University of Southern California Dissertations and Theses

Visualization and characterization of rat lymphatic vasculature in a Prox1-EGFP bacterial artificial chromosome (BAC) transgenic rat: a novel animal model for physiological and pathological lymph...

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Visualization and characterization of rat lymphatic vasculature in a Prox1-EGFP bacterial artificial chromosome (BAC) transgenic rat: a novel animal model for physiological and pathological lymph...

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Content VISUALIZATION AND CHARACTERIZATION OF RAT
LYMPHATIC VASCULATURE IN A PROX1-EGFP
BACTERIAL ARTIFICIAL CHROMOSOME (BAC)
TRANSGENIC RAT: A NOVEL ANIMAL MODEL FOR
PHYSIOLOGICAL AND PATHOLOGICAL
LYMPHANGIOGENESIS

by
Eunson Jung

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
August 2015

Copyright 2015 Eunson Jung
1

DEDICATION

To my parents

2

ACKNOWLEDGEMENTS
No words can express the amount of appreciation I have towards the numerous people that have
motivated and supported me throughout my Master’s years. This page does not hold enough space to
enumerate the names and details of their contribution that made me grow as a person and a scientist.
First and foremost, to my principal investigator/boss, Dr. Young Kwon Hong; without him, this rat
could not have been imported from China alive. It has been a hectic but very fulfilling two years, and
I thank the wonderful opportunity of having met my life-long mentor.
To Dr. Dongwon Choi and Eunkyung Park, the greatest researcher team to exist on earth. My utmost
respect goes out to their guidance through each and every step of any experimental techniques I have
laid hands on, which probably have shortened the completion of this thesis by at least five years.
To Dr. Sunju Lee, the ‘mother’ and the secret authority of our lab. She has managed to keep our lab
‘clean’ and organized, even with our last-minute need of additional supplies. Due to her, the entire
lab members are relatively healthy, and very much alive.
To my semi-family Sara Yang, Young Jin Seong, and Min Gu Hong; the greatest friends and co-
workers that have kept me sane and very much entertained throughout the hardest times.
To my actual family Wonhyeuk Jung, who has managed to keep his sanity throughout his first year
as a graduate student, and our love/hate relationship continues to improve my patience in life every
single day.
Last, but definitely not the least, immense amount of gratitude goes towards my committee members,
Dr. Young Kwon Hong, Dr. Zoltan Tokes, and Dr. Agnieszka Kobielak. Thank you for sparing the
valued time from your very busy schedule to guide and support my thesis.
3

TABLE OF CONTENTS

DEDICATION……………………………………………………………….……………………1
ACKNOWLEDGEMENTS……………………………………………………………………….2
TABLE OF CONTENTS………………………………………………………………………….3
LIST OF FIGURES……………………………………………………………………………….4
ABSTRACT……………………………………………………………………………………….5
CHAPTER 1 INTRODUCTION………………………………………….………………………6
CHAPTER 2 MATERIALS AND METHODS……………………………………………..…..14
CHAPTER 3 RESULTS…………………………………………………………………………16
CHAPTER 4 DISCUSSION………………………………………………………………..……23
REFERENCES…………………………………………………………………………………..25

4

LIST OF FIGURES

Figure 1. Development of the Mammalian Lymphatic Vasculature……………..……………….7
Figure 2. Schematic View of Blood and Lymphatic Circulation…………………………………9
Figure 3. Positive Correlation Between Interstitial Pressure and Lymph Flow……………...….10
Figure 4. Tumor Metastasis via the Remodeling of the Lymphatic System………………….…11
Figure 5. Generation of the Mouse Prox1-EGFP Bacterial Artificial Chromosome (BAC)
Construct…………………………………………………………………………………………14
Figure 6. Genotyping of EProx1-GFP Rat………………………………………………………16
Figure 7. GFP Expression in Prox1-EGFP Rat Eyes…………………………………………....17
Figure 8. GFP Expression in 5 weeks Old Prox1-EGFP Rat……………………………………18
Figure 9. GFP Expression in 2-3 Weeks Old Prox1-EGFP Rat Lymph Node……………….…19
Figure 10. GFP Expression in 6-7 Days Old Prox1-EGFP Rat…………………………………20
Figure 11. GFP Expression Colocalized with the Lymphatic Marker LYVE-1……………...…21
Figure 12. Prox1-GFP expression in lymph nodes of groin flap …………………………….…22

