Optimization and Validation of the Derivation and Use of HoxB8 Transgenic Murine Phagocytes in In Vitro Studies

Date
2022-09-16
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Abstract

The research conducted under the supervision of Dr. Robin Yates focuses on the microenvironment within phagosomes in macrophages. The research in this area is limited due to the use of bone marrow-derived macrophages that are short-lived, require the constant sacrifice of mice, and are very difficult to genetically manipulate. To overcome these shortcomings, we explored the use of another system that utilizes HoxB8 conditionally immortalized progenitor cells. The work presented in my thesis further explores the validation that conditionally immortalized phagocyte progenitors are comparable to those obtained through current methods which will ensure that this system can be used to advance research in the understudied area of phagocyte biology. To do this I finished a comprehensive tool/resource paper on HoxB8 and HoxA9-mediated immortalization. Further, I compared macrophages derived from HoxB8 conditionally immortalized progenitor cells, bone marrow-derived macrophages, and J774A.1 cells (herein referred to as J774 cells) (commonly used macrophage-like cell line) via morphology, flow cytometry, mass spectrometry-based proteomics analysis, and comparison of key phagosomal parameters (phagocytic index, phagosomal maturation (intra-phagosomal proteolysis), and phagosomal NOX2 activity). I also demonstrated the genetic mutability of macrophages derived from HoxB8 conditionally immortalized progenitor cells by assessing the transduction efficiency and generating genetically modified macrophages. Additionally, I explored a strategy where we can utilize the HoxB8 system in vivo. While the estrogen-regulated HoxB8 system works well in vitro, in vivo the endogenous estrogen interferes with the HoxB8 system, and also adding exogenous estrogen will cause physiological changes in the mice. Therefore, I explored the development of a novel HoxB8 system that uses either tamoxifen or doxycycline. Overall, the work presented in this thesis suggests macrophages derived from HoxB8 conditionally immortalized progenitor cells seem to be phenotypically and functionally equivalent to bone-marrow derived macrophages, unlike other macrophage-like cell lines (ex. J774). Taken together with previous studies, the findings reported in this body of work suggest that phagocytes derived from HoxB8 conditionally immortalized progenitor cells are more similar to primary phagocytes as compared to other immortalized phagocyte-like cell lines.

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Keywords
HoxB8, conditional immortalization, differentiation, macrophages, mass spectrometry-based proteomics, flow cytometry, phagosomes
Citation
Lail, S. (2022). Optimization and Validation of the Derivation and Use of HoxB8 Transgenic Murine Phagocytes in In Vitro Studies (Master thesis). University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca .