The genetic manipulation of non-model flatworms and its applications
Abstract/Contents
- Abstract
- Regeneration is a dynamic biological process that allows organisms to restore lost or injured tissues. Among flatworms, whole-body regeneration is common and has been studied extensively in the planarian Schmidtea mediterranea. However, despite interest in understanding their regenerative capacities, genetic manipulations and live imaging remain challenging. Here, I discuss advances made in the genetic manipulation and live imaging of two highly regenerative flatworm systems, Schmidtea mediterranea and Macrostomum lignano. First, I describe how we developed a novel method of transient mRNA-based expression in S. mediterranea. By leveraging nanostraws, an array of minuscule hypodermic needles, to deliver mRNA encoding nanoluciferase, an extremely bright luminescent enzyme, we observed the first instance of transgene expression in planarian cells. With a positive control for delivery, we identified additional chemical-based transfection methods that functioned both in vitro and in vivo. Using this, we identified post-transcriptional regulators of gene expression and explored sites of translation initiation using synthetic mRNAs. Finally, we constructed a specialized microscope capable of detecting the faint signals emitted by luminescing cells, enabling the quantification of transfection kinetics and permitting live imaging of animals transfected in vivo, thus paving the way for future studies dedicated towards developing longer-term DNA-based expression. Second, I introduce a comprehensive toolkit for live imaging of whole-body regeneration in the flatworm Macrostomum lignano, including a high throughput cloning pipeline, a refined injection protocol, and several advanced microscopy techniques. By driving tissue-specific reporter expression, we examine how various structures regenerate. Using a genetically encoded toxin, we ablate specific cell types to study their functions during regeneration. Through a custom luminescence/fluorescence microscope, we monitor the nervous system after cellular ablation and find it necessary for wound healing post-injury. Finally, we built a tracking microscope to continuously image freely moving animals for over a week, quantifying kinetics of wound healing, nerve cord repair, body regeneration, growth, and behavioral recovery. Notably, our findings suggest that nerve cord reconnection operates independently from primary body axis re-specification and other downstream processes of regeneration. Together, these results demonstrate how expanding the reach of our technological tools to non-model organisms can reveal new insights into their unique biology.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2024; ©2024 |
| Publication date | 2024; 2024 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Hall, Richard Nelson |
|---|---|
| Degree supervisor | Fire, Andrew Zachary |
| Degree supervisor | Wang, Bo, (Researcher in bioengineering) |
| Thesis advisor | Fire, Andrew Zachary |
| Thesis advisor | Wang, Bo, (Researcher in bioengineering) |
| Thesis advisor | Bintu, Lacramioara |
| Degree committee member | Bintu, Lacramioara |
| Associated with | Stanford University, School of Engineering |
| Associated with | Stanford University, Department of Bioengineering |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Richard Nelson Hall. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis Ph.D. Stanford University 2024. |
| Location | https://purl.stanford.edu/ky494kq2739 |
Access conditions
- Copyright
- © 2024 by Richard Nelson Hall
- License
- This work is licensed under a Creative Commons Attribution 3.0 Unported license (CC BY).
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