5

ABSTRACT

In the human body there are two fundamental circulatory networks, the blood and the
lymphatic system, which are anatomically similar and complementary in function. Despite its
significant role in drainage of excess interstitial fluid, transport of dietary lipids and execution of
precise immune responses, the lymphatic vasculature was considered subordinate to the blood
vasculature for many decades (Oliver 2004). Recent progresses in lymphatic research, such as
the discovery of lymphatic-specific molecular markers (Banerji, Ni et al. 1999), lineage tracing
of Prox1-expressing lymphatics (Srinivasan, Dillard et al. 2007), advancement of imaging
techniques (Yaniv, Isogai et al. 2006), and development of various lymphatic-specific reporter
mice have shown how the lymphatic vasculature plays an important role in the maintenance and
enhancement of human health (Choi, Lee et al. 2012; Yang and Oliver 2014). Moreover,
dysfunction in the lymphatic system can lead to multiple ailments such as lymphedema, obesity,
chronic inflammation, autoimmunity, and metastasis, emphasizing a good mammalian lymphatic
model to be essential in developing therapeutic drugs (Oliver 2004; Randolph, Angeli et al. 2005;
Wang and Oliver 2010; Alitalo 2011; Choi, Lee et al. 2012; Weitman, Aschen et al. 2013;
Stacker, Williams et al. 2014). Here, we report a bacterial artificial chromosome (BAC)-based
transgenic rat that expresses GFP under the control of a Prox1 promoter. The GFP expression of
the lymphatic vasculature was comprehensively investigated in various tissues and organs,
demonstrating that the lymphatic vasculatures in the rats were visualized to even minute details.
Our Prox1-EGFP rat implies enormous potential to be a powerful experimental tool for the better
study of the lymphatic vasculature, both in developmental and pathological aspect.
6

CHAPTER 1
INTRODUCTION

In the human body there are two fundamental circulatory networks, the blood and the
lymphatic system, that are indispensable to the functionality of nearly every tissue and organ.
The two distinctive vasculatures resemble each other anatomically, and are complementary in
function. However, despite their similarity, the blood vasculature has been an area of vigorous
research while the lymphatic system has been deemed as subordinate to the blood system.
Correspondingly, the lymphatic system was disregarded the attention it deserves, with a
significantly less amount of scientific and medical research done in comparison to the blood
vasculature. In 1902, Florence Sabin investigated the development of lymphatic vasculature by
injecting dye into the pig embryos. She was the pioneer of lymphatic research, being the first one
to propose that the lymphatic system forms from embryonic cardinal veins (Sabin 1902). After
more than 100 years of debate, the discovery of lymphatic-specific molecular markers such as
lymphatic vessel endothelial hyaluronan receptor (LYVE)-1 (Banerji, Ni et al. 1999), lineage
tracing of prospero homeobox 1 (PROX1)-expressing lymphatics (Srinivasan, Dillard et al. 2007)
and advancement of imaging techniques (Yaniv, Isogai et al. 2006), has confirmed the origin of
lymphatic vessels as blood vessels. These recent progresses in lymphatic research have provided
the tools to prove to the scientific community that the lymphatic system deserves much more
credit for the maintenance and enhancement of human health (Choi, Lee et al. 2012; Yang and
Oliver 2014).

