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High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes09:44

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3’...
9.9K
Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter11:32

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single...
7.4K
In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells08:58

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5’-untranslated region (UTR) and 3’-UTR. Different translation initiation modes can also be examined using this...
15.6K
A Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-Infected Cells04:07

A Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-Infected Cells

This video demonstrates an assay to study the translation regulation in poxvirus-infected cells. Uninfected and virus-infected cells were co-transfected with mRNAs encoding for firefly luciferase (Fluc) and Renilla luciferase (Rluc), with the Fluc mRNA containing a 5' Poly(A) leader sequence. Upon adding bioluminescent substrates on lysed cells, measure the signal from both the luciferases to identify the translational advantage of the Fluc mRNA in virus-infected cells, conferred by the leader...
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siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells10:43

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Here, we describe a methodology to perform a targeted siRNA “ubiquitome” screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.  This can be adapted to any biological pathway where a robust read out of reporter activity is...
11.8K
Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response12:50

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during...
6.9K