Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A genomic clone for human tumor necrosis factor (TNF-alpha) was isolated using synthetic oligonucleotide probes. The genomic DNA was cleaved to remove 5' regulatory sequences and cloned in a PSVE3 expression vector containing the SV40 early promoter. The plasmid was co-transfected with a selectable dihydrofolate reductase (DHFR) gene into DHFR-deficient Chinese hamster ovary cells. Efficient expression of TNF mRNA was established by Northern analysis. Expression of TNF protein was assayed for by cytotoxic activity for cycloheximide-treated SV80 fibroblasts. Selected transfected cultures secreted as much as 50,000 units of TNF activity/ml of culture medium. Synthesis of TNF protein was confirmed by immunofluorescence of transfected cells with a monoclonal antibody to TNF and immunoprecipitation of 17 kD protein from transfected CHO culture supernates. The efficient expression of TNF from genomic DNA in transfected mammalian cells may be advantageous for biologic uses.
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PMID:Cloning of genomic DNA for tumor necrosis factor and efficient expression in CHO cells. 321 Aug 13

The efficient genetic modification of CD34+ cell-derived dendritic cells (DC) will provide a significant advancement towards the development of immunotherapy protocols for cancer, autoimmune disorders and infectious diseases. Recent reports have described the transduction of CD34+ cells via retrovirus- and lentivirus-based gene transfer vectors and subsequent differentiation into functional DC. Since there is significant apprehension regarding the clinical uses of HIV-based vectors, in this report, we compare a murine leukemia virus (MLV)- and a human immunodeficiency virus (HIV)-based bicistronic vector for gene transfer into human CD34+ cells and subsequent differentiation into mature DC. Each vector expressed both EGFP and the dominant selectable marker DHFR(L22Y) allowing for the enrichment of marked cells in the presence of the antifolate drug trimetrexate (TMTX). Both MLV-based and HIV-based vectors efficiently transduced cytokine mobilized human peripheral blood CD34+ cells. However, in vitro expansion and differentiation in the presence of GM-CSF, TNF-alpha, Flt-3L, SCF and IL-4 resulted in a reduction in the percentage of DC expressing the transgene. Selection with TMTX during differentiation increased the percentage of marked DC, resulting in up to 79% (MLV vector) and up to 94% (lentivirus-vector) transduced cells expressing EGFP without loss of DC phenotype. Thus, MLV-based vectors and in vitro selection of transduced human DC show great promise for immunotherapy protocols.
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PMID:Pre-clinical evaluation of an in vitro selection protocol for the enrichment of transduced CD34+ cell-derived human dendritic cells. 1157 83