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)

Stable transformants of mammalian cells from gene transfer often show extreme variability in expression of the introduced transgene. This occurs from the highly variable number of copies integrated into the genome and from position effects on gene expression due to random integration. We have eliminated both of these constraints on predictable gene expression by use of a lox recombination vector. The positive selection vector system is designed to directly select Cre-mediated DNA integration at a lox target previously placed into the genome of cultured mammalian cells. Proper targeting activates expression of a defective lox-neomycin phosphotransferase (neo) fusion gene target. With CHO cell lines containing this target, almost all of the selected transformants (54 of 56 independent G418-resistant colonies) were simple single-copy integrants of the targeting DNA. To monitor gene expression at a single chromosomal site, we used a beta-actin promoter-lacZ reporter construct. Independent G418-resistant colonies from site-specific integration of the reporter gene all showed nearly identical levels of beta-galactosidase activity when the reporter construct integrated at a particular chromosomal position. The same construct integrated at a second chromosomal position exhibited a slightly different level of activity, characteristic of that second position. These results show that Cre-mediated site-specific integration can facilitate the construction of isogenic cell lines and thereby permit reproducible gene expression in stably transformed cell lines.
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PMID:Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells. 151 11

The bacterial neomycin phosphotransferase gene driven by the Moloney mouse leukemia virus long terminal repeat (LTR) or SV40 early region promoter was introduced into the human promonocyte-macrophage cell line, U937, and into the pluripotential human embryonic teratocarcinoma cell line, NT2/D1. Clonally derived cell lines capable of growing in 2-4 mg/ml of the aminoglycoside antibiotic, G418 (Geneticin), were established and transfected with pHIVCat, a plasmid expressing the bacterial chloramphenicol acetyl transferase (CAT) activity under the control of the human immunodeficiency virus (HIV-1) LTR. All of the G418 resistant (neo(r)) U937 cell lines and 10 of 14 neo(r) NT2/D1 cell lines exhibited reduced basal levels of CAT expression or impaired responses to activation of the HIV-1 LTR by phorbol 12-myristate 13-acetate (PMA) when compared to the parental lines. Other differences included inhibition of tat activation of the HIV-1 LTR and increased sensitivity of U937 cells to human tumor necrosis factor alpha. The expression of other eukaryotic promoters including the HTLV-1 LTR, SV40 ori sequences, and the human beta-actin gene promoter was similarly affected. However, differentiation of the neo(r) U937 cells into macrophages was neither delayed nor impaired. Because PMA is an activator of protein kinase C (PKC) and a potent inducer of HIV-1 directed gene expression, the amounts, sensitivity to G418, and cytosol to membrane translocation of this enzyme were determined in the wild type and neo(r) U937 cells. G418 at concentrations too low to affect cell growth (12-150 micrograms/ml) inhibited PMA-induced transactivation responses in wild type cells but did not inhibit PKC-dependent protein phosphorylation in vitro. PKC activities in the wild type and neo(r) cells were similar in absolute amounts and in the cytosol-membrane distribution of the enzyme. In contrast with wild type cells, however, all of the cytosolic Ca(2+)-phospholipid-dependent form of PKC disappeared from the neo(r) cells within 30 min after PMA induction. The results suggested that, depending upon the cell type, gene cotransfer using aminoglycoside resistance as a selectable marker may seriously perturb important cellular control mechanisms such as the PKC pathway leading to activation of gene expression.
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PMID:Gene activation mediated by protein kinase C in human macrophage and teratocarcinoma cells expressing aminoglycoside phosphotransferase activity. 166 Apr 86

We have developed a new expression vector which allows efficient selection for transfectants that express foreign genes at high levels. The vector is composed of a ubiquitously strong promoter based on the beta-actin promoter, a 69% subregion of the bovine papilloma virus genome, and a mutant neomycin phosphotransferase II-encoding gene driven by a weak promoter, which confers only marginal resistance to G418. Thus, high concentrations of G418 (approx. 800 micrograms/ml) effectively select for transfectants containing a high vector copy number (greater than 300). We tested this system by producing human interleukin-2 (IL-2) in L cells and Chinese hamster ovary (CHO) cells, and the results showed that high concentrations of G418 efficiently yielded L cell and CHO cell transfectants stably producing IL-2 at levels comparable with those previously attained using gene amplification. The vector sequences were found to have integrated into the host chromosome, and were stably maintained in the transfectants for several months.
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PMID:Efficient selection for high-expression transfectants with a novel eukaryotic vector. 166 Aug 37

Primary Schwann cells were infected in vitro with a recombinant retrovirus expressing a dominant selectable marker, neomycin phosphotransferase (conferring resistance to the drug G418), and antisense P0 RNA under the control of the human beta-actin promoter. A proportion of the G418-resistant cells failed to form myelin when cocultured with dorsal root ganglion neurons under conditions that promote Schwann cell differentiation. These cells expressed high levels of P0 antisense RNA. Among the impaired cells, the majority had segregated and ensheathed individual axon but had not differentiated further. They did not express P0 but did express myelin- associated glycoprotein and galactocerebroside. A minority of partially inhibited Schwann cells were also observed that elaborated thin myelin sheaths containing variable numbers of compacted and noncompacted lamellae. These data indicate that restricting the level of P0 expression inhibits spiralling of the Schwann cell membrane and subsequent compaction.
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PMID:Expressing antisense P0 RNA in Schwann cells perturbs myelination. 172 20

We have examined the chicken cytoskeletal actin gene for promoter activity and for the presence of cis-acting gene transcription activating sequences. Plasmids were constructed with the beta-actin promoter or other fragments of the beta-actin gene adjacent to either the truncated Herpes simplex virus (HSK)-tk promoter driving the neo gene or the enhancerless simian virus 40 (SV40) promoter driving the chloramphenicol acetyltransferase (CAT) gene. The neo plasmids were tested for frequency of transformation of mammalian cells to G418 resistance. The CAT constructions were tested for CAT expression in both transient and stable expression systems. We find that the beta-actin promoter is very strong in all of the assay systems and is as strong as any promoter we have tested. Also, we find that there are sequences in the vicinity of the beta-actin promoter which act like an enhancer sequence in activating transcription from the truncated HSV-tk promoter and the enhancerless SV40 promoter. Constructs with these actin sequences augment the transformation frequency of the neo plasmids, and stimulate the level of CAT expression from the CAT plasmids after stable chromosome insertion but not during the transient expression phase.
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PMID:Activating elements in the promoter region of the chicken beta-actin gene. 347 Feb 37

Heat shock protein 70 (HSP70) has been shown to play a fundamental role in the induction of thermotolerance in many biological systems. Elevated synthesis of HSP70 in response to diverse stresses such as heat, anoxia, ischaemia, ethanol and heavy metals has been correlated with protection against subsequent more severe stress and cross-tolerance to differing stresses. In this respect, exposure of the mammalian heart to sublethal heat treatment or ischaemia has been shown to protect against subsequent myocardial ischaemia with concomitant elevation of HSP70. However, direct demonstration that HSP70 can alone confer thermotolerance has until recently been restricted to transfection of fibroblasts with an HSP70 gene, although preliminary data from others suggests that transfection of H9c2 myocytes with an HSP70 gene can confer tolerance to substrate-free hypoxia. The purpose of this study was, therefore, to test directly whether a myocyte cell line which retains certain features of cardiac cells (as opposed to non-myocyte cells) can be protected against lethal thermal stress by transfection with a single HSP70 gene. Rat heart-derived H9c2 cells were transfected either with a plasmid from which high level expression of a human HSP70 gene is driven by a strong, heterologous (human) beta-actin promoter (APr-HS70), or with an analogous control plasmid containing no HSP70 gene (pAPr-1 neo). Following selection with the neomycin analogue G418, several clones were isolated which either expressed no HSP70 (control: pAPr-1 neo-derived) or stably expressed high constitutive levels of an inducible isoform of HSP70 (HSP70i) (APr-HS70-derived) as determined by Western blotting with a specific monoclonal antibody to HSP70i.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stable high level expression of a transfected human HSP70 gene protects a heart-derived muscle cell line against thermal stress. 808 50

Codon 248 in domain iv of the highly conserved region of the p53 gene is a frequent site of mutations associated with sporadic cancers and the familial cancer syndrome (Li-Fraumeni syndrome). Therefore, a characterization of the functional significance of a codon 248 mutation is of interest. We used antisense RNA methodology to study the role of the wild-type and mutated p53 gene in cell growth and tumorigenesis. We introduced wild-type p53 complementary DNA in sense or antisense orientation under control of a beta-actin promoter into human non-small cell lung cancer cell line H322a which has a codon 248 mutation (G to T) and WTH226b which has wild type p53. The biological properties and p53 expression of stable G418-resistant clones were analyzed. We observed that in both cell lines antisense RNA expression significantly reduced p53 mRNA and protein production; it also caused increases in growth rate in cell cultures and in tumorigenicity in nu/nu mice for both cell types, suggesting that the mechanism by which p53 suppresses cell proliferation and tumorigenesis is not always abrogated by a codon 248 mutation.
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PMID:A codon 248 p53 mutation retains tumor suppressor function as shown by enhancement of tumor growth by antisense p53. 836 31

