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Identification and characterization of the enhancer elements for lymphatic-specific expression of Prox1
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Identification and characterization of the enhancer elements for lymphatic-specific expression of Prox1
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Content
Identification and Characterization of the Enhancer Elements for
Lymphatic-Specific Expression of Prox1
By
Wonhyeuk Jung
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
Dr. Young-Kwon Hong
Dr. Zoltan A. Tokes
Dr. Alex K. Wong
In Partial Fulfilment of the
Requirements for the Degree
MASTER OF SCIENCE
Department of Biochemistry and Molecular Biology
August 2016
Copyright 2016 Wonhyeuk Jung
ACKOWLEDGEMENTS
I would like to thank members of Dr. Hong’s lab and Dr. Hong for the knowledge and
skills I gained throughout my training. I would like to thank my family for their warm support.
Finally, I would like to thank Maxwell and Daniel who have helped with the revision of this
thesis.
Table of Contents
Chapter 1. Abstract page 1
Chapter 2. Introduction page 2
Prox1, the master regulator of lymphatic development page 2
Prox1-EGFP mouse: a transgenic mouse model for lymphatic system
visualization page 3
Prox1-EGFP bacterial artificial chromosome page 5
Gs1783, an Escherichia. coli strain for bacterial artificial chromosome
engineering page 7
Chapter 3. Objectives page 8
Chapter 4. Overall Scheme of the Project page 9
Chapter 5. Results page 10
Mouse Prox1-EGFP bacterial artificial chromosome engineering page 10
In vitro functional screening of engineered bacterial artificial chromosomes
constructs page 22
Transgenic mouse generation with engineered bacterial artificial chromosomes
page 29
Chapter 6. Discussion page 35
Chapter 7. Materials and Methods page 39
Reference page 50
- 1 -
Chapter 1. Abstract
Prospero homeobox 1 or Prox1 is a mammalian homeobox transcription factor that is
critical for lymphatics development and lymphatic endothelial cell differentiation [3]. A
transgenic mouse model called Prox1-EGFP mouse has been developed and its lymphatics show
robust EGFP expression [1]. However, Prox1 is expressed in various other types of cells,
including neurons, hepatocytes and myocytes [5]. Thus, it is difficult to visualize lymphatics in
tissues rich with these cell types.
Approximately 80% of mouse enhancers show tissue-specific expression [6] and it is
likely that Prox1-EGFP BAC, the bacterial artificial chromosome that was used to generate
Prox1-EGFP mouse, has multiple enhancer regions that direct Prox1 promoter-coupled EGFP
expression for various tissues. Based on human ENCODE data, the putative enhancer region of
the BAC was postulated and the region was deleted. The effects of the deletion have been
screened in vitro and in vivo. The result of this project will assist in the identification and the
characterization of enhancers for Prox1 gene.
- 2 -
Chapter 2. Introduction
The fluid homeostasis of vertebrates is maintained by blood and lymphatic systems
distributed throughout the body. In addition to the important physiological roles these systems
play, they are also critically involved in the proliferation and metastasis of tumors [7, 8]. For
instance, lymphatic vessels have been shown to be the main metastatic route for oral squamous
cell carcinoma [9]. Finding out how lymphatic development is regulated, then, is of critical
importance to understanding both normal physiology and tumor formation and metastasis.
Prox1, the master regulator of lymphatic development
The centrifugal theory suggested by Dr. Sabin postulated that lymphatic vessels sprout
from blood vessels [10]. Observation of lymphatic development during mouse embryogenesis
has confirmed this. Around embryonic day (E) 9.0, a distinct group of blood endothelial cells
(BECs) of the anterior cardinal vein begins to express LYVE-1, a key marker of lymphatic
endothelial cells. Around E 9.75, this population of LYVE-1-positive cells starts to express
Prox1 gene, another marker of lymphatic endothelium [11].
The mammalian transcription factor Prox1, whose homologue in Drosophila is prospero,
belongs to the family of homeobox transcription factors [5]. Homeobox transcription factors are
characterized by a conserved homeodomain that binds DNA and allows transcriptional
regulation of other genes. Apart from their crucial role in embryonic development of various
organs, homeobox genes are thought to play a role in oncogenesis [12].
Prox1-deficient mice do not develop lymphatic systems [3]. Instead, their endothelial
cells remain as BECs. When Prox1 cDNA is ectopically expressed in human BECs by adenoviral
- 3 -
gene transfer, lymphatic endothelial cell markers such as podoplanin and vascular endothelial
growth factor receptor-3 (VEGFR-3) are up-regulated. Conversely, genes associated with the
blood vascular endothelial cell phenotype, such as laminin, vascular endothelial growth factor-C,
neuropilin-1, and intercellular adhesion molecule-1, are down-regulated [3]. Thus, Prox1 is
postulated to be a key player in the molecular pathway for development and differentiation into
lymphatic endothelial cells.
Prox1-EGFP mouse, a transgenic mouse model for lymphatic system visualization
In 2011, Dr. Hong’s group reported on the generation of the Prox1-EGFP mouse, which
constitutively expresses EGFP in lymphatic vessels [1]. Lymphatic vascular structure and
networks can be conveniently and non-invasively visualized in this transgenic mouse (Figure 1.).
Although the Prox1-EGFP mouse has served as a useful experimental tool in the field of
lymphatic research, the molecular mechanism underlying lymphatic-specific expression of Prox1
is unknown. Furthermore, because Prox1 is also expressed in other cell types, including neurons,
myocytes, and hepatocytes, it is difficult to visualize lymphatics in tissues rich in these cells.
- 4 -
Figure 1. Prox1-EGFP mouse EGFP expression pattern (modified from Choi et al., 2011 [1])
A(i-ii) Morphology of the transgenic embryos is indistinguishable from wild-type embryos (E14.5). However,
clear EGFP expression can be observed in the eye and along the spine.
A(iii-v) Dorsal skin of Prox1-EGFP embryo (E14.5) shows clear EGFP expression along its dermal lymphatic
plexus and lymphangiogenic front.
B(i-ii) Prox1-EGFP adult mouse’s overall morphology and phenotype does not differ from wild-type littermate.
However, the green eye phenotype can be seen even under non-fluorescent lighting.
B(v-vii) Adult mouse’s lymphatic vessels can be observed without immunostaining in trachea, skin and ear,
respectively [1].
- 5 -
Prox1-EGFP bacterial artificial chromosome
The bacterial artificial chromosome (BAC) used to generate Prox1-EGFP mouse is
Prox1-EGFP BAC. Prox1-EGFP BAC was generated by homologous recombination between
mouse Prox1 BAC and a targeting vector with enhanced green fluorescence protein (EGFP)
(Figure 2.). Mouse Prox1 BAC was originally generated by the Gene Expression Nervous
System Atlas (GENSAT) BAC Transgenic Project [2]. This BAC (RP23-360I16, vector:
pBACe3.6) holds a 196,257 bp-long mouse genomic fragment spanning the entire Prox1 gene
along with the 5’ upstream sequence (114,893 bp before the initiation ATG, chr1:190,366,888-
190,366,937) and the 3’ downstream sequence (42,337bp after the stop codon,
chr1:190,006,884-190,006,937). The targeting vector was made by cloning 5′-homologous DNA
sequence (~540 bp) from the promoter of mouse Prox1 gene into a vector (pLD53.SC2). The
recombination vector (pSV1.RecA) and the targeting vector were introduced into E. coli with
mouse Prox1 BAC. Successfully targeted BAC was isolated from ampicillin-resistant,
chloramphenicol-resistant, and tetracycline-sensitive bacterial colonies [1].
