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.
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.
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