A novel, membrane receptor-based retroviral vector for Fanconi anemia group C gene therapy
| Autor: | Andreas W Machl, Manfred Kubbies, Simone A Planitzer |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
Reporter gene
Fanconi anemia complementation group C DNA repair Cell Survival Genetic Vectors Genetic Therapy Cell cycle Biology medicine.disease Flow Cytometry Hematopoietic Stem Cells Molecular biology Viral vector Transduction (genetics) medicine.anatomical_structure Fanconi Anemia Retroviridae Fanconi anemia Genetics medicine Cancer research Molecular Medicine Humans Molecular Biology B cell |
| Zdroj: | Gene therapy. 4(4) |
| ISSN: | 0969-7128 |
| Popis: | Retroviral vectors are effective shuttle systems by introducing therapeutically relevant genes stably into the genome of proliferating cells. The majority of vectors applied for research or clinical applications use neomycin for cell selection and identification. To circumvent the time consuming and potentially toxic G418 selection process in transduction studies we constructed a novel marker vector using I-NGFR as a cell surface marker to identify DNA repair defective Fanconi anemia cells complemented with the FAC gene. The new vector constructed is based on a MoMLV backbone, a signal peptide-deleted I-NGFR receptor gene under control of a LTR promoter and the therapeutically relevant FAC gene placed downstream of a SV40 promoter. Supernatants containing high titers of amphotropic viruses from FACS cloned cell cultures were obtained and tested for primary transduction rates, rapid detection of transduced cells within 48 h and correction of mitomycin C-induced cell cycle G2 phase accumulation in a single assay using multiparameter, dual laser flow cytometry. Primary transduction efficiency detected via (I-NGFR) antibody was between 5% and 30% with Fanconi cell lines, 5% with CD34+ cells and 15% with PBLs. MMC-induced G2 phase cell cycle disturbances were fully complemented in Fanconi anemia B cell lines of complementation group C but not in B cell lines of another FA complementation group (D). In addition to the normalization of the G2 phase arrest, induction of cell death in the FAC cell line was also decreased three to 10-fold at different MMC concentrations. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|