- 6 -
Figure 2. Generation of Prox1-EGFP BAC (modified from Choi et al., 2011 [1])
5’ homologous portion of the promoter region of the mouse Prox1 gene was cloned into a targeting vector
(pLD53.SC2). Mouse Prox1 BAC was originally generated by GENSAT project [2]. Recombination vector that
expresses RecA and the targeting vector were co-transformed into the bacteria that have mouse Prox1 BAC.
Correctly targeted BAC was screened by testing for ampicillin, chloramphenicol resistance and tetracycline
sensitivity.
- 7 -
Gs1783, an Escherichia. coli strain for bacterial artificial chromosome engineering
In order to analyze the prospective enhancer region of Prox1-EGFP BAC, deletions along
the BAC were made with Gs1783 E. coli strain. Gs1783 E. coli strain’s Red gene is under the
control of a temperature-inducible promoter and I-SceI gene is under the control of an arabinose-
inducible promoter [13, 14].
The Red recombination system is often used for engineering large constructs like BACs.
The system originates from λ phages and allows double-stranded linear DNA to be inserted into
a target BAC through homologous recombination [15]. The Red recombination system consists
of Gam, Exo and Beta proteins [16]. The Gam protein blocks the E. coli RecBCD helicase and
prevents degradation of linear DNA [17]. The Exo protein produces 3’ single strand extension
[18]. The Beta protein promotes and protects the extension and annealing of the newly-formed
single strand with the complementary sequence on the target DNA [19]. When the target DNA
undergoes replication, the single strand DNA is integrated [20].
I-SceI is a homing endonuclease that was isolated from Saccharomyces cerevisiae and
has an 18bp recognition site [21]. I-SceI expression in E. coli can be induced readily and the
homing endonuclease’s activity has been confirmed to be robust in vitro [21]. Thus, BACs inside
Gs1783 can be modified through homologous recombination when the bacteria are given a heat
shock. Subsequently, if the DNA that was introduced into the BAC has an I-SceI recognition site,
newly incorporated DNA can be removed by culturing the bacteria in arabinose-treated media
and giving a second heat shock. The overall process does not leave a trace and greatly simplifies
the BAC engineering process by foregoing co-transformation.
- 8 -
Chapter 3. Objectives
The goal of the project is to identify and characterize a putative lymphatic-specific
enhancer for Prox1-EGFP BAC using BAC engineering technology and in vitro functional
assays.
The Encyclopedia of DNA Elements (ENCODE) project has mapped putative enhancer
elements upstream of Prox1 in human cells. Mouse Prox1 enhancer elements, however, have not
yet been identified. Based on the human ENCODE data (histone H3K4 mono- and tri-
methylation and H3K27 acetylation), the primary hypothesis is that distinct regulatory elements
upstream of the Prox1 gene exist that act as lymphatic-specific enhancers. By deleting potential
regulatory regions that may be unnecessary for lymphatic-specific Prox1 expression, we may be
able to achieve both reduction of overall BAC size and true lymphatic-specific EGFP expression
in transgenic mice generated from the newly made BAC constructs.
To date, two large deletions in Prox1-EGFP BAC were made using Gs1783. The
functionality of these BAC constructs was investigated in vitro and the phenotypes of the
transgenic mice generated from the modified BACs were screened. The outcome of the initial
deletions will aid in the development of further deletions and/or substitutions in the upstream
sequence of Prox1.
- 9 -
Chapter 4. Overall scheme of the Project
Deletion of prospective enhancer region
of Prox1-EGFP BAC
In vitro functional screening of
engineered BAC constructs
Transgenic mouse generation from
functional bacterial artificial
chromosome constructs
Transgenic mice phenotype screening
- 10 -
Chapter 5. Results
Prox1-EGFP bacterial artificial chromosome engineering
In order to identify prospective enhancer regions that control Prox1 expression, putative
enhancer region that spans from 3687bp to 89965bp (refer to Figure 3. legend for designation of
relative position) of Prox1-EGFP BAC was deleted by using Gs1783 E.coli cell line. First,
pEPKan-s2 DNA fragment that contains I-SceI restriction site and kanamycin resistance was
amplified with forward and reverse primers that have 60bp-long 5’ and 3’ overhang, respectively.
Each 60bp-long overhang consists of three 20bp-long blocks that are homologous to Prox1-
EGFP BAC (Figure 4.).
Figure 3. Prox1-EGFP BAC structure
The vector of the BAC, pBACe3.6 spans from 1bp to 5129bp.
pLD53.sc2: 105006bp to 108691bp (vector from the targeting vector used to introduce EGFP into the BAC)
Prox1 promoter and EGFP: 22005bp to 91005bp
5’ homologous region that is approximately 540bp long was used to introduce EGFP into original mouse Prox1
BAC which holds a 196,257 bp-long mouse genomic fragment, which spans the entire Prox1 gene along with the
5’ upstream sequences (114,893 bp before the initiation ATG) (chr1:190,366,888-190,366,937) and the 3’
downstream sequences (42,337bp after the stop codon) (chr1:190,006,884-190,006,937) [1].
Vector (pBACe3.6)
540 bp of 5’ Prox1
promoter
EGFP AMP Poly A
Prox1-EGFP BAC
Prox1
- 11 -
A brief heat shock was given to the Gs1783 with Prox1-EGFP BAC to activate Red
recombination system. Then, the amplified linear DNA fragment was electroporated into Gs1783.
Homologous recombination was induced to replace 85kb-long putative enhancer region with
1kb-long pEPKan-s2 amplicon (Figure 5.). Successful recombination results in acquirement of
kanamycin resistance, generation of novel I-SceI restriction site, and generation of novel
homologous regions within the BAC.
Figure 4. PCR amplification of pEPKan-s2
A, B, C and D represent 20bp-long sequences that are homologous to Prox1-EGFP BAC, respectively. pEPKan-s2
contains an I-SceI restriction site and kanamycin resistance. Forward primer consists of 60bp 5’ overhang and
22bp of pEPKan-s2 5’end. Reverse primer consists of 60bp 3’ overhang and 24bp of pEPKan-s2 3’end. PCR
amplification generated elongated pEPKan-s2 amplicon that is 120bp longer than the original fragment.
pEPKan-s2
Kan
+
I-
SceI
22bp of pEPKan-s2 5’ end
24bp of pEPKan-s2 3’ end
PCR
pEPKan-s2
Kan
+
I-
SceI
A 20bp B 20bp C 20bp
B 20bp C 20bp D 20bp
A 20bp B 20bp C 20bp B 20bp C 20bp D 20bp
- 12 -
In the final step, kanamycin resistant colonies are cultured in arabinose-treated media. I-
SceI is induced by the arabinose-controlled promoter and the restriction enzyme cuts the newly
introduced restriction site. Then, the bacteria are given a second heat shock. Successful
homologous recombination results in scar-less deletion and the loss of kanamycin resistance
(Figure 6.).
Figure 5. Introduction of novel homologous sites into Prox1-EGFP BAC by heat shock
Gs1783 Red recombination system can be turned on through a brief heat shock. PCR product of pEPKan-s2
carries 40bp homologous region on the 5’ and 3’end respectively. Successful homologous recombination gives the
BAC kanamycin resistance, an I-SceI restriction site and novel homologous region.