7

In mammals, the lymphatic vasculature is derived from the venous veins directed by the
sequential expression of three crucial transcription factors, COUP-transcription factor (TF) II,
Sox18 and Prox1. Initially, mouse embryonic veins at ~E8.5 are regulated by COUP-TFII that
facilitates venous endothelial cell fate and lymphatic endothelial cell (LEC) specification. At
~E9.0 LEC competence factor Sox18 is expressed in a subpopulation of venous endothelial cell
destined for LEC fate. Previous studies have presented that the cooperative expression of these

Figure 1. Development of the Mammalian Lymphatic Vasculature Embryonic veins that express
COUP-TFII become competent for lymphatic differentiation at embryonic day 9.0 with cooperative
expression of Sox18. Following the expression of the lymphatic master regulator Prox1, a portion of the
venous endothelial cell (EC) becomes committed to become lymphatic endothelial cells (LECs). These
cells bud off to form lymph sacs via the aid of VEGF-C/VEGFR-3 signaling, and eventually sprout and
mature into complete lymphatic vasculature (Oliver and Srinivasan 2010).

8

two genes, COUP-TFII and Sox18, is sufficient to induce the homeodomain transcription factor
Prox1 expression (Srinivasan, Geng et al. 2010), the master regulator of lymphatic vasculature,
at a subpopulation of cells at ~E9.75. This subpopulation is specified to suppress the blood
vasculature endothelial cell (BEC) phenotype and differentiate into LECs. This subset of
endothelial cells determined for LEC fate migrate away from the embryonic veins with the aid of
lymphatic-specific receptors such as the vascular endothelial growth factor receptor 3 (VEGFR3)
following the chemotaxis guidance of VEGF-C, to form primitive lymph sac. As LECs
differentiate and mature, LECs sprout to form the extensive lymphatic network that spreads
throughout the whole body (Figure 1) (Oliver and Srinivasan 2010; Bautch and Caron 2015).

The blood vasculature is a closed, circular system where the blood leaves the heart, flows
through the entire body via the arteries, capillaries, and veins, and returns to the heart in
repetitive cycles. On the contrary, the lymphatic vasculature is composed of a blind-ended linear
system and secondary lymphoid organs including the lymph nodes, tonsils, Peyer’s patches,
spleen and thymus. Protein-rich interstitial fluids from tissues and organs, collectively called
lymph, are drained into larger collecting ducts that are nested within lymph nodes, and returns to
the blood circulation via the thoracic duct (Figure 2) (Oliver 2004; Choi, Lee et al. 2012). There
are three foremost functions of the lymphatic system: drainage of excess interstitial fluid,
transport of dietary lipids and execution of precise immune responses (Oliver 2004). In
concordance, both the blood and the lymphatic system have to balance their roles as barriers that
block unnecessary or detrimental flow of materials across the vasculature wall, and as crossroads
that exchange required macromolecules, cells, and fluid (Figure 3) (Bautch and Caron 2015).
9

A dysfunction in the lymphatic system can lead to multiple ailments. Most commonly, a
defective lymphatic vasculature due to genetic inheritance (primary) or damage to the lymphatic
vessels by surgery or radiation (secondary) leads to lymphedema, a condition of excessive fluid
accumulation that result in tissue swelling (Choi, Lee et al. 2012). Approximately 20-30% of
breast cancer patients that undergo radical axillary lymph node dissection suffer from
discomforting to severe lymphedema on the same side of the arms where their lymph nodes were
removed (Alitalo 2011). Furthermore, recent studies have even indicated that a malfunctioning
lymphatic system can be correlated with the onset of obesity due to the increase in fat disposition.
Reversely, obesity has been shown to inhibit smooth lymph flow, which leads to the increase in

Figure 2. Schematic View of Blood and Lymphatic Circulation While the blood vasculature
is a closed, circular system, where blood flows from the heart to the arteries, veins, and then
back to the heart, lymphatic vasculature is an open system where lymph is drained from the
interstitial space, and transported to the blood circulation.

