Luciferase Gene Expression In Cells Research Articles

Up-Scaled Synthesis and Characterization of Nonviral Gene Delivery Particles for Transient In Vitro and In Vivo Transgene Expression.

Polyethylenimine-based polyplexes are promising nonviral gene delivery systems for preclinical and clinical applications. Pipette-based polyplexing is associated with several disadvantages, such as batch-to-batch variability, restriction to smaller volumes, and variable gene delivery results. The present protocol describes syringe-pump-mediated upscaled synthesis of well-defined gene delivery nanoparticles capable of efficient in vitro and in vivo gene delivery. Syringe-pump-based synthesis ensures controlled mixing, upscaling, and reproducible gene delivery. Nanoparticle tracking analysis of the upscaled formulations involved single nanoparticle tracking, thereby generating highly resolved biophysical characterization. Gene delivery performance was investigated by luciferase gene expression in cells and three-dimensional bioluminescence imaging in mice.

MiR-15/16 complex targets p70S6 kinase1 and controls cell proliferation in MDA-MB-231 breast cancer cells

BackgroundMicroRNAs are small non-coding RNAs that regulate post-transcriptional mRNA expression by binding to 3′ untranslated region (3′-UTR) of the complementary mRNA sequence resulting in translational repression and gene silencing. They act as negative regulators of gene expression and play a pivotal role in regulating apoptosis and cell proliferation. Studies have shown that miRNAs interact with p53 by regulating the activity and function of p53 through direct repression or its regulators. Mammalian target of rapamycin (mTOR) is an evolutionary conserved check point protein kinase that plays a major effect in the control of cell division via protein synthesis regulation. mTOR regulates protein synthesis through phosphorylation and inactivation of 4E-BP1 and through phosphorylation and activation of S6 kinase 1 (S6K1). These two downstream effectors of mTOR control cell growth and metabolism. In mammals, mTOR protein kinase is the central node in the nutrient and growth factor signaling and p53 plays a critical role in sensing genotoxic stress. Activation of p53 inhibits mTOR activity, which in turn regulates its downstream targets providing a cross talk among both the signaling machinery. MicroRNA-15 and 16 belong to a common precursor family and are highly conserved. Deletion or downregulation of these two microRNAs has been shown to accelerate cell division by modulating the expression of the genes involved in controlling cell cycle progression. These microRNAs may function as tumor suppressors and act on the downstream targets of p53 signaling pathway. To have a better insight of the role of miR-15/16 in regulating the cross talk of p53 and mTOR, we performed an in depth study in MDA-MB-231 breast cancer cells by performing a gain-of-function analysis with lentiviral plasmids expressing microRNA-15 and 16. MethodsThe effect of individual microRNAs on RPS6KB1 was examined by using 3′-UTR clones via luciferase based assays. The cell cycle effects were observed by flow-cytometric analysis. Reverse transcription PCR was used to explore the expression of mTOR and RPS6KB1 in cells transfected with miR-15/16. ResultsOverexpression of miR-15/16 led to inhibition of cell proliferation causing G1 cell cycle arrest as well as caspase-3 dependent apoptosis. Forced expression of miR-15/16 might lead to decrease in mRNA level of RPS6KB1, mTOR. The effect was a complete reversal after treatment with anti-miRs against miR-15/16 proving the specificity of the expression. In addition, the dual luciferase reporter assays indicated a clear decrease in luciferase gene expression in cells transfected with lentiviral based miR-15 and 16 plasmids indicating that miR-15/16 directly targets RPS6KB1 through its 3′-UTR binding. Further, these microRNAs also inhibit epithelial to mesenchymal transition (EMT) by targeting key proteins such as Twist1 and EZH2 clearly demonstrating its crucial role in controlling cell proliferation. ConclusionThis study suggests that exogenous microRNA-15/16 can target RPS6KB1, control cell proliferation and cause apoptosis in caspase-dependent manner even in the absence of functional p53.

Role of nuclear factor-kappaB in the expression by tumor necrosis factor-alpha of the human polymeric immunoglobulin receptor (plgR) gene.

We analyzed the mechanism of human polymeric immunoglobulin receptor (pIgR) gene upregulation by tumor necrosis factor (TNF)-alpha. Northern blot analysis showed that the expression of pIgR mRNA was enhanced by TNF-alpha stimulation. This activation was completely inhibited by RNA polymerase or protein synthesis inhibitors, suggesting that the regulation of pIgR gene expression depends on de novo RNA and protein synthesis. Furthermore, the stimulation of pIgR mRNA by TNF-alpha was decreased by pyrrolidinedithiocarbamate and L-1-4'-tosylamino-phenylethyl-chloromethyl ketone, which are known nuclear factor (NF)-kappaB inhibitors. For further analysis of gene regulation, we cloned and sequenced the 1.5-kb 5'-flanking region of the pIgR gene. In the upstream region, we found two NF-kappaB-binding motifs (named kappaB1 and kappaB2 from the 5' region). An electrophoretic mobility shift assay indicated that two components of the NF-kappaB/Re1 family, p50 and p65, bound with higher affinity to the KB2 element than to the kappaB1 element. We also analyzed plgR gene expression using reporter plasmids expressing the firefly luciferase gene. Stimulation by TNF-alpha significantly activated the pIgR gene promoter, as a 775-bp upstream region of the pIgR gene increased luciferase gene expression in cells treated with TNF-alpha. The activation of promoter activity by TNF-alpha was abolished when a mutation was inserted into kappaB1 or kappaB2. These data indicated that pIgR gene expression induced by TNF-alpha is transcriptionally regulated via activation of NF-kappaB. In addition, there is a possibility that another factor may act in concert with NF-kappaB.

Up-regulation of glutamate-cysteine ligase gene expression by butylated hydroxytoluene is mediated by transcription factor AP-1.

Several regulatory elements, including AP-1 and NF-kappa B, are present in the 5'flanking region of the human glutamatex-cysteine ligase (EC 6.3.2.2, gamma-glutamyl-cysteine synthetase) catalytic subunit (GLCLC) gene. In this study, we investigated the role of redox-sensitive transcription factors in the regulation of GLCLC gene expression in LLC-PK1 cells that were exposed to the antioxidant butylated hydroxytoluene (BHT). Exposure of LLC-PK1 cells to 100 microM BHT induced expression of transcription factor AP-1, as demonstrated by an electrophoretic mobility shift assay. Peak AP-1 induction occurred after 3 h of incubation with BHT, BHT increased luciferase gene expression in cells that were transfected with a luciferase reporter vector containing an AP-1 element upstream of a SV40 promoter. Northern analysis showed that transcription of GLCLC gene in cells after incubation with BHT was increased 30% compared with control cells. Cellular glutathione concentrations were also significantly increased in cells exposed to BHT. In contrast, exposure of LLC-PK1 cells to 100 microM BHT did not alter expression of the transcription factor NF-kappa B. These results show that induction of transcription factor AP-1 by BHT is involved in transactivation of GLCLC gene expression.