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)

Gene therapy for the beta thalassemias and sickle cell anemia will require high levels of expression of human beta globin genes. One method to achieve this goal is amplification of globin genes transferred into the stem cells in the bone marrow of these patients. If the amplified genes remain normally regulated, they will then further increase their expression on being induced to differentiate along an erythroid pathway. To begin this study, we constructed a plasmid containing a neomycin resistance gene, a human beta globin gene, and a wild-type DHFR cDNA, and transfected it into mouse erythroleukemia cells. All the G418-resistant clones analyzed acquired and expressed the human beta globin gene. By serial passage of the cells in increasing concentrations of methotrexate, the exogenous human beta globin genes were stably amplified in all lines, and all increased their globin mRNA expression roughly proportional to their augmented copy number. Most of the clones further increased their beta globin expression on addition of an erythroid stimulus (dimethylsulfoxide). These results indicate that globin gene amplification may be useful in increasing globin mRNA expression in further experiments whose goal is gene therapy.
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PMID:Regulated expression of amplified human beta globin genes. 244 79

Hybrid genes containing human gamma or beta globin gene promoters linked to a neomycin resistance (neoR) gene were transfected into erythroid (K562) and nonerythroid (HeLa) cells. The number of clones resistant to G418, a neomycin analogue, was used to assay promoter strength. The results indicate that in K562 cells both promoters are active, and the gamma gene promoter is much stronger than the beta. By contrast, neither gene promoter is active in HeLa cells. These experiments indicate that these globin gene promoters are tissue-specific and sufficient for activity.
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PMID:The role of human globin gene promoters in the expression of hybrid genes in erythroid and non-erythroid cells. 347 Dec 19

Human globin genes can be transferred into mouse and human erythroid cells in culture, and can be appropriately expressed at the mRNA level in these cells. A plasmid containing a human beta globin gene is expressed in mouse erythroleukemia cells (MELC), and another containing a human epsilon or gamma gene is expressed in human erythroleukemia (K562) cells. A neomycin resistance (neoR) gene on the plasmids has been used to select for those cells containing the transferred globin genes; this selection may favor the expression of the globin genes by providing chromosomal positions requiring neoR expression. Analyzing clones resistant to G418, a neomycin analogue, demonstrated globin mRNA expression and induction. Retroviral vectors have also been used to transfer and appropriately express human beta genes in MELC. In addition, a plasmid containing a dihydrofolate reductase (DHFR) gene as well as neoR and beta globin genes has been used to amplify and express beta globin mRNA in MELC. These experiments suggest that high level appropriate expression of human beta globin genes is feasible and provides potentially useful approaches to the long-range goal of gene therapy for sickle cell anemia and beta thalassemia.
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PMID:Human globin gene expression after gene transfer. 347 10

Helper-free double recombinant adenoviruses containing a genomic human globin gene and the neomycin resistance gene (neoR) have been constructed. The inserted globin and neoR genes are stable and transcription of two human globin genes (beta and a hybrid gamma-beta gene) is correctly initiated at the respective globin promoter during lytic infection in 293 cells. The neoR gene driven by the SV40 early promoter confers G418 resistance to human fibroblasts and K562 human erythro-leukemia cells transformed with these viruses. Most neoR clones contain the entire recombinant viral genome, including the inserted globin gene, integrated into their chromosomes. Normally, K562 cells express their gamma but not their beta globin genes. The transferred human beta globin gene was not expressed in either K562 cells or fibroblasts. However, the hybrid gamma-beta globin gene was expressed in all K562 clones that contained the gene whereas gamma-beta mRNA was barely detectable in the fibroblasts. This demonstrates tissue-specific expression of the adenovirus-transferred globin gene. Furthermore, the two transferred genes, globin and neoR, which are situated more than 20 kb apart in the viral genome appear to be independently regulated.
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PMID:Stable gene transfer and tissue-specific expression of a human globin gene using adenoviral vectors. 378 Jun 79