10

susceptibility to breast cancer-related lymphedema development (Wang and Oliver 2010;
Weitman, Aschen et al. 2013). In addition, an intact and stable lymphatic system is essential for
the proper functioning of our immune system. When encountering foreign antigens that invade
the human body, dendritic cells uptake the antigens, travel through the afferent lymphatic vessels
to lymph nodes, and present the antigens to residing immune cells. The immune cells suitable for
the eradication of the foreign invaders massively proliferate, which results in our body
undergoing an inflammatory response, an active interaction between the microbes and our
defensive immune cells. If smooth lymph flow is inhibited, it will reduce the efficacy of our
immune cell response, increasing the possibility of chronic infection (Oliver 2004; Randolph,
Angeli et al. 2005).

Figure 3. Positive Correlation Between Interstitial Pressure and Lymph Flow With low
interstitial pressure, there is limited lymph flow into the lymphatic vessels. In the case of high
interstitial pressure, there is a smooth lymph flow that induces the intake of macromolecules,
fluid and cells into the lymphatic vessels.

11

The major source of death in cancer patients is the metastasis of their tumor cells into
distal sites of the body via the lymphatic and blood vessels. Poor prognosis of cancer patients is
often based on the existence of tumor cells in regional or sentinel lymph nodes (SLNs). This
indicates how the lymphatic system is one of the primary devices for tumorigenesis and tumor
metastasis. Amid the tumor growth, many cancer cells produce growth factors that stimulate
lymphangiogenesis. The lymphatic vessels adjacent to the primary tumor site undergo active
remodeling; main occurrences being lymphangiogenesis, lymphatic enlargement, and
enlargement of the collecting lymphatic. Lymphatic remodeling greatly facilitates the invasion of
tumor cells into the lymphatic vasculature, which eventually results in dissemination of these
tumor cells into distal sites, and metastasis as its outcome. Over the years, the role of the
lymphatic system in tumor metastasis has been more and more acknowledged. Accordingly, the

Figure 4. Tumor Metastasis via the Remodeling of the Lymphatic System Lymphatic vasculature
nearby the primary tumor site undergoes remodeling such as lymphangiogenesis and lymphatic
enlargement. These proximal lymphatic vessels promote the spread of tumor cells into distal sites,
resulting in metastasis (Stacker, Williams et al. 2014).

12

scientific community is actively searching for a therapeutic tactic to block the lymphatic growth
proximate to the primary tumor site for metastasis inhibition and better prognosis of cancer
patients (Figure 3) (Stacker, Williams et al. 2014).
Attributable to flourishing research of lymphangiogenesis, several transgenic mice were
derived to fluorescently visualize the lymphatic vasculature. Gene-targeted BAC transgenic
constructs that express fluorescent protein include Lyve-1 EGFP-hCre knock-in mice crossed
with Rosa26-YFP mice (Pham, Baluk et al. 2010), Prox1-mOrange2 (Hagerling, Pollmann et al.
2011), and Prox1-tDTomato (Truman, Bentley et al. 2012). Additionally, a Vegfr3 promoter-
driven dual reporter for fluorescence and luminescence (EGFPLuc) has been reported in 2012
(Martinez-Corral, Olmeda et al. 2012). Despite the increasing variety of lymphatic reporter mice,
a lymphatic-specific fluorescent reporter rat has yet been established. Previously, our lab has
reported a bacterial artificial chromosome (BAC)-based transgenic mice that expresses GFP
under the control of Prox1 promoter. In these mice, GFP is fluorescently active only in lymphatic
endothelial cells and not blood endothelial cells, which conveniently distinguishes the complete
lymphatic vasculature from the blood vasculature (Choi, Lee et al. 2012). However, despite the
convenient availability and manipulation of mice, their small size results in experimental and
surgical limitations. Technological advancement has made researchers re-visit the efficacy of rat
models due to their significant advantage for examining physiological changes in both macro and
microscopic perspective (Iannaccone and Jacob 2009). Owing to their large body size, rats allow
easier and less invasive surgical procedures, and due to their closer genomic relationship to
humans, they also permit a more accurate clinical implication. Here we report a BAC-based
transgenic rat that expresses GFP under Prox1 promoter. The GFP expression of the lymphatic
vasculature was comprehensively investigated in various tissues and organs, showing that the
13

lymphatic vasculature is visualized even in minute details. Our Prox1-EGFP rat implies
enormous potential to be a powerful experimental tool for better study of lymphatic vasculature,
both in developmental and pathological aspect.