Prox1-EGFP BAC
A
B
C
D
1kb
85kb
Gs1783 Red
recombination
system
activated by
heat shock
A
B
C
B
C
D
I-
SceI
Kan
+
Novel homologous region
- 13 -
85kb deletion BAC construct’s integrity was checked with restriction enzyme digestion
and PCR (Figure 7., Figure 8.). Figure 9. describes the orientation of the primers used for PCR
screening.
Figure 6. Scar-less deletion is achieved through I-SceI expression induction and a second heat shock
Gs1783’s I-SceI gene expression is induced by the arabinose-controlled promoter in arabinose-treated media.
Once the DNA is linearized by I-SceI, the bacteria are given a heat shock to remove the DNA fragment that has
been inserted. Kanamycin resistance is lost when the inserted DNA is deleted by the homologous recombination.
A
B
C
B
C
D
I-
SceI
Kan
+
Cultured in arabinose-
treated media
Induced I-SceI cuts the
new restriction site
Gs1783 Red
recombination
system
activated by
heat shock
A
D
Scar-less deletion product
- 85kb deletion BAC
construct
B
C
- 14 -
Figure 7. XmaI digestion of 85kb deletion BAC construct
85kb deletion BAC construct was digested with XmaI. The expected digestion pattern on 1% agarose gel was
generated by pDRAW32. The BAC was purified by CsCl prep and was dialyzed afterwards. Pmax GFP was used
as a control to calculate the BAC’s concentration. The digestion pattern was confirmed by 1% agarose gel run.
- 15 -
After confirming 85kb deletion BAC construct’s integrity, another deletion was made.
Putative enhancer region that spans from 137095bp to 208669bp of Prox1-EGFP BAC (refer to
Figure 3. legend for the designation of relative position) was deleted. The same procedure that
was used to make 85kb deletion was used to make 71kb deletion (Figure 10., Figure 11., Figure
12.) . Restriction analysis was done with EcoRI (Figure 13.). Also, deleted regions were
screened by PCR (Figure 14.). Figure 15. describes the orientations of the primers used for the
screening process.
Figure 8. Integrity check of 85kb deletion BAC construct by PCR
*YH3041 is 85kb deletion BAC construct.
Lane 1, 2 – Forward primer HP4002, reverse primer HP4004 were used to screen 85kb deletion of Prox1-EGFP
BAC. 267bp expected.
Lane 3, 4 – Forward primer HP4174, reverse primer HP4175 were used to screen the vector of Prox1-EGFP BAC
(pBACe3.6). 236bp expected.
85kb deletion BAC construct’s integrity was checked with PCR. HP4002, HP4004, HP4174, and HP4175 are
names of the primers used for the PCR. The orientations of the primers are described in Figure 9.
ThermoFisher
Scientific
GeneRuler 100bp
DNA ladder
- 16 -
Figure 9. Screening of 85kb deletion BAC construct
Schematic representation of the orientations of the primers for the PCR in Figure 8. HP4002, HP4174 are forward
primers and HP4004, HP4175 are reverse primers. HP4002/4004 set was used to screen 85kb deletion.
HP4174/4175 set was used to screen the vector of Prox1-EGFP BAC (pBACe3.6).
HP4002
HP4004
5’ 3’
A
D
Scar-less deletion product
- 85kb deletion BAC
construct
A D
pBACe3.6
pBACe3.6
HP4174
HP4175
3’ 5’
B
C
B C
- 17 -
Figure 10. PCR amplification of pEPKan-s2 with 5' and 3' overhang
E, F, G and H represent 20bp-long sequences that are homologous to Prox1-EGFP BAC, respectively. pEPKan-s2
contains an I-SceI restriction site and kanamycin resistance. Forward primer consists of 60bp 5’ overhang and
22bp of pEPKan-s2 5’end. Reverse primer consists of 60bp 3’ overhang and 24bp of pEPKan-s2 3’end. PCR
amplification generated elongated pEPKan-s2 amplicon that is 120bp longer than the original fragment.
pEPKan-s2
Kan
+
I-
SceI
22bp of pEPKan-s2 5’ end
24bp of pEPKan-s2 3’ end
PCR
pEPKan-s2
Kan
+
I-
SceI
E 20bp F 20bp G 20bp
F 20bp G 20bp H 20bp
E 20bp F 20bp G 20bp F 20bp G 20bp H 20bp
- 18 -
Figure 11. Introduction of novel homologous sites into Prox1-EGFP BAC by heat shock
Gs1783 Red recombination system can be turned on through a brief heat shock. PCR product of pEPKan-s2
carries 40bp homologous region on the 5’ and 3’end respectively. Successful homologous recombination gives the
BAC kanamycin resistance, an I-SceI restriction site and novel homologous region.
A
D
Scar-less deletion product
- 85kb deletion BAC
construct
E
F
G
H
71kb
1kb
Gs1783 Red
recombination
system
activated by
heat shock
E
F
G
F
G
H
Novel homologous region
Ka
n+
I-
Sc
eI
B
C
- 19 -
Figure 12. Scar-less deletion is achieved through I-SceI expression induction and a second heat shock
Gs1783’s I-SceI gene expression is induced by an arabinose-controlled promoter in arabinose-treated media. Once
the DNA is linearized by I-SceI, the bacteria are given a heat shock to remove the DNA fragment that has been
inserted. Kanamycin resistance is lost when the inserted DNA is deleted by the homologous recombination
E
F
G
F
G
H
Ka
n+
I-
Sc
eI
Cultured in arabinose-
treated media
Induced I-SceI cuts the
new restriction site
Gs1783 Red
recombination
system
activated by
heat shock
A
D
Scar-less deletion product
- 85kb+71kb deletion BAC
construct
E
H
B
C
F
G
- 20 -
Figure 13. EcoRI digestion of 85kb+71kb deletion BAC construct
85kb+71kb deletion BAC construct was digested with EcoRI. The expected digestion pattern on 1% agarose gel
was generated by pDRAW32. The BAC was purified by CsCl prep and was dialyzed afterwards. Pmax GFP was
used as a control to calculate the BAC’s concentration. The digestion pattern was confirmed by 1% agarose gel
run.
- 21 -
Figure 14. Integrity check of 85kb +71kb deletion BAC construct by PCR
85kb deletion and 71kb deletion of Prox1-EGFP BAC was screened with PCR. HP4002/4004 set screens the 85kb
deletion. HP3085/4016 set screens the 71kb deletion. The orientations of the primers are described in Figure 15. A
BAC with pBACe3.6 vector that was generated by human male BAC library project was used as a negative
control [4].
A1 – 85kb deletion BAC construct CsCl prep, A2 – DW, A3 – A BAC from RPCI-11 Human Male BAC Library
(negative control BAC with pBACe3.6 vector), A4 – 85kb+71kb deletion BAC construct colony, B1 – 85kb+71kb
deletion BAC construct colony, B2 – DW, B3 – A BAC from RPCI-11 Human Male BAC Library (negative
control BAC with pBACe3.6 vector)
A
1 2 3 4
HP3085/4016 210bp expected
1 2 3
B
ThermoFisher
Scientific
GeneRuler 100bp
DNA ladder
ThermoFisher
Scientific
GeneRuler 100bp
DNA ladder
Negative
control
Negative
control
- 22 -
In vitro functional screening of modified bacterial artificial chromosomes
constructs
After successful screening of 85kb deletion BAC construct and 85kb+71kb deletion BAC
construct, both BACs were transfected into Chinese hamster ovary (CHO) cells with
Lipofectamine 2000 (ThermoFisher, catalog number: 12566014) to check their EGFP expression
under the control of Prox1 promoter. Prox1-EGFP BAC, and pEGFP-C2 plasmid (pEGFP-C2
plasmid (Takara Clontech, catalog number: 63248) were used as positive controls. Prox1-EGFP
BAC, pEGFP-C2, 85kb deletion BAC construct and 85kb+71kb deletion BAC construct all
showed EGFP expression (Figure 16.).
