This Molecular Biology & Genetics Core (Core B) will provide essential, cutting edge, molecular biology and genetics expertise to facilitate the translational impact of this highly integrated PPG focused on ventilator- induced lung injury (VILI) / acute respiratory distress syndrome (ARDS). PPG Core B will be responsible for the generation of promoter luciferase reporter constructs, as well as modified promoter luciferase reporter constructs (serially deleted or point mutated). Core B will also generate expression DNA plasmids for key proteins studies in all three Projects for different over-expression systems including the E. Coli. system (for Core D to generate recombinant protein), mammalian cell system (for projects to study protein function in endothelial cells or murine lung delivery). We will also perform site-directed mutagenesis (SDM) on these expression constructs to introduce mutations representing prioritized single nucleotide polymorphisms (SNPs) or post-translational modifications (PTMs) of interest. Core B will also provide epigenetic analysis on DNA methylation and acetylation with routine molecular biology methods. As a validation step, Core B will perform DNA-protein interaction characterizations utilizing electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). In addition, Core B will provide Project investigators with a well-characterized ARDS biobank (i.e. DNA from European descent and African descent subjects), a complete list of selected SNPs for each PPG candidate gene, mid-throughput genotyping services, and data analysis tools to test the association between PPG selected SNPs and susceptibility and severity of VILI/ARDS. Thus, overall Core B will utilize these technologies to provide a molecular biology and genetics study platform for all three Projects and generate novel information on the genes targeted for investigation in this PPG. All three Projects and Core D will utilize this centralized core for the molecular biology and genetics analyses planned in this PPG.
Each of the PPG project focuses on specific ventilator-induced lung injury (VILI) /acute respiratory distress syndrome (ARDS) gene targets. Core B will serve as a centralized infrastructure to provide molecular biology and genetic expertise in evaluating the regulation of PPG target genes via genetic, epigenetic and transcriptional mechanisms.