Assay Optimization for a “CRISPR” Understanding of Immune Cell Migration

Neutrophils are specialized immune cells that lead the first wave of host defense during an innate immune response, sensing chemotactic signals and rapidly migrating to sites of infection or injury. Migration must be regulated by a series of signals that prevent excessive neutrophil recruitment, which can result in unresolved infections, chronic inflammation, and tissue damage. Abnormalities in neutrophil function can stem from genetic mutations that result in diminished or absent neutrophil response. Understanding genes that play a role in regulating neutrophil migration is crucial for understanding the mechanisms behind this innate immune response.

This project relies on a preliminary screening of genes of interest that have been identified to affect neutrophil migration. Our preliminary screening utilizes a PLB-985 cell line, a model for neutrophil-like cells, in which we have induced silencing mutations of various genes using CRISPR. After identifying genes of interest, knockout lines of individual gene mutations have been generated, and we must now validate their effect on neutrophil migration via a secondary screening. Neutrophil migration is assessed by inducing cell migration through a transwell assay using a chemoattractant, and we expect the silencing mutations of our genes of interest to supress or inhibit this process.

This study investigates the optimization of the transwell assays used in our screenings, which will help ensure accurate results when confirming the effects of various genetic mutations in neutrophil migration. Our optimization of transwell assays explored changes in various components of our experimental procedure, this was done with the goal of obtaining results that align with the expected neutrophil migration trends described by literature. Various factors such as coating and release methods, use of different chemoattractants, and changes in migration period were tested to enhance the reproducibility and accuracy of our screenings, ensure consistency in our migration trends, and establish a reliable system for assessing our individual knockout cell lines.

The primary goal of this project to ensure that the observed changes in neutrophil migration is impacted solely by the effects of various gene silencing mutations, and to prevent our results from being influenced by our experimental conditions. Before we proceed with screening individual knockout lines, we must continue to refine our screenings to align with the migration patterns reported in literature. Exploring candidate migration factors is critical for broadening our molecular understanding of neutrophil function, which will in turn provide a much-needed road map for understanding how and why neutrophil migration defects occur in diseases including infection, cancer, and autoimmunity