Plasmid plus midi kit

pCMV-dR8.2 dvpr (plasmid #8455), pMD2.G (a kind gift from Didier Trono (plasmid #12259), and AB.pCCL.sin.cPPT.U6.miR-210-decoy.hPGK.GFP.WPRE (a kind gift from Brian Brown (plasmid # 6599) [44 (link)] were purchased from Addgene (Cambridge, MA). Plasmids were isolated using Qiagen Plasmid Plus Midi Kit (Qiagen, Germany) as per the manufacturer's instructions. HEK-293T cells were seeded on 6 cm plates (Sarsted, Germany) one day prior to transfection. Calcium phosphate-mediated transfection was performed using packaging:envelope:transfer plasmids at a ratio of 0.259:0.087:0.345 μg/cm², and virus was harvested 42, 46, 50 and 66 hours post transfection. The virus containing supernatant passed through 0.45μM low protein-binding filter (Sarstedt, Germany) filters.
A375, MML-1, and SK-MEL-2 cells were transduced with 1 ml of virus containing 2–4 μg/ml of polybrene (Sigma-Aldrich) per cell line and seeded on 6-well plates (Sarstedt, Germany). 12 h post transduction, fresh media was added and cells were allowed to grow for another 24 h. All cell lines were analyzed with BD Accuri C6 FCM (BD Biosciences, USA) for GFP expression before further experiments were performed (data not shown).

An AAVS1 transgene knock-in vector kit, consisting of the pCas-Guide-AAVS1 (GE100023) and pAAVS1-puro-DNR (GE100024) plasmids, was purchased from OriGene Technologies, Inc. (Rockville, MD). A “cassette” containing the bioengineered fVIII transgene “lcoET3” under the transcriptional control of the constitutively active human EF1α promoter was cloned into the multiple cloning site (MCS) of the pAAVS1-puro-DNR plasmid using standard restriction enzyme digestion and ligation protocols and reagents (New England Biolabs/NEB, Ipswich, MA). lcoET3 is a liver-codon-optimized chimeric human/porcine fVIII transgene containing a designed to produce bioengineered fVIII with increased FVIII secretion efficiency and activity (22 (link), 36 (link)). Both plasmids contain the ampicillin resistance gene. Therefore, they were transfected into competent E. coli cells (NEB) using heat shock, and the transformants were plated on LB agar plates containing ampicillin (100 μg/mL). Ampicillin-resistant colonies were plucked 24 hours later and inoculated into LB broth. Once the cultures were expanded, the plasmids from the E. coli were isolated using the Plasmid Plus Midi kit (Qiagen, Valencia, CA), and restriction enzyme digestion was performed followed by 2% agarose gel electrophoresis to verify the identity and integrity of the plasmid.