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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
1P01HL134610-01A1
Application #
9417439
Study Section
Special Emphasis Panel (ZHL1)
Program Officer
Reineck, Lora A
Project Start
Project End
Budget Start
2017-09-01
Budget End
2018-08-31
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Arizona
Department
Type
DUNS #
806345617
City
Tucson
State
AZ
Country
United States
Zip Code
85721