Figure 15. Screening of 85kb+71kb deletion BAC construct
Schematic representation of the primers for the PCRs in Figure 14. HP4002, HP3085 are forward primers and
HP4004, HP4016 are reverse primers. HP4002/4004 set was used to screen 85kb deletion. HP3085/4016 set was
used to screen 71kb deletion.
A
D
Scar-less deletion product
- 85kb+71kb deletion BAC
construct
E
HP3085
HP4016
5’ 3’
E H
H
F
G
HP4002
HP4004
5’ 3’
A D B C
F G
B
C
- 23 -
Figure 16. In vitro functional screening of engineered BAC constructs in CHO cells
Prox1-EGFP BAC, pEGFP-C2, 85kb deletion BAC construct and 85kb+71kb deletion BAC construct was
transfected into CHO cells with Lipofectamine 2000.
A: pEGFP-C2 1ug, Lipofectamine 2000 3ug
B: Prox1-EGFP BAC 1ug, Lipofectamine 2000 3ug
C: 85kb deletion BAC construct 1ug, Lipofectamine 2000 4ug
D: 85kb+71kb deletion BAC construct 1ug, Lipofectamine 2000 3ug
20um
20um 20um
20um
20um 20um
20um
20um 20um
20um
20um 20um
- 24 -
pEGFP-C2, and 85kb+71kb deletion BAC construct showed better transfection efficiency
and greater cytotoxicity. In contrast, Prox1-EGFP BAC and 85kb deletion BAC construct did not
show transfection-associated cytotoxicity but had low transfection efficiency.
All transfections showed EGFP expression, thus it is highly likely that deletions do not
harm the Prox1 promoter controlled expression of EGFP. Next, Prox1-EGFP BAC, pEGFP-C2,
85kb deletion BAC construct and 85kb+71kb BAC deletion construct were transfected into
human colon cancer cell line (SW620) with Lipofectamine 2000. Prox1-EGFP BAC, pEGFP-C2,
85kb deletion BAC construct and 85kb+71kb deletion BAC construct all expressed EGFP in
SW620 cell line as well (Figure 17.).
Also, CHO cell line and SW620 cell line’s Prox1 expression was confirmed with
immunostaining to assure that EGFP expression by the BAC constructs are coming from Prox1
expression (Figure 18., Figure 19.).
- 25 -
Figure 17. In vitro functional screening of engineered BAC constructs in SW620 cells
Prox1-EGFP BAC, pEGFP-C2, 85kb deletion BAC construct and 85kb+71kb deletion BAC construct was
transfected into SW620 cells with Lipofectamine 2000.
A: pEGFP-C2 1ug, Lipofectamine 2000 3ug
B: Prox1-EGFP BAC 1ug, Lipofectamine 2000 3ug
C: 85kb deletion BAC construct 1ug, Lipofectamine 2000 4ug
D: 85kb+71kb deletion BAC construct 1ug, Lipofectamine 2000 3ug
A
B
C
D
Bright field GFP Bright field+GFP
- 26 -
Figure 18. Prox1 immunostaining of CHO cell line
CHO cell line’s Prox1 expression was checked with immunostaining. Lymphatic endothelial cells were used as
positive control. Anti-Prox1 antibody from ReliaTech (product No. 102-PA32AG) was used as the primary
antibody. Goat anti-Rabbit IgG conjugated with Alexa Fluor 594 from ThermoFisher (product No. A-11037) was
used as the secondary antibody.
LEC
DAPI DsRed DAPI+DsRed
CHO
- 27 -
Finally, Prox1-EGFP BAC, pEGFP-C2, 85kb deletion BAC construct and 85kb+71kb
deletion BAC construct were transfected into human lymphatic endothelial cells. For the primary
cell line, the transfection efficiency was generally lower. The EGFP expression of pEGFP-C2
and 85kb deletion BAC construct was observed but Prox1-EGFP BAC and 85kb+71kb deletion
BAC construct’s EGFP expression could not be detected (Figure 20.).
Figure 19. Prox1 immunostaining of SW620 cell line
SW620 cell line’s Prox1 expression was checked with immunostaining. Lymphatic endothelial cells were used as
positive control. Anti-Prox1 antibody from ReliaTech (product No. 102-PA32AG) was used as the primary
antibody. Goat anti-Rabbit IgG conjugated with Alexa Fluor 594 from ThermoFisher (product No. A-11037) was
used as the secondary antibody.
LEC
DAPI DsRed DAPI+DsRed
SW620
- 28 -
Figure 20. In vitro functional screening of engineered BAC constructs in LECs
Prox1-EGFP BAC, pEGFP-C2, 85kb deletion BAC construct and 85kb+71kb deletion BAC construct was
transfected into LECs with Lipofectamine 2000.
A: pEGFP-C2 1ug, Lipofectamine 2000 3ug
B: Prox1-EGFP BAC 1ug, Lipofectamine 2000 3ug
C: 85kb deletion BAC construct 1ug, Lipofectamine 2000 4ug
D: 85kb+71kb deletion BAC construct 1ug, Lipofectamine 2000 3ug
- 29 -
Transgenic mouse generation with engineered bacterial artificial chromosomes
In vitro screening of the BAC constructs showed that 85kb deletion BAC construct is
functional in CHO cell line, SW620 cell line and human LECs. Thus, 85kb deletion BAC
construct was used to generate transgenic mice. CsCl prep of the BAC was prepared and the
DNA was dialyzed to lower the salt concentration.
The first round of microinjection yielded one female mouse (No. 2) with 85kb deletion
BAC construct integrated in its genome (Figure 21.).
Figure 21. Genotyping transgenic mouse from 85kb deletion BAC construct microinjection
The same primer set (HP4002/4004) that was used to screen for 85kb deletion (Figure 8.) was used to screen for
transgenic mice that have 85kb deletion BAC construct integrated into their genome. Lane 1 – DW, lane 2 –
pseudo-pregnant mouse genomic DNA, lane 3 – pup No.1, lane 4 – pup No.2
ThermoFisher
Scientific
GeneRuler 100bp
DNA ladder
- 30 -
First positive mouse (No. 2) was housed with its two male littermates. Second round of
microinjection also produced one positive pup (No. 14) (Figure 22., Figure 23.) Also, No. 2
mouse gave birth to 6 pups. Among 6 pups, 3 pups (No.22, 24, 25) were confirmed as positive
for 85kb deletion BAC construct (Figure 24.).
Figure 22. 85kb deletion BAC construct transgenic mice from second microinjection genotyping
Transgenic mice were generated with 85kb deletion BAC construct. The second round of microinjection resulted
in 10 pups from 4 pseudo-mothers. Eight pups (No. 10-17) that were produced from the No. 1 and No. 2 pseudo-
mothers were genotyped through PCR.
Ladder – ThermoFisher Scientific Generuler 100bp DNA ladder (catalog number: SM0241)
A – HP4004/4004 primer set for 85kb deletion screening was used. 267bp expected.
B – HP726/727 primer set for EGFP gene screening was used. 360bp expected.
C – HP4174/4175 primer set for pBACe3.6 screening was used. 236bp expected.