14

CHAPTER 2
MATERIALS AND METHODS
Prox1-EGFP BAC transgenic rat
All experimental protocols were reviewed and approved by the University of Southern
California Institutional Animal Care and Use Committee.

Figure 5. Generation of the Mouse Prox1-EGFP Bacterial Artifical Chromosome (BAC)
Construct
Mouse Prox1 gene ~540bp in length was PCR-amplified and cloned into the targeting vector
containing EGFP. Prox1-EGFP targeting vector was co-transformed into bacteria with a
15

recombination vector containing tetracyclin resistance for homologous recombination. Bacterial
colonies that were resistant to ampicillin and chloramphenicol, and sensitive to tetracycline were
selected, isolating successful BAC. The Prox1-EGFP BAC construction was injected via
pronuclear injection into female recipient rat. (Choi, Chung et al. 2011) This diagram was
modified from Gong et al (Gong, Zheng et al. 2003).

Whole Mount Staining
Rats were sacrificed and small pieces of their tails were harvested. The hair was removed with
Sally Hansen Simply Smooth hair remover crème and fixed in 4% paraformaldehyde (PFA) in
phosphate buffered saline (PBS) overnight at 4 ℃. Tissues were embedded in Tissue-Tek O.C.T.
Compound and sectioned in 6 micron thickness. Cryosectioned tissues were permeabilized in 0.5%
Triton X-100 in PBS for 20 minutes in room temperature (RT), blocked with bovine serum
albumin (BSA)/goat serum in 0.5% Triton-X100 for 1 hour in RT, and incubated in primary
LYVE-1 Ab (Angiobio Company) O/N at 4 ℃. After secondary antibody incubation at room
temperature for 1 hour, the tissues were mounted with DAPI. Images were acquired using a
fluorescent stereomicroscope (Leica).

Groin Flap
Rat was euthanized and the groin flap was surgically removed from its hind leg. The flap was
visualized under a fluorescence stereoscope (Zeiss).

16

CHAPTER 3
RESULTS

Figure 6. Genotyping of EProx1-GFP Rat
Prox1-EGFP transgenic rat was genotyped with PCR. Lane 1 Prox1-EGFP mouse, lane 2 non-
transgenic negative control; lane 3-5 Prox1-EGFP negative pup, lane 6-8 Prox1-EGFP positive
pup; lane 9 water.
17

Figure 7. GFP Expression in Prox1-EGFP Rat Eyes
(A) On the left is Prox1-EGFP mouse, right is Prox1-EGFP rat. (B) GFP expression in the eyes
of Prox1-EGFP mouse and rat. (C) Approximately 4-5 week old Prox1-EGFP rat eyes dissected
into clover-leaf shape. GFP is expressed in Schlemm’s canal and limbal lymphatics. (D) Close-
up view of GFP expression in Schlemm’s canal and limbal lymphatics.
Evidently, Prox1-EGFP rat has a much larger body size compared to a Prox1-EGFP mouse, with
a fully grown adult mouse the size of a fully grown rat’s head. Prox1-EGFP rat can be
conveniently distinguished from wild-type rat under UV light due to the expression of Prox1 in
its eyes making their eyes shine a vivid green light. Whole mount of a Prox1-EGFP rat cornea
18

dissected into a clover-leaf shape clearly visualized the vasculature of Schelmm’s canal and its
surrounding limbal lymphatic in the circular edges (Figure 7).

