-hTERT-puro and pcA3-PBase-Tomato using AmaxaTM 4D NucleofectorTM (Lonza) with the P2 Kit for primary mammalian fibroblasts (Lonza). For each nucleofection, 1 x 10⁶ cells were suspended in 100μL buffer P2 together with 9 μg of pTT-PB-hTERT-puro and 6μg pcA3-PBase-Tomato plasmid and nucleofected using program CA-137. The cells with integration of pTT-PB-hTERT-puro were selected with Puromycin (1 μg/ml) for the first 10 passages.

sgRNA design: sgRNAs were designed (http://crispor.tefor.net/, accessed on 22 June 2022) and their efficacies were validated in NIH/3T3 cells. sgRNA was synthesized in vitro (IDT DNA, Leuven, Belgium) and used for electroporation into mES cells.
Electroporation protocol: NIH/3T3 or WT mES cells were mixed with CRISPR-Cas9 components (IDT-DNA, Leuven, Belgium) and electroporated via Amaxa^TM 4D-Nucleofector^TM (Lonza, Cologne, Germany) using the manufacturer’s guide. The extended electroporation protocol is included in the Supplementary Methods.
Surveyor nuclease assay: sgRNAs were first validated in NIH/3T3 cells via hetero-duplexes formed by CRISPR-Cas9 mediated gene editing, reaction buffer, and T7E1 endonuclease (NEB, Frankfurt, Germany). sgRNA with the highest efficacy was used for WT mES cell gene edit.
After electroporation, mES cells were seeded, single colonies were picked, expanded, and further assessed via PCR and restriction digestion. This was done by the Hind III restriction enzyme (NEB, Frankfurt, Germany). Positive clones were sent for Sanger sequencing (LGC genomics, Berlin, Germany).