Lane 1 – DW, lane 2, 3 – pseudo-mother No. 1 and No. 2, lane 4 – CsCl prep of 85kb deletion BAC construct,
lane 5 – positive female (No. 2) from the first microinjection, lane 6 to 13 – Eight pups (No. 10-17) from pseudo-
mother No. 1 and No. 2
No. 14 pup was confirmed as a positive.
- 31 -
Figure 23. 85kb deletion BAC construct transgenic mice from second microinjection genotyping
Transgenic mice were generated with 85kb deletion BAC construct. The second round of microinjection resulted
in 10 pups from 4 pseudo-mothers. Two pups (No. 18, 19) that were produced from the No. 3 and No. 4 pseudo-
mothers were genotyped through PCR.
Ladder – ThermoFisher Scientific Generuler 100bp DNA ladder (catalog number: SM0241)
A – HP4004/4004 primer set for 85kb deletion screening was used. 267bp expected.
B – HP726/727 primer set for EGFP gene screening was used. 360bp expected.
C – HP4174/4175 primer set for pBACe3.6 screening was used. 236bp expected.
Lane 1 – DW, lane 2, 3 – pseudo-mother No. 3 and No. 4, lane 4 – CsCl prep of 85kb deletion BAC construct,
lane 5 – positive female (No. 2) from the first microinjection, lane 6, 7 – Two pups (No. 18, 19) from pseudo-
mother No. 3 and No. 4
Both No. 18 and No. 19 were confirmed as negative.
A
300 bp
1 2 3 4 5 6 7
B
300 bp
1 2 3 4 5 6 7
C
300 bp
1 2 3 4 5 6 7
- 32 -
Figure 24. 85kb deletion BAC construct transgenic mice from the first microinjection genotyping (second
generation from No. 2 mouse)
Transgenic mice were generated with 85kb deletion BAC construct. The first round of microinjection resulted in
one positive female mouse (No. 2 mouse). The positive mouse was housed with two male littermates. 6 pups were
genotyped.
Ladder – ThermoFisher Scientific Generuler 100bp DNA ladder (catalog number: SM0241)
A – HP4004/4004 primer set for 85kb deletion screening was used. 267bp expected.
B – HP726/727 primer set for EGFP gene screening was used. 360bp expected.
C – HP4174/4175 primer set for pBACe3.6 screening was used. 236bp expected.
Lane 1 – DW, lane 2, 3 – two negative male littermates of No. 2 mouse, lane 4 – CsCl prep of 85kb deletion BAC
construct, lane 5 – positive female (No. 2) from the first microinjection, lane 6-11 – 6 pups (No. 21-26) from No. 2
mouse.
Three pups (No. 22, 24 and 25) were confirmed as positive.
A
1 2 3 4 5 6 7 8 9 10 11
300bp
B
300bp
1 2 3 4 5 6 7 8 9 10 11
C
1 2 3 4 5 6 7 8 9 10 11
300bp
- 33 -
The ears of two mice with 85kb deletion BAC construct (No. 2, 14) were punched to
observe the lymphatics vessels. Both of the mice’s lymphatics did not express EGFP. Next, one
negative mouse (No. 21) and three positive mice from No. 2 (No. 22, 24 and 25) were euthanized
to observe their organs’ EGFP expression. Clear EGFP expression in the eyes was observed for
positive mice (Figure 25.). However, no lymphatic EGFP expression could be observed.
- 34 -
Figure 25. EGFP expression of the eyes of 85kb deletion construct transgenic mice
Initial microinjection of 85kb deletion construct resulted in one positive female mouse (No. 2). The progenitors of
No. 2 mouse were genotyped and the EGFP expression of the eyes of positive mice (No. 24 and 25) and a negative
littermate mouse (No. 21) were observed under a stereoscope.
From left, No. 24, 25 and 21.
- 35 -
Chapter 6. Discussion
A promoter is defined as a gene’s start site and the region immediately upstream (up to
several 100bp) from a core promoter [22]. A core promoter can be the DNA segment where
transcription initiates or the region ±40–50 bp from the transcription start site [23].
In contrast to promoters, enhancers often lie at varying distances from their target
promoters. In metazoans, enhancers range from 100 bp to 1 Mb away from their regulated gene
on the same chromosome, and can be found either upstream or downstream from their targets.
They can even be found in the body of another gene, and their regulation can bypass other genes
independently of orientation [24]. Thus, it is more difficult to precisely define an enhancer region
for a certain gene.
Enhancer activity is often restricted to a certain cell type or tissue [24], and
approximately 80% of mouse enhancers show tissue-specific expression [6]. A potential
approach to analyze enhancers includes introducing regional deletions and observing the effects
on a certain cell or tissue type. In order to identify the enhancer region of the Prox1-EGFP BAC,
two large deletions were made using Gs1783, an E. coli cell line.
Although targeted scar-less deletions along BACs can be made using Gs1783, this
bacterial strain is not ideal for long term storage. Additionally, large scale BAC isolation can
result in random homologous recombination due to uncontrolled Red gene activation. As a result,
85kb deletion BAC construct and 85kb+71kb deletion BAC construct were introduced to the
DH10B E. coli cell line (ThermoFisher Scientific catalog number: 18297010).
However, the DH10B E. coli cell line does not have pir gene. This gene is required to
activate the R6K origin of replication found in Prox1-EGFP BAC construct, 85kb deletion BAC
construct, and 85kb+71kb deletion BAC construct [25]. As a result, the replication of the new
- 36 -
BAC constructs was inefficient in DH10B, and the yield of CsCl prep of the BACs was low. To
address this problem, 85kb deletion BAC construct and 85kb+71kb deletion BAC construct were
transferred to the Pir2 E. coli cell line (ThermoFisher Scientific catalog number: C111110). After
the transfer, CsCl prep yield of both constructs were increased by 5 fold.
Prior to the microinjection to generate the transgenic mouse, 85kb deletion BAC
construct and 85kb+71kb deletion BAC construct were screened for adequate EGFP expression
in vitro. The initial transfection into both CHO and SW620 cell failed due to severe cytotoxicity.
PEI (Sigma-Aldrich product number: 181978), PEI-adenovirus complex, Lipofectamine 2000
(ThermoFisher catalog number 12566014) and electroporation were used to deliver the BACs
into the CHO cell line or the SW620 cell line, but all methods failed to achieve efficient
transfection.
Since the CsCl prep method separates BACs from E. coli genomic DNA by density using
a highly-concentrated solution, it was thought that the failure was due to the high salt
concentration. To lower the salt concentration of the solution used in the CsCl prep, both BAC
constructs were dialyzed against 10mM TRIS (pH 8.0), 1mM EDTA solution and then 10mM
TRIS (pH 8.0), 0.1mM EDTA solution. This dialysis lowered the salt concentration and cleared
most of sheared DNA fragments (Figure 6., 13.).
When dialyzed 85kb deletion BAC constructs and 85kb+71kb deletion BAC constructs
were used to transfect CHO and SW620 cell line, the transfection efficiency increased, and
EGFP expression from both BACs was clearly observed (Figure 16., 17.).
The SW620 cell line’s Prox1 expression has been previously reported [26], however, the
CHO cell line’s Prox1 expression has yet to be confirmed by our group. In order to assure that
BACs’ EGFP expression is connected to Prox1 expression, both cell lines’ Prox1 expression was
- 37 -
verified through immunostaining (Figure 18., 19.). Both cell lines showed robust Prox1
expression using ReliaTech anti-Prox1 antibody (ReliaTech, product number 102-PA32AG) and
SantaCruz anti-Prox1 antibody (SantaCruz, catalog number sc-22135-R). (Data not shown for
SantaCruz antibody).