Fig 8. GFP Expression in 5 weeks Old Prox1-EGFP Rat
Sprouting of lymphatic vessels visualized in the (A) whole diaphragm of a 5 weeks old Prox1-
GFP rat, and the (B-C) enlarged diaphragm. (D-E) The bladder and (F) trachea also displayed
intricate lymphatic vasculature that surrounds the whole tissue.
In concurrence with the visualization of the lymphatic vasculature in the eye, 5 weeks old adult
Prox1-EGFP rat organs showed intricate and highly dispersed lymphatic vessels in the
diaphragm, bladder and the trachea, with the lymph valves having a brighter GFP expression
(Figure 8).
19

Figure 9. GFP Expression in 2-3 Weeks Old Prox1-EGFP Rat Lymph Node
In 2-3 weeks old Prox1-EGFP rat, the entire lymph node expressed GFP, with the lymphatic
vessels retaining a stronger and more defined GFP expression. The ends of the lymphatic vessels
in the lymph nodes showed active lymphangiogenesis with thinner, more branched lymphatic
vessels sprouting at the tips (Figure 9).
20

Fig 10. GFP Expression in 6-7 Days Old Prox1-EGFP Rat
GFP expression in Prox1-GFP pup (A) back skin, (B) ear, (C) mesentery, (D-F) diaphragm, (G-
H) heart, (I) kidney, (J) bladder, (K) testes, and (L) liver.
The GFP signals of the lymphatic vasculature were even stronger and more specified in the
organs of a young pup. Prox1-EGFP rats 6 to 7 days old showed elaborate and highly
21

disseminated lymphangiogenesis in the skin region shown in the back skin and the ear. The
intestine showed thick and highly branched lymphatic vessels in the mesentery, and very thin,
numerous lymphatic vessels surrounding the colon. Although Prox1 is also expressed in muscles,
the lymphatic vasculature could be distinguished from the diaphragm muscle and the heart
muscle due to the higher expression of GFP, with bright, discernible lymph valves. While the
kidney, bladder, and testes all showed tissue-specific lymphatic vasculature, GFP was expressed
entirely in the liver. (Figure 10)

Figure 11. GFP Expression Colocalized with the Lymphatic Marker LYVE-1
(A) GFP expression in the Prox1-EGFP rat tail, (B) whole mount staining with LYVE-1 antibody
in red, and (C) merged image show co-localization of the GFP expression with the lymphatic
marker LYVE-1.
LYVE-1 staining showed that the expression of GFP in the Prox1-EGFP rat was colocalized with
the expression of the lymphatic-specific marker, LYVE-1 in the rat tail section. This confirmed
that the GFP expression was specific to the lymphatic vasculature.
22

Figure 12. Prox1-GFP expression in lymph nodes of groin flap
(A-B) Prox1-EGFP rat groin flap was dissected out, and its (C-D) GFP expression was
visualized.
The groin flap of the Prox1-EGFP rat was surgically removed to characterize the GFP expression
in the vascularized groin lymph node. The GFP expression analysis showed that the flap was
successfully isolated with intact lymph nodes and surrounding blood vessels.

23

CHAPTER 4
DISCUSSION
The generation of Prox1-EGFP BAC transgenic rat was successful by the utilization of
the BAC used previously to generate the Prox1-EGFP mouse (Choi, Chung et al. 2011). This
indicated that the genomic regulation of mice was well conserved in the rat. The main advantage
of our Prox1-EGFP rat, in addition to its large body size, was the expression of GFP in the eyes
that readily distinguished it from wile-type rats under UV light. In the eyes, the two different
types of vasculature that expresses Prox1, Schlemm’s canal and the limbal lymphatics, were
visibly distinct in structure and abundance. Schlemm’s canal (SC) is a distinctive ring-shaped
vascular structure that encircles the anterior of the eye, and functions in maintaining fluid
homeostasis by draining aqueous humor from the eye to the systemic circulation. A defect in the
drainage of aqueous humor potentially leads to intraocular pressure (IOP) elevation, an infamous
cause factor for the development of glaucoma - a group of eye conditions that result in optic
nerve damage and gradual loss of vision - which affects more than 70 million people worldwide.
(Karpinich and Caron 2014) SC displays similar properties to the lymphatic vasculature in that it
is a blind-ended tube that functions as a drainage site where aqueous humor and antigen-
presenting cells are released into the venous circulation, and that the master regulator of the
lymphatic system, Prox1, is expressed (Park, Lee et al. 2014). Because the monolayer endothelial
cell lining of Schlemm’s canal serves as the principal blockade against aqueous humor outflow,
extensive research has been conducted to characterize this unique vasculature in effort to find a
prospective therapeutic target. Since our Prox1-EGFP rat easily visualizes SC with GFP, our
lymphatic reporter rat show enormous potential for the study of this newly developing research
field.
24

