Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

As a step toward using gene targeting for gene therapy, we have corrected a human beta S-globin gene to the normal beta A allele by homologous recombination in the mouse-human hybrid cell line BSM. BSM is derived from a mouse erythroleukemia cell line and carries a single human chromosome 11 with the beta S-globin allele. A beta A-globin targeting construct containing a unique oligomer and a neomycin-resistance gene was electroporated into the BSM cells, which were then placed under G418 selection. Then 126 resulting pools containing a total of approximately 29,000 G418-resistant clones were screened by PCR for the presence of a targeted recombinant: 3 positive pools were identified. A targeted clone was isolated by replating one of the positive pools into smaller pools and rescreening by PCR, followed by dilution cloning. Southern blot analysis demonstrated that the isolated clone had been targeted as planned. The correction of the beta S allele to beta A was confirmed both by allele-specific PCR and by allele-specific antibodies. Expression studies comparing the uninduced and induced RNA levels in unmodified BSM cells and in the targeted clone showed no significant alteration in the ability of the targeted clone to undergo induction, despite the potentially disrupting presence of a transcriptionally active neomycin gene 5' to the human beta A-globin gene. Thus gene targeting can correct a beta S allele to beta A, and the use of a selectable helper gene need not significantly interfere with the induction of the corrected gene.
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PMID:Correction of a human beta S-globin gene by gene targeting. 203 73

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

We investigated the effect of the v-erbA oncogene product, an altered thyroid hormone receptor, in chicken erythrocyte progenitor cells. Bone marrow cells were infected with a retrovirus vector (XJ12) carrying the v-erbA gene in association with the neoR gene. XJ12-infected erythrocyte progenitor cells gave rise to G418-resistant clones. Some were composed of blast cells identified as transformed CFU-Es blocked in their differentiation. These cells could be grown in culture for at least 25 generations and required anemic chicken serum as a source of erythropoietic growth factors. XJ12 can infect erythrocyte progenitor cells in vivo but is not sufficient to induce erythroleukemia. These data suggest that the activation of a nuclear hormone receptor might represent one step toward the development of neoplasms.
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PMID:Expression of the v-erbA product, an altered nuclear hormone receptor, is sufficient to transform erythrocytic cells in vitro. 256 87

Conditions were developed for stable introduction of foreign DNA into human lymphoid cell lines by electroporation. To introduce stably the p40 gene of human T-cell leukemia virus type I (HTLV-I) into the human lymphoid cell line Jurkat, the p40 expressing plasmid, pMAXRHneo-1, which carries the neo resistant gene, was transfected into Jurkat cells at a voltage of 2500 V and capacitance of 21.7 microF, and stable transformants were screened for neo (G418) resistance. The frequency of transformants was more than one per 2 x 10(5) cells used initially. Clones that were resistant to G418 were shown to have the p40 gene integrated into the host genome and to express mRNA and protein from the introduced plasmid. Expression of p40 in the transformed Jurkat cells was also confirmed by testing the trans-activating effect of HTLV-I enhancer by p40. High frequencies of stable transformations of 10(-4) to 10(-6) were also reproducibly obtained by electroporation of the human T cell lines HSB-2 and TALL-1, a human B cell line Raji, a human monocytic cell line U937, and a human erythroleukemia cell line K562. These results demonstrate that electroporation is a very efficient method for introducing foreign DNA into human lymphoid cell lines.
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PMID:Electroporation: application to human lymphoid cell lines for stable introduction of a transactivator gene of human T-cell leukemia virus type I. 278 6