In order to check 85kb deletion BAC construct and 85kb+71kb deletion BAC construct’s
EGFP expression in lymphatic endothelial cells, both BAC constructs were transfected into
human LECs with Lipofectamine 2000. Dialyzed BACs were used and the cytotoxicity was not
high. However, the transfection efficiency was significantly lower for the primary cell line
(Figure 20.). Prox1-EGFP BAC and 85kb deletion BAC construct showed EGFP expression in
few cells, but 85kb+71kb deletion BAC construct did not express EGFP. In summary, Prox1-
EGFP BAC and 85kb deletion BAC construct expressed EGFP in CHO, SW620 cell lines, and in
lymphatic endothelial cells. Thus, transgenic mice were created from 85kb deletion BAC
construct.
Two rounds of microinjection were performed, and each injection yielded one positive
mouse (No. 2 from the first microinjection Figure 21., No. 14 from the second microinjection
Figure 22.). Both mice had their ears punched and lymphatic vessels observed under a
stereoscope (Leica, catalog number: MDG41). However, there was no EGFP expression along
their lymphatic structures.
The No. 2 mouse gave birth to 6 pups after being placed in breeding pairs. Three mice
(No. 22, No.24, and No. 25, Figure 24.) among 6 pups were confirmed positive for 85kb deletion
BAC construct. Their lymphatic structure did not show any EGFP expression. They were
euthanized along with one negative control littermate (No. 21), and their eyes, large intestine,
hearts and livers were removed in order to screen their EGFP expression.
- 38 -
Interestingly, No. 22, 24 and 25’s eyes showed clear EGFP expression (Figure 25., data
not shown for No. 22), but the EGFP signal was considerably weaker than the Prox1-EGFP
mouse. The Prox1-EGFP mouse’s EGFP expression in the eyes is strong enough to be observed
under normal lighting. In contrast, No. 22, 24 and 25’s EGFP expression in the eye was
observable only under fluorescence lighting. Thus, it is likely that the 85kb deletion made on
Prox1-EGFP BAC removed a portion of the enhancer region for most of the organs except for
the eyes and negatively affected overall Prox1 expression.
Therefore, this data suggest that the region spanning 3687bp to 89965bp (refer to Figure
3. legend for designation of relative position) serves as the enhancer in Prox1-EGFP BAC. Also,
it is possible that along the non-deleted region, there is an enhancer region that controls the
Prox1 expression in the lens. It has been previously reported that Prox1 is crucial for lens
differentiation during embryogenesis [27], yet the specific enhancer elements for the murine
Prox1 gene have not been described. This result could potentially help in identifying the
regulatory sequence for Prox1 lens expression.
In conclusion, lymphatic-specific EGFP expression was not achieved. The EGFP
expression along the lymphatic structures was lost with the deletion indicating that the enhancer
for Prox1 expression along lymphatic vessels is likely to be located within 85kb deletion. The
outcome of this large initial deletion will allow our group to refine our further deletions and/or
substitution mutations into the upstream sequence of Prox1. These results will serve as the first
step for identifying and characterizing the enhancers for the Prox1 gene in the mouse genome.
- 39 -
Chapter 7. Materials and Methods
Bacterial strains
DH10B (ThermoFisher Scientific catalog number: 18297010), Pir2 (ThermoFisher
Scientific catalog number: C111110) were incubated in Luria Bertani (LB) medium with
appropriate antibiotics at 37°C. Gs1783 cell line was a gift from Dr. Jae U. Jung and was
incubated in LB medium with appropriate antibiotics at 32°C.
Isolation and culture of LECs
Human LECs were isolated as previously described [28]. LECs were cultured as in
conditions that were previously described [29].
Culture of SW620 and CHO cells
SW620 (ATCC, catalog number: CCL-227) and CHO (ATCC, catalog number: CCL-61)
cells were propagated in DMEM medium (Sigma-aldrich, catalog number: D5796) supplemented
with 5ml of penicillin/streptomycin (penicillin: 10,000 U/mL, streptomycin: 10 mg/mL, VWR,
catalog number: 45000-652) and 50ml of fetal bovine serum (Omega, catalog number: FB-11).
- 40 -
PCR amplification
All PCR amplification used the same condition except for pEPKan-s2 amplification. For
pEPKan-s2 amplification, Vent® DNA Polymerase (NEB catalog number: M0254S) was used
instead of L. Taq and the elongation step of the heat cycle was prolonged to 1 minute 30 seconds.
Target DNA 1ul of each sample
[F/R primer]
0.5ul of each (final concentration: 0.125uM)
*L. Taq 1ul
2.5mM dNTP 1.5ul (final concentration: 187.5uM)
25mM MgCl2 1.2ul (final concentration: 1.5mM)
10x buffer 2ul
DW 12.3ul
Temperature Cycle [30cycles] 94C 2min/94C 30sec/55C 30sec/72C 30sec/30cycles/72C
5min/4C forever
Ladder: 5ul of GeneRuler 100bp DNA Ladder, 1% agarose gel
Total reaction volume: 20ul
*L. Taq is a Taq polymerase purified by Eunson Jung (eunsonju@usc.edu). The protocol for the
procedure was established by Dr. Engelke [30].
Primers
For 85kb deletion (3687bp to 89965bp (refer to Figure 3. legend for designation of
relative position)) of Prox1-EGFP BAC, HP4005/4006 primer set was used to amplify pEPKan-
s2. For 71kb deletion, HP4013/4014 primer set was used.
HP4007/4008 primer set was used to check for correct pEPKan-s2 amplicon homologous
recombination for 85kb deletion BAC construct generation.
HP4002/4004 primer set was used to check 85kb deletion for Prox1-EGFP BAC and for
genotyping transgenic mouse from microinjection of 85kb deletion BAC construct.
HP3085/4016 primer set was used to check 71kb deletion of Prox1-EGFP BAC.
HP726/727 primer set was used to check EGFP gene.
HP4174/4175 primer set was used to check pBACe3.6 vector.
- 41 -
Primers sequence:
>HP4007 sequence (forward)
ACACAACGTGGCTTTGTTGA
>HP4008 sequence (reverse)
TTGAGACACAACGTGGCTTT
>HP4005 sequence (forward)
GGTTTTTACGTGGGCTCTGCAGATTTGAGCACAAGTCCTCCTTGCTGCAGACATGCTGTGAGGATGACGACGATAAGTAGGG
>HP4006 sequence (reverse)
AATACACCGAAGTCCCCTGTCACAGCATGTCTGCAGCAAGGAGGACTTGTGCTCAAATCTAACCAATTAACCAATTCTGATTAG
>HP4013 sequence (forward)
CTTTTACACCCGCTACCCCAGCTCCAACATGCTGAAGACCACAGTCTATCACTAGCCTGA AGGATGACGACGATAAGTAGGG
>HP4014 sequence (reverse)
CTTAGCCTCATAGGTGAGCTTCAGGCTAGTGATAGACTGTGGTCTTCAGCATGTTGGAGCAACCAATTAACCAATTCTGATTAG
>HP4002 sequence (forward)
CGGTCGAGCTTGACATTGTA
>HP4004 sequence (reverse)
GGCCCCTGGAGTTATAGACC
>HP3085 sequence (forward)
GAGGGCCACACTCTTTTGTT
>HP4016 sequence (reverse)
GTCTCTGGGTCTCTGCCAAG
>HP726 sequence (forward)
TGCTCAGGTAGTGGTTGTCG
>HP727 sequence (reverse)
CACATGAAGCAGCACGACTT
>HP4174 sequence (forward)
GTTCCCACTCCCTGTGAAGG
>HP4175 sequence (reverse)
CCGTTTCGATCCTCCCGAAT
pEPKan-s2 PCR amplification
pEPKan-s2 was originally generated by Dr. Osterrieder’s group in 2006 [14]. The DNA
fragment contains I-SceI restriction site and kanamycin resistance. Dr. Jae U. Jung kindly
provided us the DNA. The full sequence of pEPKan-s2 is as follows.
