Molecular Biology Core

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Molecular Biology Core Project Summary Molecular biology approaches are utilized to understand the mechanisms underlying respiratory diseases caused by respiratory pathogens. The overall objective of the Molecular Biology Core (MBC) is to provide centralized services and technical expertise in molecular biology for Phase III CoBRE and non-CoBRE investigators. Under the Phase I CoBRE award, the MBC was established to provide services involving molecular biology technologies, including RNAi gene silencing, transgene expression, mutagenesis, gene and pathway reporter assays, real-time PCR, and DNA microarray analyses for gene profiling. State-of-the-art technologies and other services based on the needs of the CoBRE investigators have been continuously developed and added to the Core since its inception. In Phase II, the MBC was expanded to include new techniques, including CRISPR/Cas9 technology and bulk RNA sequencing, and to offer reagents for respiratory and infectious disease research, including cell lines, primary lung cells, and respiratory viruses and bacteria. This Phase III application describes additional cutting-edge technologies and valuable reagents that will be offered to Center investigators, including single-cell RNA sequencing, spatial gene profiling, genome- wide CRISPR screening, human lung tissue models (lung organoids) and clinical samples from respiratory infectious disease patients. The specific aims of the MBC are to provide services and support for 1) functional studies using RNAi and CRISPR/Cas9, transgene overexpression, mutagenesis and deletion, lentiviral, adenoviral and nonviral vectors for gene transfer, and stable knockout and transgenic cell lines, 2) genome- wide gene profiling using bulk RNA sequencing, single-cell RNA sequencing, and spatial gene profiling, 3) genome-wide CRISPR gene activation, knockout and interference screens, 4) molecular assays including reporter assays, real-time PCR and droplet digital PCR, 5) reagents for respiratory and infectious disease research including respiratory viruses and bacteria, and reporter viruses, cell lines, primary lung cells, human lung organoids and clinical samples from respiratory infectious disease patients, and 6) advice, consultation and training on various aspects of molecular biology, developing or adapting new methods as needed and disseminating core resources and technologies.