Piggybac transposon vector

Fresh buffy coats from healthy donors were collected from the Blood Donation Center of the University Hospital Freiburg, and PBMCs were isolated by Pancoll (Pan Biotech) density gradient centrifugation. We followed recently published protocols [12 (link), 50 (link)] for stable transfection of PBMCs with piggyBac vectors, except for the methods for stimulation and expansion of T cells. Briefly, 10 μg AC133-CAR piggyBac transposon vector and 5 μg Super piggyBac transposase (System Biosciences) plasmid were used for one nucleofection reaction. To stimulate T cells, on days 1 and 14, allogeneic PBMCs from five donors were irradiated with 40 Gy and added to the T cell culture at a ratio of 10:1 and the cell culture was supplemented with 50 ng/mL of the anti-CD3 mAb OKT3 and 300 IU/mL IL-2. At 2 days after stimulation, the T cells were washed and cultured in T cell medium (RPMI 1640 plus 10% FBS, penicillin-streptomycin, and GlutaMAX) supplemented with 300 IU/mL IL-2 for expansion. The medium was refreshed every 2–3 days. AC133-CAR-expressing T cells were selected with 1 μg/mL puromycin starting 5 days after transfection. Transfected T cells were analyzed on day 24 (10 days after the second stimulation). Nontransfected T cells were generated as described above, but without nucleofection.

To create a stable COL7A1-dTS-MG expressing cell line the MG was cloned into a PiggyBac transposon vector (System Biosciences, Mountain View, CA, USA) according to Koller et al. using the following primer pair for PCR: (forward primer: 5′-GATCTCTAGACACCATGGTGAGCAAGGGCGC-3′, reverse primer: 5′-GATCTCTAGATCACTTGTACAGCTCATCC-3′). The stable COL7A1-dTS-MG expressing cell line was created according to a previously published protocol [16 (link)].

Sp1, TBP and H2B cDNA was amplified from ES cell cDNA libraries. Foxp2 cDNA was obtained from GE Dharmacon (Cat. #: MMM1013-202798679). Htt-94Q and Htt-25Q cDNA were obtained from Addgene (Plasmid #23966; Htt-94Q and Plasmid #1177; Htt-25Q). Subsequently, full-length and truncated protein fragments were cloned into Piggybac transposon vector (PB533A-2, System Biosciences) or a modified Piggybac transposon vector with PuroR using indicated primers or gBlock fragments (IDT) (Supplementary file 1). HaloTag (Promega, Madison, WI) or mEOS3.2 (Addgene: Plasmid #54525) was further cloned to fuse with fragments at the N-terminus (Sp1, Sp1 fragment, TBP) or C-terminus (H2B, Htt-25Q and Htt-94Q). The primer information for cloning is in Supplementary file 1.