scholarly journals A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy

2004 ◽  
Vol 32 (21) ◽  
pp. e172-e172 ◽  
Author(s):  
Michael Lindenbaum ◽  
Ed Perkins ◽  
Erika Csonka ◽  
Elena Fleming ◽  
Lisa Garcia ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Protein Production ◽  
Artificial Chromosome ◽  
Engineering System ◽  
Chromosome Engineering ◽  
Mammalian Artificial Chromosome

scholarly journals Development of a Safeguard System Using an Episomal Mammalian Artificial Chromosome for Gene and Cell Therapy

2015 ◽  
Vol 4 ◽  
pp. e272 ◽  
Author(s):  
Narumi Uno ◽  
Katsuhiro Uno ◽  
Shinya Komoto ◽  
Teruhiko Suzuki ◽  
Masaharu Hiratsuka ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Artificial Chromosome ◽  
Mammalian Artificial Chromosome ◽  
Gene And Cell Therapy

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

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 498-498 ◽  
Author(s):  
Sandra L. Vanderbyl ◽  
Brent Sullenbarger ◽  
Nicole White ◽  
Carl F. Perez ◽  
G. Neil MacDonald ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cord Blood ◽  
Hematopoietic Cells ◽  
Artificial Chromosome ◽  
Human Umbilical Cord ◽  
Engineering System ◽  
Site Specific ◽  
Post Transfection ◽  
Mammalian Artificial Chromosome ◽  
Targeting Vector

Abstract 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 in situ hybridization (FISH) and contained autonomous ACEs (one ACE/cell). In recent gene-base cell therapy clinical trials, hematopoietic cells have been transduced with retroviral vectors, with at least two patients contracting leukemia due to viral insertional oncogenesis. ACEs may provide greater safety as an autonomous replicating non-integrating platform that can be loaded and characterized in vitro with multiple and/or large therapeutic genes and transferred ex vivo to hematopoietic cells to treat hematological, immunodeficiency, and genetic diseases. We believe that this is the first report of the transfer of a functioning mammalian artificial chromosome into human hematopoietic cells.

scholarly journals Development of Caco-2 cells expressing four CYPs via a mammalian artificial chromosome

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Yumi Ohta ◽  
Kanako Kazuki ◽  
Satoshi Abe ◽  
Mitsuo Oshimura ◽  
Kaoru Kobayashi ◽  
...  
Keyword(s):  
Artificial Chromosome ◽  
Mammalian Artificial Chromosome

scholarly journals Induction of spontaneous human neocentromere formation and long-term maturation

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
Marina Murillo-Pineda ◽  
Luis P. Valente ◽  
Marie Dumont ◽  
João F. Mata ◽  
Daniele Fachinetti ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
De Novo ◽  
Histone H3 ◽  
Engineering System ◽  
Chromosome Engineering ◽  
Histone H3 Variant ◽  
Chromosomal Passenger ◽  
Long Read ◽  
Functional Kinetochore

Human centromeres form primarily on α-satellite DNA but sporadically arise de novo at naive ectopic loci, creating neocentromeres. Centromere inheritance is driven primarily by chromatin containing the histone H3 variant CENP-A. Here, we report a chromosome engineering system for neocentromere formation in human cells and characterize the first experimentally induced human neocentromere at a naive locus. The spontaneously formed neocentromere spans a gene-poor 100-kb domain enriched in histone H3 lysine 9 trimethylated (H3K9me3). Long-read sequencing revealed this neocentromere was formed by purely epigenetic means and assembly of a functional kinetochore correlated with CENP-A seeding, eviction of H3K9me3 and local accumulation of mitotic cohesin and RNA polymerase II. At formation, the young neocentromere showed markedly reduced chromosomal passenger complex (CPC) occupancy and poor sister chromatin cohesion. However, long-term tracking revealed increased CPC assembly and low-level transcription providing evidence for centromere maturation over time.

Transgene expression after stable transfer of a mammalian artificial chromosome into human hematopoietic cells

2005 ◽  
Vol 33 (12) ◽  
pp. 1470-1476 ◽  
Author(s):  
Sandra L. Vanderbyl ◽  
Brent Sullenbarger ◽  
Nicole White ◽  
Carl F. Perez ◽  
G. Neil MacDonald ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Hematopoietic Cells ◽  
Artificial Chromosome ◽  
Mammalian Artificial Chromosome

Generation of induced pluripotent stem cells by using a mammalian artificial chromosome expression system

2014 ◽  
Vol 65 (3) ◽  
pp. 331-345 ◽  
Author(s):  
Anna Tóth ◽  
Katalin Fodor ◽  
P. Blazsó ◽  
I. Cserpán ◽  
Tünde Praznovszky ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Expression System ◽  
Artificial Chromosome ◽  
Induced Pluripotent ◽  
Mammalian Artificial Chromosome
Sign in / Sign up

Export Citation Format

Share Document