Santa Clara UniversityUnited States of America
Targeted nucleases have recently emerged as a powerful genome editing tool. The ability of introducing targeted, desired changes into mammalian genome makes them an invaluable tool to unravel functions of miRNAs in biology and disease. In combination with homologous donor vector, targeted nucleases can achieve high efficiency and precision, enabling bi-allelic ablation of miRNA in cultured somatic cells. Here we review the structure and function of miRNA as well as the unique implementation of genome editing technology in modifying miRNA sequences in mammalians. This chapter discusses the four mainstay genome editing technologies: meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat-associated nuclease Cas9 (CRISPR-Cas9), focusing on TALEN.
Part of the book: Modern Tools for Genetic Engineering
Genetically encoded reporter circuits have been revolutionizing our ability to monitor, manipulate, and visualize specific cellular responses to a variety of environmental stimuli. However, the development of genetic circuits that enable both high throughput (HTP) application and laboratory automation remains challenging. In this report, we describe a novel dual-reporter circuit that utilizes a secretory Gaussia luciferase (Gluc) and a green fluorescent protein (GFP) for monitoring inflammatory signaling, a fundamental process in many life events. We designed and built this genetic circuit into a simple adeno-associated viral (AAV) vector, which is suitable for both simple transfection and efficient transduction protocols. We demonstrated high sensitivity and specificity of this new circuit and its ability to monitor a broad range of inflammatory response in various human cell models. Importantly, this novel system is simple, robust, and readily adaptable to HTP applications and laboratory automation including fluorescence activated cell sorting (FACS) and microplate reader analysis. By combining both GFP and Gluc in one genetic circuit, our new dual-reporter circuit provides an easy and powerful tool for monitoring and quantifying inflammatory signals in various mammalian cells.
Part of the book: Cell Signalling