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)

An efficient procedure for the insertion of genetic markers into a large proportion of the mouse haemopoietic system was developed, based on the in vitro expansion of retrovirally infected bone marrow and selection of the transduced cells. Bone marrow cells harvested 4 d after 5-FU treatment were incubated under IL-3/SCF stimulation and their growth dynamic, susceptibility to retroviral infection and reconstitution capacity evaluated throughout the incubation period. On the third day of culture a maximum expansion in the CFU-GM and CFU-S12 progenitor pools was observed (130- and 15-fold, respectively), with no apparent impairment in long-term repopulating precursors. This expansion was, however, accompanied by a net decrease in the CFU-GM susceptibility to the infection by supernatants containing a Moloney-derived ecotropic retroviral vector carrying the neor gene. The designed protocol thus involved the infection of freshly harvested 5-FU-treated bone marrow, followed by expansion under IL-3/SCF stimulation and selection for resistance to G418. This procedure allowed us to harvest up to 780 CFU-GM and 50 CFU-S12 per 10(5) bone marrow cells, free from non-genetically marked progenitors. Most of the animals reconstituted with the transduced marrow bore, for at least 5 months, a very high proportion of bone marrow, spleen and thymus cells tagged with the reporter gene. These results, together with the high percentage of haemopoietic precursors bearing the neor gene and expressing resistance to G418 5 months after the transplantation indicates that long-term lympho-haemopoietic repopulating cells were efficiently transduced and selected in vitro under conditions that preserve their self-renewal and differentiation properties. This gene-transfer methodology may improve the development of gene therapy protocols where the purging of non-transduced precursors would guarantee a lasting and uniform expression of exogenous genes.
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PMID:Ex vivo expansion and selection of retrovirally transduced bone marrow: an efficient methodology for gene-transfer to murine lympho-haemopoietic stem cells. 752 19

Retroviral vectors provide an efficient means to introduce genes into hematopoietic stem cells. In order to develop retroviral infection protocols which preserve the radioprotective capacity of CFU-S, we designed a clonal hematopoietic reconstitution assay. In this assay, single CFU-S-derived derived colonies from bone marrow cells of 5-FU-treated mice were tested for their capacity to prevent radiation-induced mortality. Three parameters which may modify stem cell potential were tested in infection protocols using a retroviral vector containing the gene for neomycin resistance: (1) the partition of stem cells between the adherent and nonadherent fraction; (2) the replacement of the packaging cell line by a "competent' stromal cell line; and (3) the effects of G418 selection. All CFU-S having radioprotective capacity were found in the adherent fraction when the packaging cell line or the stromal cell line (MS-5) chosen for its capacity to maintain long-term bone marrow culture were used during the co-culture. The neo resistance gene was transduced into CFU-S with the same efficiency using co-culture with the packaging cell line or co-culture with the MS-5 cell line plus viral supernatant. However, in the presence of MS-5, a much higher proportion of CFU-S (70% versus 30%) had radioprotective properties, suggesting an important role for the stromal cells in the maintenance of hematopoietic reconstituting ability. Finally, G418 selection, even for a limited period (24 h), significantly decreased the radioprotective capacities of CFU-S (56% versus 18%). Subsequently, hematopoietic reconstitution by single CFU-S was quantified in recipient mice. The progeny of CFU-S were found at a significant level in the blood, spleen and bone marrow in 38% and 15% of mice, 1 and 3 months after transplantation, respectively. These results demonstrate that we have substantially improved the infection protocol. Under these conditions of infection, it is possible to conserve CFU-S properties and to transduce a gene into a stem cell with short-term hematopoietic reconstitution potential.
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PMID:Stromal cells maintain the radioprotective capacity of CFU-S during retroviral infection. 887 23

Recent studies indicate that wild-type p53 can trigger cell apoptosis induced by many chemotherapeutic agents which induce DNA damage or cause disruptions of DNA metabolism, such as ADM, 5-FU, VP-16 and radiation. We introduced the wild-type p53 gene into a MDR cell line KBV200 in which the endogenous p53 was found to be rearranged. By G418 selection and Northern blot analysis, a G418-resistant clone named KBV200-p53 was obtained which continuously expressed the exogenous wild-type p53 mRNA. After treatment with Vincristine(VCR), the wild type p53-expression cells presented typical morphology characteristic of apoptosis analysed under electron and fluorescence microscopes. Flow cytometer analysis showed that the KBV200-p53 cells were more readily undergo apoptosis than their parental cells KBV200. After treatment with VCR 600 nmol.L-1 for 24 h, the apoptotic percentage of KBV200-p53 and KBV200 cells was about 42.4% and 8.4%, respectively. This result indicates that wild-type p53 stimulates VCR-induced apoptosis in KBV200 cells.
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PMID:[Wild-type p53 stimulates vincristine-induced apoptosis]. 1159 2

The recombinant retroviral vector pLCDSN containing E. coli cytosine deaminase gene was constructed. After packaging with PA317 cell line, the infectious particles were used to infect human colon carcinoma cell line LoVo. A single clone harbouring EC-CD gene was picked after G418 selection. There was no significant difference in cell growth curve or morphology between the LoVo/LCDSN and LoVo cells. Both of them were very sensitive to 5-FU in vitro (IC(50), approximately 0.5 &mgr;mol/L). However, the expression of the CD gene did increase the sensitivity of these cells to the nontoxic prodrug, 5-FC, decreasing the IC(50) for 5-FC from 22 000 &mgr;mol/L for parental LoVo cells to 13 &mgr;mol/L for LoVo/LCDSN cells. Obvious by side effect was also observed. When cells transduced with CD gene were mixed with wild type cells at a ratio of 30:70, above 80% of the cancer cells could be killed after treatment with a nontoxic concentration of 5-FC.
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PMID:Expression of Cytosine Deaminase Gene in Human Colon Carcinoma Cells by Recombinant Retroviral Vector. 1221 18