In addition to the eye, other organs such as the skin, heart, diaphragm, intestine and
mesentery, kidney, bladder, testes, and lymph nodes present tissue-specific sprouting of
lymphatic vessels, increasingly specific with rats of a younger age. To show the utilization of our
lymphatic-specific reporter rat, we dissected out the groin flaps and analyzed the GFP expression
in the vascularized lymph nodes. As mentioned previously, breast cancer patients often undergo
sentinel and lymph node dissection to check for metastasis, and as a consequence, suffer from
lymphedema. There is no cure for lymphedema currently, and treatments to reduce the
discomfort include massages, laser therapy, surgical removal of the lymph, or lymphatic
reconstructive procedures which often have a short-term outcome. Vascularized lymph node
transfer is one of the newest surgical therapies to treat lymphedema. Simply put, the procedure
involves cutting out vascularized groin flaps with intact lymph nodes and transferring them to the
sites of disrupted lymph flow. Presumably, either by secreting growth factors to induce
lymphangiogenesis or performing as lymphatic pumps, the healthy transplanted lymph nodes
will effectively mitigate lymphedema. However, the exact method of this surgical therapy is still
to be understood. This semi-invasive surgical procedure will be difficult in mice with a smaller
body size, in concurrence very small lymph nodes that are not readily visible. As seen in Figure
9 the flaps show the intact structure of lymph nodes with the lymphatic vasculature with a bright
GFP expression. In the near future, we plan to transplant the Prox1-EGFP rat flaps to wild-type
rats to elucidate how the flap incorporates to the recipient lymphatic bed, and analyze the how it
induces lymphatic flow. Therefore, this study shows that our lymphatic-reporter rat model hold
enormous potential for molecular and surgical studies of lymphangiogenesis and lymphatic
diseases. (Raju and Chang 2015)

25

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Abstract (if available)

Abstract In the human body there are two fundamental circulatory networks, the blood and the lymphatic system, which are anatomically similar and complementary in function. Despite its significant role in drainage of excess interstitial fluid, transport of dietary lipids and execution of precise immune responses, the lymphatic vasculature was considered subordinate to the blood vasculature for many decades (Oliver 2004). Recent progresses in lymphatic research, such as the discovery of lymphatic-specific molecular markers (Banerji, Ni et al. 1999), lineage tracing of Prox1-expressing lymphatics (Srinivasan, Dillard et al. 2007), advancement of imaging techniques (Yaniv, Isogai et al. 2006), and development of various lymphatic-specific reporter mice have shown how the lymphatic vasculature plays an important role in the maintenance and enhancement of human health (Choi, Lee et al. 2012

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Asset Metadata

Creator Jung, Eunson (author)

Core Title Visualization and characterization of rat lymphatic vasculature in a Prox1-EGFP bacterial artificial chromosome (BAC) transgenic rat: a novel animal model for physiological and pathological lymph...

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 07/10/2015

Defense Date 05/11/2015

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag BAC,bacterial artificial chromosome,lymphatic reporter,OAI-PMH Harvest,Prox1 reporter rat,Prox1-EGFP,transgenic rat

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Hong, Young Kwon (committee chair), Kobielak, Agnieszka (committee member), Tokes, Zoltan A. (committee member)

Creator Email ej484@nyu.edu,eunsonju@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-592422

Unique identifier UC11301974

Identifier etd-JungEunson-3590.pdf (filename),usctheses-c3-592422 (legacy record id)

Legacy Identifier etd-JungEunson-3590.pdf

Dmrecord 592422

Document Type Thesis

Format application/pdf (imt)

Rights Jung, Eunson

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA