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Engineered Viral Vectors and Developed Tissue Clearing Methods for Single-cell Phenotyping in Whole Organs


Chan, Ken Yee (2017) Engineered Viral Vectors and Developed Tissue Clearing Methods for Single-cell Phenotyping in Whole Organs. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9NC5Z7H.


A central question in biology is how different cell types interact with each other and their native environment to form complex functional systems and networks. Although our ability to investigate this question has considerably expanded from the development of genetically encoded tools, some limitations still persist. For instance, we are limited in our ability to visualize the native three dimensional environments of whole organs. Additionally, it is challenging to efficiently deliver transgenes into difficult-to-target areas through direct-injections, such as the cardiac ganglia, or broadly distributed networks, such as the myenteric nervous system, which limits our ability to extensively study these areas. Therefore, tools and methods that overcome these limitations are needed. Towards this end, my thesis work has been focused on developing tools for single-cell resolution phenotyping in whole organs. I have been developing tissue clearing technologies to render whole organs transparent for optical interrogation and characterizing viral capsids and engineering viral vectors for noninvasive widespread gene delivery to the central and peripheral nervous system.

Tissue clearing techniques for three dimensional optical interrogation were invented over a century ago. However, these earlier methods used harsh organic chemicals and failed to retain the tissue’s native fluorescence or epitopes. These earlier methods eventually became incompatible to the hundreds of newly generated transgenic mouse lines that allowed for cell type-specific expression of fluorescent transgenes or to fluorescent labeling techniques, such as immunohistochemistry (IHC). The first part of my dissertation is aimed at addressing these limitations by further developing and standardizing a tissue clearing method that utilizes the vasculature to perfuse clearing reagents. This technique, called perfusion assisted agent release in situ (PARS) enables (i) whole organ clearing of soft tissue, (ii) preservation of native fluorescence, and (iii) preservation of epitopes compatible with IHC.

Although PARS allows us to optically investigate whole soft tissue organs, it is unsuitable for clearing bone tissue. The clearing of bone is important as it may provide optical access to delicate environments, such as the lymphatic vessels lining the dural sinuses beneath the skull that would otherwise be damaged through traditional methods. However, clearing bone tissue is challenging since it is composed of both soft (bone marrow) and hard (mineral) tissue. To overcome this challenge, I developed a clearing method that rendered intact bone tissue transparent by using EDTA to decalcify bones and by constructing a convective flow chamber to efficiently clear bones. This method, called Bone CLARITY, is able to preserve native fluorescence and epitopes. In order to demonstrate the utility of Bone CLARITY, I collaborated with colleagues to quantitatively access a rare and non-uniformly distributed population of osteoprogenitor cells in their native three dimensional environment. Bone CLARITY in conjunction with light-sheet microscope enabled the early detection of an increase to this osteoprogenitor population after administration of a novel anabolic drug, which may have been undetected with traditional techniques.

Towards my second goal, I have been working on characterizing adeno-associated viruses (AAVs) for non-invasive widespread gene delivery across the central or peripheral nervous system. Through systemic delivery, these novel AAVs are able to efficiently deliver transgenes to (i) difficult-to-target areas, such as the dorsal root ganglia; (ii) cellular populations that are widely distributed across the mouse body, such as neurons in the myenteric nervous system, and (iii) through highly selective barriers, such as the blood-brain barrier. These viruses enable rapid expression of transgenes for perturbing and monitoring cellular circuits, or for potentially treating neurological diseases. In addition, I worked on engineering or validating several different gene regulatory elements to achieve cell type restricted expression in transgenic and non-transgenic animals with AAVs. These viral vectors may prove useful in rapidly testing newly developed genetic tools. Finally, I developed and characterized two different two-component viral vector systems to control the density of labeling when systemically delivering genes with our novel engineered viruses. I utilized this two-component system to perform single-cell morphology studies in the CNS and PNS. Collectively, these capsids and vectors expand the AAV toolbox and enable efficient and versatile gene delivery into the CNS and PNS of transgenic and non-transgenic animals.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Adeno-associated viruses, tissue clearing, Bone CLARITY, gene delivery.
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Biology
Awards:Demetriades-Tsafka-Kokkalis Prize in Entrepreneurship or Related Fields, 2017. Lawrence L. and Audrey W. Ferguson Prize, 2017.
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Gradinaru, Viviana
Thesis Committee:
  • Lester, Henry A. (chair)
  • Cai, Long
  • Allman, John Morgan
  • Gradinaru, Viviana
Defense Date:18 May 2017
Additional Information:Supplemental material held in CaltechDATA.
Funding AgencyGrant Number
NIH Director's New Innovator IDP20D017782
Heritage Medical FoundationUNSPECIFIED
Curci FoundationUNSPECIFIED
Pew Charitable TrustUNSPECIFIED
Kimmel FoundationUNSPECIFIED
SPARC 1OT2OD023848-01
NIH/National Institute on Aging 1R01AG047664
NIH BRAIN 1U01NS090577
NIH/ National Institute of Mental Health 1R21MH103824
Gordon and Betty Moore FoundationGBMF2809
Caltech Programmable Molecular Technology InitiativeUNSPECIFIED
NIH U01 MH109147 02S1
NIH NS085910
Beckman Institute and Rosen Center UNSPECIFIED
Record Number:CaltechTHESIS:05302017-222932176
Persistent URL:
Related URLs:
URLURL TypeDescription material (dataset) in CaltechDATA: Visualizing endogenous fluorescence throughout a cleared mouse femur material (dataset) in CaltechDATA: Visualizing endogenous fluorescence throughout a cleared mouse tibia material (dataset) in CaltechDATA: Visualizing endogenous fluorescence throughout a cleared mouse vertebral body material (dataset) in CaltechDATA: AAV-PHP.S transduction across multiple layers of the small intestine adapted for Chapter 3 in support of Chapter 4 to work related to thesis training in support of Chapter 4 adapted for Chapter 2 to work related to thesis training to work related to thesis training
Chan, Ken Yee0000-0002-8853-5186
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10226
Deposited By: Ken Chan
Deposited On:02 Jun 2017 19:45
Last Modified:04 Oct 2019 00:16

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