> pEPKan-s2 sequence
AGGATGACGACGATAAGTAGGGATAACAGGGTAATCGATTTATTCAACAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAA
AAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCGAGG
CCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGG
GAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAA
TTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTA
GAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGC
GATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGA
ACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGG
GAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTT
CATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAAT
TGGTTAATTGGTT
- 42 -
pEPKan-s2 was PCR amplified with the 82bp long forward primer and 84bp long reverse
primer to add 5’ and 3’ 60bp overhang, respectively. For 85kb deletion (3687bp to 89965bp
(refer to Figure 3. legend for designation of relative position)) of Prox1-EGFP BAC,
HP4005/4006 primer set was used to amplify pEPKan-s2. For 71kb deletion, HP4013/4014
primer set was used.
The PCR product was digested with DpnI to get rid of the template. Then, the pEPKan-s2
amplicon was checked by running the DNA in 1% agarose gel. After confirming that PCR has
taken place correctly (120bp elongation), the DNA was eluted from the gel with a gel elution kit
(Qiagen catalog number: 28704).
Electroporation
DH10B, Pir2 and Gs1783 was made into the electrocompetent cells by following the
protocol established by Dr. Lee group [13]. Briefly, the E. coli cells were grown overnight in
Luria Bertani (LB) medium with appropriate antibiotics (i.e., 12.5 μg/mL chloramphenicol and
50ug/ml ampicillin to maintain bacterial artificial chromosome (BAC), 12.5ug/ml
chloramphenicol, 50ug/ml ampicillin and 50ug/ml kanamycin after the homologous
recombination) in a shaker at 220rpm in 37°C (32°C for Gs1783). 100 mL of warm new LB
medium (with antibiotics) was inoculated at a 1:50 ratio with overnight culture. Inoculated
culture was incubated in a shaker at 220 rpm in 37°C (32°C for Gs1783) until OD
600
reached
0.5–0.7 nm. The culture was placed in wet ice and chilled for 20 minutes. The culture was
distributed into two 50ml conical tubes and then spun down at 4°C at ≥4500× g for 5-10 minutes
and the supernatant was discarded. The bacteria were re-suspended with ice-cold autoclaved DW
and transferred to sterile round bottom 15ml tubes. The washing process was repeated 2 to 3
- 43 -
times in round bottom tubes. After the last washing step, the bacteria were re-suspended in 1ml
of ice-cold autoclaved DW. 50ul of electrocompetent cells were used for each electroporation.
The setting for electroporation was 2.5 kV with (ice cold) 2mm cuvettes (Genesee, catalog
number: 40-101).
First homologous recombination by Gs1783’s innate system
Gs1783 was cultured in Luria Bertani (LB) medium with appropriate antibiotics in a
shaker at 220rpm and at 30°C. 100 mL of warm new LB medium (with antibiotics) was
inoculated at a 1:50 volume ratio with overnight culture. Inoculated culture was incubated in a
shaker at 220 rpm in 32°C until OD
600
reached 0.5–0.7 nm. The culture was transferred to 42°C
water bath shaker and was shaken at 220 rpm for 12 minutes. Then, the culture was placed in wet
ice and chilled for 20 minutes. The culture was distributed into two 50ml conical tubes and then
spun down at 4°C at ≥4500× g for 5-10 minutes and the supernatant was discarded. The bacteria
were re-suspended with ice-cold autoclaved DW and transferred to sterile round bottom 15ml
tubes. The washing process was repeated 2 to 3 times in round bottom tubes. After the last
washing step, the bacteria were re-suspended in 1ml of ice-cold autoclaved DW. 50ul of
electrocompetent cells were used for each electroporation. 100 ng of pEPKan-s2 PCR product or
BACs were electroporated into 50 μL bacteria with the following setting: 2.5 kV with (ice cold)
2mm cuvettes (Genesee, catalog number: 40-101).
Then, the electroporated bacteria were incubated in a shaker for 1h at 220 rpm in 30-32°C
with addition of 450µl LB medium without antibiotics. The bacteria were spun down and re-
suspended in 100ul of LB medium without antibiotics. The bacteria were plated on to an agar
plate with appropriate antibiotics (i.e., 12.5 μg/mL chloramphenicol and 100ug/ml ampicillin for
- 44 -
the original BACs and 12.5 μg/mL chloramphenicol and 100ug/ml ampicillin and 100ug/ml
kanamycin after homologous recombination). The plates were incubated at 32°C for
approximately 24h (until colonies are visible).
The candidate colonies were checked by colony PCR. HP4007/4008 primer set was used
to check for correct insertion of pEPKan-s2 PCR product for 85kb deletion. For 85kb+71kb
deletion, colony PCR was not done. Instead, second homologous recombination step was taken
right after the first homologous recombination.
SceI induction and second homologous recombination by Gs1783’s innate system
Fresh overnight culture of Gs1783 that went through first homologous recombination was
inoculated into LB media at 1:50 ratio with appropriate antibiotics. The inoculated culture was
grown for for 2–4 h at 32°C and 220 rpm until bacteria reached early logarithmic phase (OD600
0.5–0.7 nm). 2mL of logarithmic phase culture was mixed with 2 mL of warm LB medium with
the appropriate antibiotics (total volume: 4ml). The mixture was cultured in a shaker at 32°C and
220 rpm for 45 minutes. Then, the culture was transferred to 42°C water bath shaker (220 rpm)
for 12 minutes. Recovery culture was done in 32°C at 220 rpm for another 1-2h. Finally, the
culture was spun down and re-suspended with 100ul LB medium and plated onto agar plates
with chlroramphenicol (12.5ug/ml) and ampicillin (50ug/ml) and 1% arabinose. The plates were
incubated at 32°C for 24–48h. The colonies’ kanamycin sensitivity was checked by transferring
the colonies onto chlroramphenicol (12.5ug/ml), ampicillin (50ug/ml) and kanamycin (100ug/ml)
plates.
- 45 -
CsCl prep of 85kb deletion BAC construct and 85kb+71kb deletion BAC construct
85kb deletion BAC construct and 85kb+71kb deletion BAC construct in Pir2 E.coli cell
line was cultured in 1L LB medium (chlroramphenicol 12.5ug/ml, ampicillin 50ug/ml) for 12- 24
hours. The culture was spun down at 4,000 rpm for 25 minutes and the supernatant was
discarded. The bacteria were re-suspended in 25ml of solution 1. Then, 50ml of solution 2 was
added gently. After mixing, 37.5ml of solution 3 was added gently. The final mixture was spun
down at 10,000 rpm for 20 minutes at 4°C. Clear supernatant was transferred clean tubes. 67.5ml
of isopropanol (VWR, catalog number: EMD-PX1835-5) was added and then the mixture was
spun down at 10,000 rpm for 20 minutes at 4°C. The supernatant was discarded and the pellet
was re-suspended with appropriate amount of DW to fully melt the pellet. Then, the solution was
treated with appropriate amount of RNaseA (Biobasic, catalog number: RB0473) and was
incubated at 37°C for 1h ~2h to get rid of RNA. Next, the solution was treated with the same
volume of phenol/chloroform (Amresco, catalog number: K169) and was mixed well by
vortexing thoroughly and then was spun down at 14,000 rpm for 15 minutes in 2°C. The upper
layer was collected and then treated with 0.6 volume of isopropanol and was spun down at
10,000 rpm for 20 minutes at 4°C. The resulting pellet was re-suspended with 500ul of DW.