Over the past five years, several new defects in the beta-thalassemias have been described from this laboratory using both restriction enzyme and sequencing analyses of cloned beta-thalassemia genes. The enzyme HphI has been shown to recognize a single nucleotide change at the 5' end of beta-IVS 2, and, using restriction enzyme analysis, demonstrated for the first time a specific defect associated with beta(0)-thalassemia. Cloning and sequencing of a beta-thalassemia gene have identified a single base change within IVS 2 at a position 705 nucleotides from the 5' end of IVS 2 that results in a beta(0)-thalassemia phenotype; no normal splicing occurs in this gene despite the fact that both the 5' and 3' ends of IVS 2 are unchanged. A unique and strong cryptic 3' acceptor splice site present in the normal gene at a position 580 nucleotides from the 5' end is used extensively in the mutant gene. Studies of this gene have indicated that there are sequences within IVS that are responsible for optimal expression of this gene; changes in these sequences can lead to markedly abnormal patterns of splicing. In addition, beta-globin gene expression has been evaluated in human erythroleukemia cells, K562 cells, and, although stable transformants with integrated beta-globin genes have been obtained, none of these transformants expressed the added beta-globin genes. This is presumably due to trans-acting factors or distal cis-acting effects that suppress the expression of these added beta-globin genes. In addition, a low epsilon-producing cell line, Bos cells, was used as a recipient for an exogenous epsilon-globin gene. A neomycin resistance gene was cotransfected into these cells, and a neomycin analogue (G418) was used to select cells containing both the neomycin resistance and epsilon-globin genes. Using Southern blotting, 10 of 11 stably transformed G418-resistant lines, which contain intact epsilon-globin genes, express epsilon-globin mRNA at much higher levels than the Bos cells into which they were transfected. Two of these lines express the epsilon-globin genes at a level comparable to that of wild-type K562 cells. These results indicate that the transfer and expression of human globin genes in human erythroid cells is feasible, and can occur at a high level.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Abnormal globin gene structure and expression in beta-thalassemia. 299 Feb 98

Electroporation has recently been shown to have advantages over the commonly used method of calcium phosphate precipitation for obtaining DNA-mediated transformation of certain types of cells. Although mouse erythroleukemia cells and other cells of hematopoietic origin are not transformed at useful frequencies by calcium phosphate-DNA precipitation methods, we obtained high frequencies of transformation (approximately 10(-5)) of these cells with electroporation. Even higher transformation frequencies (approximately 10(-3)) were obtained with human fibroblasts. Another advantage of electroporation was found when analysis of Southern blots of DNA from 243 transformed erythroleukemia cell colonies indicated that, under appropriate conditions, about 79% of the transformed cells had the exogenous DNA integrated in single copies at single sites. Under conditions of higher DNA and lower cell concentrations using fibroblasts, cotransformation was obtained with two plasmids that confer HAT or G418 resistance when integrated into cellular DNA. About 23% of the transformed cells developed both types of resistance. We describe a simple, inexpensive apparatus for carrying out electroporation.
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PMID:Efficient transformation and frequent single-site, single-copy insertion of DNA can be obtained in mouse erythroleukemia cells transformed by electroporation. 346 50

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

The contribution of the human globin gene promoters to tissue-specific transcription was studied by using globin promoters to transcribe the neo (G418 resistance) gene. After transfection into different cell types, neo gene expression was assayed by scoring colony formation in the presence of G418. In K562 human erythroleukemia cells, which express fetal and embryonic globin genes but not the adult beta-globin gene, the neo gene was expressed strongly from a fetal gamma- or embryonic zeta-globin gene promoter but only weakly from the beta promoter. In murine erythroleukemia cells which express the endogenous mouse beta genes, the neo gene was strongly expressed from both beta and gamma promoters. In two nonerythroid cell lines, human HeLa cells and mouse 3T3 fibroblasts, the globin gene promoters did not allow neo gene expression. Globin-neo genes were integrated in the erythroleukemia cell genomes mostly as a single copy per cell and were transcribed from the appropriate globin gene cap site. We conclude that globin gene promoter sequences extending from -373 to +48 base pairs (bp) (relative to the cap site) for the beta gene, -385 to +34 bp for the gamma gene, and -555 to +38 bp for the zeta gene are sufficient for tissue-specific and perhaps developmentally specific transcription.
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PMID:Human globin gene promoter sequences are sufficient for specific expression of a hybrid gene transfected into tissue culture cells. 356 96

