Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have utilized highly purified hematopoietic progenitor and stem cells (HPCs, HSCs) from normal peripheral blood to develop methodology for: (a) efficient transfer into HPCs of a non-hematopoietic membrane reporter, i.e., the nerve growth factor receptor complementary DNA; and (b) effective gene transduction of putative HSCs, i.e., cells initiating Dexter-type long-term culture (LTC-ICs). Purified HPCs induced into cycling by growth factors (interleukin 3, interleukin 6, c-kit ligand) were transduced with the N2 retroviral vector containing the neomycin resistance (neor) gene. More than 80% of transduced HPCs were resistant to the toxic G418 level. Thereafter, the HPCs were effectively transduced with the LNSN retroviral vector containing a nerve growth factor receptor complementary DNA; the nerve growth factor receptor was detected on > or = 18% of the transduced HPCs. These experiments provide a new tool from which (a) to monitor expression of a transduced membrane report on hematopoietic cells, particularly at the level of HPCs/HSCs, and (b) to characterize the transduced cells by double- and triple-labeling membrane antigen analysis. Purified HPCs/HSCs grown in Dexter-type LTC were transduced at 1 week by exposure to supernatant N2 retroviral particles in the absence of exogenous hematopoietic growth factors. The procedure, devoid of toxic effects, allowed an efficient neor transduction into LTC-ICs. Thus, we consistently detected neomycin-resistant mRNA in the clonal progeny of HPCs produced in LTC at 5-8 weeks in both the nonadherent and adherent fractions; this timing of expression coincides with that of HPC production by LTC-ICs, thereby indicating the effective transduction of the LTC-ICs. These experiments represent a first step toward development of preclinical models for gene transfer into human peripheral blood HSCs by complex retroviral vectors.
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PMID:Efficient transfer of selectable and membrane reporter genes in hematopoietic progenitor and stem cells purified from human peripheral blood. 804 88

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.
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PMID:A novel, membrane receptor-based retroviral vector for Fanconi anemia group C gene therapy. 917 20

Efficient gene transfer into hematopoietic stem cells offers a number of potential therapeutic applications. However, the relatively low titer of retroviral supernatants and the requirement for cell division to ensure integration have meant that transduction efficiency has been low. We have modified a flowthrough approach to cell transduction and have been able consistently to increase gene transfer efficiency into human hematopoietic progenitor cells. We transduced CD34 cells with retroviral vectors encoding a truncated nerve growth factor receptor (NGFR) or neo. Retroviral supernatant was pulled through 0.2-micron polycarbonated membranes, followed by placement of cells on the filter. In the absence of cytokines, the transduction efficiency of CD34 cells with a NGFR vector was increased 3-11-fold over that obtained at an identical MOI in liquid culture to produce 11%-44% transduction. Furthermore, both Thy1+ and Thy1- subsets in a total CD34 population were transduced with similar efficiency, and transduction with a neo vector, as measured by G418 resistance in clonogenic assays, increased 1.5-5-fold. The mechanism by which gene transfer is improved may reflect colocalization of cells and retrovirus. Costaining of cells transduced on the filter with an NGFR retrovirus with both an NGFR antibody and a gp70 antibody that recognizes viral coat protein revealed high-level coexpression. The levels of in vitro gene transfer we obtain are equivalent to those observed when CD34 cells are cocultured in liquid culture with cytokines. However, culture with cytokines may commit CD34 cells to differentiation and has produced disappointingly low levels of subsequent in vivo gene transfer. Gene marking studies using distinguishable retroviral vectors will provide a means of learning whether the effects of flowthrough transduction genuinely enhance the efficiency of gene transfer to human marrow-repopulating cells.
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PMID:Increased transduction efficiency of primary hematopoietic cells by physical colocalization of retrovirus and target cells. 962 Dec 55