Adiponectin (also known as 30-kDa adipocyte complement-related protein; Acrp30) is a hormone secreted by adipocytes that acts as an antidiabetic and anti-atherogenic adipokine. Levels of adiponectin in the blood are decreased under conditions of obesity, insulin resistance and type 2 diabetes. Administration of adiponectin causes glucose-lowering effects and ameliorates insulin resistance in mice. Conversely, adiponectin-deficient mice exhibit insulin resistance and diabetes. This insulin-sensitizing effect of adiponectin seems to be mediated by an increase in fatty-acid oxidation through activation of AMP kinase and PPAR-alpha. Here we report the cloning of complementary DNAs encoding adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) by expression cloning. AdipoR1 is abundantly expressed in skeletal muscle, whereas AdipoR2 is predominantly expressed in the liver. These two adiponectin receptors are predicted to contain seven transmembrane domains, but to be structurally and functionally distinct from G-protein-coupled receptors. Expression of AdipoR1/R2 or suppression of AdipoR1/R2 expression by small-interfering RNA supports our conclusion that they serve as receptors for globular and full-length adiponectin, and that they mediate increased AMP kinase and PPAR-alpha ligand activities, as well as fatty-acid oxidation and glucose uptake by adiponectin.
Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.
A potent retrovirus packaging cell line named Platinum-E (Plat-E) was generated based on the 293T cell line. Plat-E is superior to existing packaging cell lines regarding efficiency, stability and safety. The novel packaging constructs utilized in establishment of Plat-E ensure high and stable expression of viral structural proteins. Conventional packaging constructs made use of the promoter of MuLV-LTR for expression of viral structural genes gag-pol and env, while our packaging constructs utilized the EF1␣ promoter, which is 100-fold more potent than the MuLV-LTR in 293T cells in combination with the Kozak's consensus sequence upstream of the initiation codon resulting in high expression Keywords: packaging cell; retroviruses; ecotropic; EF1␣ promoter Retroviral vectors and packaging cells are important tools for gene transfer applications. Introduction of retroviral vectors containing the gene of interest into suitable packaging cells enables production of infectious retroviruses, and these particles can infect target cells and stably transmit the gene of interest into chromosomes. In conventional strategies, stable high producers of a retrovirus vector harboring a gene of interest were established by transducing the retrovirus construct into NIH3T3-based packaging cells such as PA317, 1 and 2-3 months were usually needed to acquire high producers. Pear et al 2 developed a unique packaging system by which high titer retroviruses can be obtained in 3 days by transient transfection. The expression of viral structural genes was driven by the MuLV LTR in Bosc23 cells. For transient transfection, the combination of Bosc23 cells and the pMX-neo vector 3 produced 1-3 × 10 6 /ml viruses, assessed based on the number of neomycin resistant colonies of the infected NIH3T3 cells (data not shown). Since Bosc23 cells carry the large T antigen, we attempted to increase titers of the retroviruses by introducing the SV40 origin to the pMX vector for amplification of the vector. However, this proved unfeasible (data not shown), suggesting that the limiting factor was the expression level of the viral structural proteins in the packaging cells. of virus structural proteins in Plat-E cells. To maintain the high titers of retroviruses under drug selection pressure, we inserted the IRES (internal ribosome entry site) sequence between the gene encoding gag-pol or env, and the gene encoding a selectable marker in the packaging constructs.Plat-E cells can stably produce retroviruses with an average titer of 1 × 10 7 /ml for at least 4 months. In addition, as we used only the coding sequences of viral structural genes to avoid inclusion of unnecessary retrovirus sequences in the packaging constructs, the probability of generating the replication competent retroviruses (RCR) by recombination can virtually be ruled out. Gene Therapy (2000) 7, 1063-1066. together with the plasmid encoding another selection gene GPT (guanine phosphoribosyl transferase), one after the other. Therefore, expression of selectable markers did not guarantee ...
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