Scaffold Design, Function and Over-expression of Lentiviral-based microRNAs
Poster Aug 25, 2015
Angela Schoolmeesters, Melissa L. Kelley, Annaleen Vermeulen, Anja Smith, *Mayya Shveygert, *Xin Zhou, *Robert Blelloch Dharmacon, now part of GE Healthcare, 2650 Crescent Drive, Suite #100, Lafayette, CO 80026, USA
MicroRNAs are short non-coding RNAs that regulate gene expression through targeted degradation of one or more genes and have been shown to play an important role in development, differentiation and disease. For example, microRNAs are critical for stem cell function and differentiation. Also, dysregulation of microRNA expression has been associated with the development and progression of many types of cancers. To better understand the functional roles of microRNAs in phenotypes of interest, gain-of-function experiments can be performed by introducing microRNA mimics into cells, which mirror the effects of endogenous microRNAs. Studies with synthetic microRNA mimics are useful in easy-to-transfect cell types as well as instances where phenotypes are readily observed over short time periods. However, additional delivery strategies are required for cells that are refractory to transfection and for phenotypes that develop over longer time points. Here we describe the strategy for scaffold design and highlight the importance of choosing an optimal promoter in cells of interest for the over-expression of lentiviral-based microRNA mimics. We demonstrate down-regulation of gene targets in difficult-to-transfect primary, immune and neuronal cells (HuVEC, K562, SH-SY5Y), and show mesenchymal to epithelial transition markers in MDA-MB-231 breast cancer cells by over-expression of miR-429.
Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes. The majority of these are located in the intergenic or intragenic regions suggesting that the implicated variants may alter chromatin conformation. This, in turn, is likely to influence the expression of nearby or more remotely located genes to alter beta cell function. At present, however, detailed molecular and functional analyses are still lacking for most of these variants. We recently analysed one of these loci and mapped five causal variants in an islet-specific enhancer cluster within the STARD10 gene locus. Here, we aimed to understand how these causal variants influence b-cell function by alteration of the chromatin structure of enhancer clusterREAD MORE
The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are closely related transcription factors that regulate the expression of phase I (cytochrome P450s), phase II metabolizing enzymes and transporter genes in response to xenobiotics, including prescription drugs.READ MORE
Early life stress (ELS) is highly associated with development of psychopathology
and mood disorders in adulthood. Genetic studies have identified variation in the gene calcium voltage-gated channel subunit alpha1C (CACNA1C) to increase risk for several psychiatric disorders. This poster assessed the expression of Cacna1c following prepubertal stress.