Transgene Expression after Stable Transfer of a Mammalian Artificial Chromosome into Human Hematopoietic Cells

• Published in: Blood 2004-11, Vol.104 (11), p.498-498
• Main Authors: Vanderbyl, Sandra L., Sullenbarger, Brent, White, Nicole, Perez, Carl F., MacDonald, G. Neil, Stodola, Tom, Bunnell, Bruce, Ledebur, Harry C., Lasky, Larry C.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V104.11.498.498

The ACE System, consisting of a pre-engineered mammalian artificial chromosome containing multiple site-specific integration sites (acceptor sites); an ACE Targeting Vector containing a Platform ACE specific donor site and the gene(s) of interest; and the ACE Integrase, a proprietary integrase that catalyzes the site-specific recombination of the ACE Targeting Vector onto the Platform ACE, is a versatile biological engineering system for the genetic modification of cells for gene-based cell therapies, the generation of high expressing cell lines for the production of recombinant proteins, and the generation of transgenic animals. ACEs are promising gene delivery vehicles as they are stably maintained, autonomous, non-integrating chromosomes that are easily purified by flow cytometry and readily transfected into a variety of cell types. We developed and optimized a procedure for transfecting human cord blood CD34+ cells with ACEs using LipofectAMINE PLUS (Invitrogen) and iododeoxyuridine-labeled (IdU) ACEs and ACEs encoding genes for humanized Renilla green fluorescence protein (hrGFP) and zeomycin resistance (zeoR, which confers resistance to bleomycin). CD34+ positively selected cells were isolated from human umbilical cord blood using a Ficoll-Hypaque (Pharmacia) density gradient followed by two positive selection steps using magnetic beads (Miltenyi). Prior to transfection, the cells were stimulated with thrombopoietin, stem cell factor, flt-3 ligand, and IL-6, all at 100 ng/mL, for 1–3 days. We quantified the delivery of IdU-labeled ACEs, 24–48 hours post-transfection, by a screening technique that utilizes a FITC-conjugated anti-BrdU B44 clone antibody (Becton Dickinson) that cross-reacts with IdU, and flow cytometry. We detected IdU-labeled ACEs in 2.5–4.0 % of the cells 24–48 hours post-transfection. CD34+ positively selected cells transfected with hrGFP-zeoR-ACEs were plated into methycellulose culture with or without bleomycin. Using these conditions we were able to detect hrGFP expression in up to 3% of the total transfected cells grown without bleomycin. Untransfected negative control CD34+ positively selected cells did not grow in methycellulose culture with bleomycin; however there was significant CFU-GM colony growth with CD34+ positively selected cells transfected with hrGFP-zeoR-ACEs in the presence of bleomycin (3 to 12 cfu/10,000 cells plated, in 3 experiments). The bleomycin-resistant colonies (100% hrGFP+) were analyzed via fluorescent