500ul of re-suspended solution was transferred into ultracentrifuge tubes (BeckmanCoulter,
catalog number: 361621) and 30ul of EtBr (Sigma, catalog number: E7637-1G, 10ug/ul) was
added. Then, CsCl solution was added until the ultracentrifuge tube is completely full. The
solution was spun down at 80k for over 16 hours at room temperature.
The lower red band after the ultracentrifuge spin was retrieved with 25g needle with 3-
5ml syringe. The DNA solution’s EtBr was removed with TE-saturated butanol by mixing the
solution with TE-saturated butanol in 1:3 volume ratio and removing the top layer. 2.2 volume of
- 46 -
DW was added. 1 volume (after DW addition) of isopropanol was added and then the solution
was spun down at 7,000 rpm at 10°C for 15 minutes. The supernatant was discarded and the
pellet was washed with 70% ethanol. Finally, the BACs were re-suspended in 500ul DW.
Solutions for CsCl prep
Sol’n I (100ml)
50 mM Glucose : 5ml of 1M Glucose (Sigma, catalog number: G8270-1KG) (autoclaved or
filtered)
25 mM Tris-Cl (pH 8.0): 2.5 ml of 1 Tris-Cl (pH 8.0)
10 mM EDTA (pH 8.0): 2 ml of 0.5M EDTA (pH 8.0)
Store at 4 ℃.
Sol’n II (100ml)
0.2 N NaOH: 2 ml of 10N NaOH (EMD Millipore, catalog number: SX0607N)
1 % SDS (VWR, catalog number: PI28365): 5 ml of 20% SDS
Store at 25 ℃.
Sol’n III (100 ml)
5M Potassium acetate (Sigma, catalog number: P1190-1KG)(49g/100 ㎖): 60 ml
Glacial acetic acid (Sigma, catalog number: 242853): 11.5 ml
dH2O : 28.5 ml
Store at 4 ℃.
CsCl solution
Dissolve 350g of CsCl (Sigma, catalog number: C3032-1KG) in 300ml DW and then autoclave.
Add autoclaved DW until the final volume is 400ml.
- 47 -
TE-saturated butanol
Mix 10 mM Tris, 1 mM EDTA (bring to pH 8.0 with HCl) with butanol (Sigma, catalog
number : 360465) in 1:3 volume ratio overnight.
Dialysis
4L of 10mM TRIS (pH 8.0), 1mM EDTA solution and 2L of 10mM TRIS (pH 8.0),
0.1mM EDTA solution was prepared with autoclaved DW. CsCl prep of 85kb deletion BAC
construct and 85kb+71k deletion BAC construct was loaded into Slide-A-Lyzer Dialysis
Cassettes 10k Mwco 0.5-3ml (Pierce, product number: 66425) and then was washed in 2L of
10mM TRIS (pH 8.0), 1mM EDTA solution for 2 hours. Next, the samples were washed in 2L of
10mM TRIS (pH 8.0), 1mM EDTA solution overnight. Finally, the samples were washed in 2L
of 10mM TRIS (pH 8.0), 0.1mM EDTA solution for 2 hours.
TRIS (Bioland, catalog number: CT01-5KG), EDTA (Biopioneer, catalog number: C0119)
Restriction analysis
1ug of dialyzed CsCl prep of 85kb deletion BAC construct (2ul of 500ng/ul solution) was
mixed with 0.5ul of XmaI restriction enzyme (NEB, catalog number: R0180) and 2ul of
CutSmart buffer (NEB, catalog number: B7204). The total volume was brought up to 20ul with
DW. The mixture was incubated in 37°C for 2 hours. 85kb+71kb deletion BAC construct was
digested with EcoRI (NEB, catalog number: R3101) with the same condition.
- 48 -
Transfection
CHO, SW620 and LEC were cultured in 12 well culture dish (Genesee, catalog number:
25-106) so that each well is about 70% confluent. 1ug of dialyzed CsCl prep of 85kb deletion
BAC construct (2ul of 500ng/ul solution) and 1ug of dialyzed CsCl prep of 85kb deletion BAC
construct (2ul of 500ng/ul solution) was first suspended in 50ul of Opti-MEM media
(ThermoFisher Scientific, catalog number: 31985062). Next, 3ug or 4ug of Lipofectamine 2000
(3ul of 1ug/ul solution, Invitrogen, catalog number: 11668) was suspended in 50ul of Opti-MEM
media. Both solutions were incubated in room temperature for 5 minutes. DNA solution was
mixed into Lipofectamine 2000 solution and was incubated in room temperature for 20 minutes.
Each well was washed with PBS (Sigma, D8537) once and was filled with 200ul culture medium.
Then, the DNA-Lipofectamine 2000 complex was treated to each well.
Immunofluorescence analysis
LEC, CHO and SW620 was grown in 12 well on round glass coverslips (Warner, 64-
0384). Then, the cells were washed twice with PBS and were fixed in the 1:1 mixture of
aceton/methanol for 10 minutes at room temperature. The fixed cells were stained with anti-
Prox1 antibody (ReliaTech, product number 102-PA32AG). Corresponding secondary antibodies
were labeled with Alexa Fluor 594 (ThermoFisher, product No. A-11037).
Mice’s eyes, large intestines, hearts and livers were isolated following mouse
experimental guidelines of the USC Department of Animal Resources. After observing each
organ for EGFP expression with a stereoscope, frozen sections of the organs were fixed in cold 4%
paraformaldehyde for 20 minutes and were observed again for EGFP expression. Images were
- 49 -
acquired and processed using a 20x objective on a Zeiss microscope and AxioVision Digital
Imaging software.
- 50 -
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Abstract (if available)
Abstract
Prospero homeobox 1 or Prox1 is a mammalian homeobox transcription factor that is critical for lymphatics development and lymphatic endothelial cell differentiation [3]. A transgenic mouse model called Prox1-EGFP mouse has been developed and its lymphatics show robust EGFP expression [1]. However, Prox1 is expressed in various other types of cells, including neurons, hepatocytes and myocytes [5]. Thus, it is difficult to visualize lymphatics in tissues rich with these cell types. ❧ Approximately 80% of mouse enhancers show tissue-specific expression [6] and it is likely that Prox1-EGFP BAC, the bacterial artificial chromosome that was used to generate Prox1-EGFP mouse, has multiple enhancer regions that direct Prox1 promoter-coupled EGFP expression for various tissues. Based on human ENCODE data, the putative enhancer region of the BAC was postulated and the region was deleted. The effects of the deletion have been screened in vitro and in vivo. The result of this project will assist in the identification and the characterization of enhancers for Prox1 gene.
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Jung, Wonhyeuk
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Identification and characterization of the enhancer elements for lymphatic-specific expression of Prox1
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Biochemistry and Molecular Biology
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07/05/2016
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