RANKL is a multifunctional cytokine that is essential for the differentiation of bone resorbing osteoclasts under physiological and pathological conditions. The central role of RANKL in pathological bone loss has led to investigation of the mechanisms that control transcription of the Tnfsf11 gene, which encodes RANKL, as well as identification of the cells that produce it. While traditional forms of mouse genetics have provided important information on these topics, the recent development of CRISPR-based tools for gene editing and transcriptional control provides an opportunity to approach these questions with increased depth and precision. Herein I describe two different studies in which we used CRISPR-based tools to provide new information on the role of RANKL in skeletal biology. In the first, we determined the role of a potential transcriptional regulatory region near the proximal promoter of the Tnfsf11 gene. To accomplish this we used CRISPR-based gene editing to delete the region from the mouse genome. We found that this maneuver had no measurable impact on Tnfsf11 gene expression, RANKL protein levels, and bone mass. In the second study, we explored the use CRISPR interference as an alternative approach for conditional gene deletion. We generated a series of transgenic mice in which expression of the Tnfsf11 gene was suppressed to different degrees. We demonstrated that this approach effectively suppressed Tnfsf11 transcription in mice and that this suppression negatively correlated with the level of transgene expression. Based on this result, we propose that CRISPR interference may constitute a more cell type-specific approach for loss-of-function studies in mice.
|Advisor:||O'Brien, Charles A|
|Commitee:||Biris, Alexandru, Bumpass, David, Rusch, Nancy, Smeltzer, Mark|
|School:||University of Arkansas for Medical Sciences|
|Department:||Interdisciplinary Biomedical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 81/5(E), Dissertation Abstracts International|
|Keywords:||Bone remodeling, CRISPR, CRISPRi, CRISPR interference, Gene regulation, RANKL|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be