A commonly encountered problem in orthopedics is bone and cartilage tissue injury which heals incompletely or without full structural integrity. This necessitates development of improved methods for treatment of injuries which are not amenable to treatment using current therapies. An already large and growing number of growth factors which play significant roles in bone remodeling and repair have been identified in the past few years. It is well established that bone morphogenic proteins induce the production of new bone and cartilage. An efficient method of delivery of these growth factors by conventional pharmacological means has yet to be elucidated. We wished to evaluate the use of retroviral vector-mediated gene transfer to deliver genes of therapeutic relevance for bone and cartilage repair. To determine the feasibility of using amphotropically packaged retroviral vectors to transduce primary rabbit mesenchymal stem cells of periosteal origin, primary periosteal cells were isolated from New Zealand white rabbits, transduced in vitro with a retroviral vector bearing both the nuclear localized lacZ marker gene and the neo(r) gene, and selected in G418. We used a convenient model for analysis of in vivo stability of these cells which were seeded on to polymer scaffold grafts and implanted into rabbit femoral osteochondral defects. The nuclear localized beta-galactosidase protein was expressed in essentially 100% of selected cells in vitro and was observed in the experimental explants from animals after both 4 and 8 weeks in vivo, while cells transduced with a retroviral vector bearing only the neo(r) gene in negative control explants showed no blue staining. We extended our study by delivering a gene of therapeutic relevance, human bone morphogenic protein 7 (hBMP-7), to primary periosteal cells via retroviral vector. The hBMP-7 gene was cloned from human kidney 293 cell total RNA by RT-PCR into a retroviral vector under control of the CMV enhancer/promoter. Hydroxyapatite secretion, presumably caused by overexpression of hBMP-7, was observed on the surface of the transduced and selected periosteal cells, however, this level of expression was toxic to both PA317 producer and primary periosteal cells. Subsequently, the strong CMV enhancer/promoter driving the hBMP-7 gene was replaced in the retroviral vector by a weaker enhancer/promoter from the rat beta-actin gene. Nontoxic levels of expression of hBMP-7 were confirmed at both the RNA and protein levels in PA317 producer and primary periosteal cell lines and cell supernatants. This work demonstrates the feasibility of using a gene therapy approach in attempts to promote bone and cartilage tissue repair using gene-modified periosteal cells on grafts.
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PMID:Expression of human bone morphogenic protein 7 in primary rabbit periosteal cells: potential utility in gene therapy for osteochondral repair. 1032 33

One of the underlying mechanisms of multidrug resistance (MDR) is cellular over-production of P-glycoprotein (P-gp), which acts as a drug efflux pump. P-gp is encoded by a small group of related genes termed MDR; only MDR1 is known to confer drug resistance. To overcome P-gp-mediated drug resistance, we have developed two anti-MDR1 hammerhead ribozymes driven by the beta-actin promoter. Upon transduction of the ribozymes into MDR cells, vincristine resistance was decreased. These two ribozymes were constructed, which showed different cleavage activities. In this study, to determine suitable target sites for the anti-MDR1 ribozyme, the exon 1b-intron 1 boundary, the translation-initiation site, the intron 1-exon 2 boundary and the exon 2-intron 2 boundary, codons 179 and 196 of the MDR1 gene were selected as candidates. To improve the ribozyme activity, a retroviral vector containing RNA polymerase III promoter was used. Stable retrovirus producer cells were generated by transfecting the retroviral vector plasmids carrying the ribozyme into the packaging cell line. Retroviral vector transduction of human leukemia cell lines expressing MDR1 was accomplished by co-culturing these with virus producer cells. Stably transduced cells were selected by G418 and pooled to determine the efficacy of each ribozyme. These ribozyme-transduced cells became vincristine-sensitive concomitant with the decreases in MDR1 expression, P-gp amount and drug efflux pump function. Among the ribozymes tested, the anti-MDR1 ribozyme against the translation-initiation site exhibited the strongest efficacy. This retrovirus-mediated transfer of anti-MDR1 ribozyme may be applicable to the treatment of MDR cells as a specific means to reverse resistance.
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PMID:Retrovirus-mediated transfer of anti-MDR1 hammerhead ribozymes into multidrug-resistant human leukemia cells: screening for effective target sites. 1036 43

We used DNA transfection and protein introduction techniques to investigate the pressure tolerance of cytoskeletal structures in pectoral fin cells derived from the deep-sea fish Simenchelys parasiticus (habitat depth, 366-2,630 m). The deep-sea fish cells have G418 resistance. The cell number increased until day 6 of cultivation and all cells had died by day 35 when cultured in 35-mm Petri dishes in medium containing G418. Enhanced yellow fluorescent protein-tagged human beta-actin (EYFP-actin) was stably expressed by 1 in 100,000 deep-sea fish cells. Because almost none of the EYFP-actin was incorporated into actin filaments of the cells, we replaced the relatively large EYFP tag with a chemical fluorescent compound and succeeded in incorporating fluorescently labeled rabbit actins into the deep-sea fish actin filaments. Most of the filament structure in the cells with rabbit actin inserted underwent depolymerization when subjected to pressure of 100 MPa for 20 min, in contrast to control cells. There were no differences in the tubulin filament structure between control cells and deep-sea fish cells with fluorescein-labeled bovine tubulin inserted after the application of pressure ranging from 40 to 100 MPa for 20 min.
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PMID:Piezotolerance of the cytoskeletal structure in cultured deep-sea fish cells using DNA transfection and protein introduction techniques. 1900 37


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