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)

Cardiomyocyte transplantation could offer a new approach to replace scarred, nonfunctional myocardium in a diseased heart. Clinical application of this approach would require the ability to generate large numbers of donor cells. The purpose of this study was to develop a scalable, robust, and reproducible process to derive purified cardiomyocytes from genetically engineered embryonic stem (ES) cells. ES cells transfected with a fusion gene consisting of the alpha-cardiac myosin heavy chain (MHC) promoter driving the aminoglycoside phosphotransferase (neomycin resistance) gene were used for cardiomyocyte enrichment. The transfected cells were aggregated into embyroid bodies (EBs), inoculated into stirred suspension cultures, and differentiated for 9 days before selection of cardiomyocytes by the addition of G418 with or without retinoic acid (RA). Throughout the culture period, EB and viable cell numbers were measured. In addition, flow cytometric analysis was performed to monitor sarcomeric myosin (a marker for cardiomyocytes) and Oct-4 (a marker for undifferentiated ES cells) expression. Enrichment of cardiomyocytes was achieved in cultures treated with either G418 and retinoic acid (RA) or with G418 alone. Eighteen days after differentiation, G418-selected flasks treated with RA contained approximately twice as many cells as the nontreated flasks, as well as undetectable levels of Oct-4 expression, suggesting that RA may promote cardiac differentiation and/or survival. Immunohistological and electron microscopic analysis showed that the harvested cardiomyocytes displayed many features characteristic of native cardiomyocytes. Our results demonstrate the feasibility of large-scale production of viable, ES cell-derived cardiomyocytes for tissue engineering and/or implantation, an approach that should be transferable to other ES cell derived lineages, as well as to adult stem cells with in vitro cardiomyogenic activity.
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PMID:Scalable production of embryonic stem cell-derived cardiomyocytes. 1367 53

Recently, amniotic fluid was suggested as a new source for stem-cell research and tissue engineering approaches. In order to enable isolation of stem cells and establishment of lines of such cells with an undifferentiated phenotype we have introduced green fluorescent protein regulated by the promoters of the stem cell-specific genes, Oct-4 or Rex-1, into human amniotic fluid cells. For the introduction of DNA into human amniotic fluid cells, we have optimized a specific transfection protocol. We found that human amniotic fluid contains cell populations which are able to activate these promoters. These undifferentiated cells expressing green fluorescent protein can be analysed on a flow cytometer. In addition, we have introduced a plasmid harboring a neomycin-resistance gene under the control of the Oct-4 promoter. G418 selection allowed the isolation of undifferentiated stem cells expressing Oct-4 protein out of human amniotic fluid samples. Our findings confirm the existence of stem cells within amniotic fluid. In addition, the ability to transfect human amniotic fluid cells and to isolate stem-cell marker-positive cells will provide the means to study and manipulate these cells for the purpose of basic and applied research.
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PMID:Activation of ectopic Oct-4 and Rex-1 promoters in human amniotic fluid cells. 1627 76

The pluripotency of mouse embryonic stem (ES) cells is maintained by self-renewal. To screen for genes essential for this process, we constructed an RNA interference (RNAi) library by inserting subtracted ES cell cDNA fragments into plasmid containing two opposing cytomegalovirus promoters. ES cells were transfected with individual RNAi plasmids and levels of the pluripotency marker Oct-4 were monitored 48 hours later by real time RT-PCR. Of the first 89 RNAi plasmids characterized, 12 downregulated Oct-4 expression to less than 50% of the normal level and 7 of them upregulated Oct-4 expression to more than 150% of the normal level. To investigate their long-term effect on self-renewal, ES cells were transfected by these 19 RNAi plasmids individually and G418-resistant colonies were subjected to alkaline phosphatase (AP) staining after 7 days selection. Except for 4 plasmids that caused cell death, the ratio of AP positive colonies was repressed to less than 60% of the control group by the other 15 plasmids and even below 20% by 10 plasmids. The cDNA fragments in these 10 plasmids correspond to eight genes, including Zfp42/Rex-1, which was chosen for further functional analysis. RNAi knockdown of Zfp42 induced ES cells differentiate to endoderm and mesoderm lineages, and overexpression of Zfp42 also caused ES cells to lose the capacity of self-renewal. Our results indicate that RNAi screen is a feasible and efficient approach to identify genes involved in ES cells self-renewal. Further functional characterization of these genes will promote our understanding of the complex regulatory networks in ES cells.
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PMID:Screening for genes essential for mouse embryonic stem cell self-renewal using a subtractive RNA interference library. 1696 Jan 29

The limited life span of bovine germ line stem cells in vitro is one of the obstacles to spermatogenesis analysis, genetic manipulation and generating transgenic animal. The aim of this study is to establish immortalized bovine germ line stem cells by c-myc or hTERT. We constructed pEMY and pETE expression vectors and transfected germ line cells from 5-month-old bovine. After G418 screening, four types of positive clones were obtained. The results showed that they expressed exogenous genes c-myc or hTERT at mRNA and protein level by RT-PCR and Western blotting detection. Presumable cell lines GM7, GT3, GMT5 all expressed germ-line-stem-cell-specific makers by immunocytochemical analysis, such as c-kit, Oct-4 and GFRalpha-1. The putative cell lines also had higher capacity of proliferation than freshly isolated bovine spermatogonial stem cells. So we can conclude that exogenous genes c-myc or hTERT have integrated into the genome of bovine germ cells and upregulated the expression of telomerase.
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PMID:Immortalization of bovine germ line stem cells by c-myc and hTERT. 1715 51

Porcine pluripotent cells with the capacity to generate germ line chimeras have not been developed yet. The transcription factor Oct-4 is an important marker of undifferentiating status and a central regulator of pluripotency in cells. Establishment of an Oct-4 promoter-based reporter system, such as that used in mice, will be a useful tool for monitoring the differentiating statuses of porcine cells both in vivo and in vitro. In the present study, we constructed a vector, pOGN2, in which enhanced green fluorescent protein (EGFP) was driven by the porcine Oct-4 promoter. In pigs containing this vector, EGFP was expected to be specifically expressed in pluripotent cells. We delivered the vectors into porcine fetal fibroblasts (PEFs) using liposomes. After transfected PEFs were selected with G418, we established eight cell lines containing the pOGN2 vector. When transgenic cells were used as donor nuclei to make somatic cell nuclear transfer (SCNT) embryos, SCNT embryos derived from four transgenic cell lines expressed green fluorescence. When PEFs with pOGN2 vectors were infected with retroviral vectors encoding the four transcription factors (Oct-4, Sox2, Klf4, and c-Myc), EGFP-expressing iPS cell colonies were observed at day 20. This work lays a foundation that can be used to generate a pig strain with an Oct4-EGFP reporter system, which would be greatly helpful in studying the differentiating and reprogramming mechanisms of pig embryos.
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PMID:Establishment of a porcine Oct-4 promoter-driven EGFP reporter system for monitoring pluripotency of porcine stem cells. 2125 51