Gene therapy approaches for beta-thalassemia and sickle cell anemia focus on the transfer of a human beta-globin gene into the patient's hematopoietic stem cells (HSC). Expression of the transferred sequences should be erythroid specific and match the expression of the endogenous alpha-globin genes in adult erythropoiesis. Here we explore the potential of recombinant adeno-associated virus (AAV) vectors for human beta-globin gene transfer. We have constructed a recombinant AAV-vector containing a human beta-globin gene together with the DNasel hypersensitive sites 4, 3 and 2 of the human beta-globin locus control region. The vector replicates to high titers and can efficiently transduce hematopoietic and non-hematopoietic cells. In transduced and G418 selected murine erythroleukemia (MEL) cell clones, human beta-globin gene expression was regulated and reached levels comparable to endogenous murine beta maj. These data show that AAV-vectors are promising tools in gene therapy approaches for the haemoglobinopathies.
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PMID:Regulated high-level human beta-globin gene expression in erythroid cells following recombinant adeno-associated virus-mediated gene transfer. 767 Nov 9

We have studied effects of ferric transferrin (FeTF), ferric lactoferrin (FeLF), ferric complexes of pyridoxal- or salicylaldehyde-isonicotinoyl hydrazone, (Fe-PIH, Fe-SIH), and ferric ammonium citrate (FAC) on expression of protein kinase C (PKC) mRNA transcripts in a variety of cultured cell lines. FeTF supported an increase of PKC-beta mRNA transcripts in T-lymphoblastoid (CCRF-CEM; Jurkat), B-lymphoblastoid (Daudi; Raji), promyelocyte (HL-60), erythroleukemia (K562), and monocyte (U937) cell lines. By contrast, FeLF, Fe-PIH, and Fe-SIH did not support an increase of PKC-beta mRNA transcripts in any of these cell lines. Furthermore, FAC supported an increase of PKC-beta mRNA transcripts in HL-60, K562, and U937 cells only. Preincubation of cells with desferrioxamine (DF), a cell-permeable iron chelator, abolished the increments of PKC-beta mRNA observed in response to FeTF or FAC. In contrast to results with PKC-beta, neither FeTF nor FAC caused an increase of PKC-alpha transcripts in any cell line. To locate iron-responsive DNA regulatory elements of the PKC-beta gene, we prepared genetic constructs containing various portions of the human PKC-beta 5'-flanking DNA linked to the firefly luciferase gene. Constructs were cotransfected with the neomycin resistance plasmid, Pwl-neo, into HRE H9 cells, and stable transfectants were selected in G418. Treatment with FeTF of transfectants bearing chimeric gene constructs with 2,200 bp of the PKC-beta 5'-flanking region increased luciferase activity and mRNA transcripts 2.5-fold. This increase was blocked by DF. Neither luciferase activity nor mRNA increased with FeTF in stable transfectants bearing constructs with 342 bp or 587 bp of the PKC-beta 5'-flanking region. These data provide direct confirmation that iron is involved in regulation of PKC-beta but not PKC-alpha gene expression in many cell lines. The form in which iron is presented to these cell lines appears to affect its availability for this function, and cells vary in their capabilities to use nontransferrin iron to support PKC-beta gene expression. Finally, transcriptional upregulation of PKC-beta by FeTF is mediated by DNA sequences located between -2200 bp and -587 bp in the 5'-flanking region of the human PKC-beta gene.
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PMID:Regulation of protein kinase C (PKC) expression by iron: effect of different iron compounds on PKC-beta and PKC-alpha gene expression and role of the 5'-flanking region of the PKC-beta gene in the response to ferric transferrin. 794 5


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