Non-invasive monitoring of hepatocellular carcinoma in transgenic mouse with bioluminescent imaging.
ABSTRACT A small animal imaging system for hepatocellular carcinoma (HCC)-specific reporter gene expression will enable monitoring of carcinogenesis or therapeutic intervention in vivo. Transgenic mouse was developed in which firefly luciferase (fLuc) expression was controlled by the AFP enhancer/promoter. The bioluminescent signals of the transgenic neonates were strong at their liver region and decreased after birth. Bioluminescent imaging (BLI) of a transgenic mouse treated with N-nitrosodiethylamine revealed distinct fLuc activity in the liver and an increased pattern with time. The transgenic mouse model can be used to monitor AFP producing HCC by a chemical carcinogen in a live animal by BLI.
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ABSTRACT: Optical molecular imaging, a new medical imaging technique, is developed based on genomics, proteomics and modern optical imaging technique, characterized by non-invasiveness, non-radiativity, high cost-effectiveness, high resolution, high sensitivity and simple operation in comparison with conventional imaging modalities. Currently, it has become one of the most widely used molecular imaging techniques and has been applied in gene expression regulation and activity detection, biological development and cytological detection, drug research and development, pathogenesis research, pharmaceutical effect evaluation and therapeutic effect evaluation, and so forth, This paper will review the latest researches and application progresses of commonly used optical molecular imaging techniques such as bioluminescence imaging and fluorescence molecular imaging.BioMed research international. 01/2014; 2014:429198.
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ABSTRACT: The in vivo molecular imaging method is a useful tool for monitoring carcinogenesis in various hepatocellular carcinoma (HCC) models, such as xenografted-, chemical induced- and transgenic mice. The tumor-specific gene expression strategy, such as transcriptional targeting, is essential for achieving a lower toxicity for normal liver tissue in therapy and the monitoring of tumor progression in diagnosis, respectively. The present study aimed to visualize spontaneously developing α-fetoprotein (AFP)-producing HCC through targeted gene expression in tumors using recombinant adenoviral vector. The recombinant adenovirus vector, AdAFPfLuc (containing firefly luciferase gene driven by human AFP enhancer/promoter) was prepared. After in vitro infection by adenovirus, gene expression was confirmed using the luciferase assay, semi-quantitative reverse transcriptase-polymerase chain reaction and western blotting in AFP-producing and nonproducing cells. Tumor-bearing mice were intravenously injected with adenovirus, and bioluminescent images were obtained. The expression of fLuc was efficiently demonstrated by the luciferase assay in AFP-producing cells but not in AFP-nonproducing cells. AFP-producing HCC targeted gene expression was confirmed at the mRNA and protein levels. After being injected intravenously in HuH-7 xenografts and HCC-bearing diethylnitrosamine-treated mice using adenovirus, functional reporter gene expression was confirmed in tumors by in vivo bioluminescent imaging (BLI). The recombinant adenovirus vector system can be used to monitor spontaneously developing AFP-producing HCC and to evaluate targeted gene expression in tumors by in vivo BLI in a small animal model.The Journal of Gene Medicine 07/2012; 14(8):513-20. · 2.16 Impact Factor
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ABSTRACT: Bioluminescent imaging (BLI) is a powerful noninvasive tool that has dramatically accelerated the in vivo interrogation of cancer systems and longitudinal analysis of mouse models of cancer over the past decade. Various luciferase enzymes have been genetically engineered into mouse models (GEMM) of cancer, which permit investigation of cellular and molecular events associated with oncogenic transcription, posttranslational processing, protein-protein interactions, transformation, and oncogene addiction in live cells and animals. Luciferase-coupled GEMMs ultimately serve as a noninvasive, repetitive, longitudinal, and physiologic means by which cancer systems and therapeutic responses can be investigated accurately within the autochthonous context of a living animal.Cancer Discovery 04/2013; · 15.93 Impact Factor
Non-invasive monitoring of hepatocellular carcinoma in transgenic
mouse with bioluminescent imaging
Ju Hui Parka,c, Kwang Il Kima, Yong Jin Leea, Tae Sup Leea, Kyeong Min Kima, Sang-Soep Nahmb,
Young-Seo Parkc, Gi Jeong Cheona,d, Sang Moo Limd, Joo Hyun Kanga,⇑
aMolecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences (KIRAMS), Seoul 139-706, Republic of Korea
bDepartment of Veterinary Medicine, Konkuk University, Gyeonggi-do 461-701, Republic of Korea
cDepartment of Food Science & Biotechnology, Kyungwon University, Gyeonggi-do 461-701, Republic of Korea
dDepartment of Nuclear Medicine, Korea Institute of Radiological and Medical Sciences (KIRAMS), Seoul 139-706, Republic of Korea
a r t i c l e i n f o
Received 6 April 2011
Received in revised form 27 May 2011
Accepted 9 June 2011
a b s t r a c t
A small animal imaging system for hepatocellular carcinoma (HCC)-specific reporter gene
expression will enable monitoring of carcinogenesis or therapeutic intervention in vivo.
Transgenic mouse was developed in which firefly luciferase (fLuc) expression was con-
trolled by the AFP enhancer/promoter. The bioluminescent signals of the transgenic neo-
nates were strong at their liver region and decreased after birth. Bioluminescent imaging
(BLI) of a transgenic mouse treated with N-nitrosodiethylamine revealed distinct fLuc
activity in the liver and an increased pattern with time. The transgenic mouse model can
be used to monitor AFP producing HCC by a chemical carcinogen in a live animal by BLI.
? 2011 Elsevier Ireland Ltd. All rights reserved.
Hepatocellular carcinoma (HCC) is the most common
type of liver cancer, accounting for more than 600,000
new cases of liver cancer worldwide with an increasing
incidence [1–4]. Currently, many systems for detecting
HCC are used, such as radioimmunoassay of serum sam-
ples for the detection of tumor markers, ultrasonography
(US), computed tomography (CT) and magnetic resonance
imaging (MRI) . a-fetoprotein (AFP) is a serum glycopro-
tein whose concentration decreases rapidly after birth and
its expression is repressed in adults. In the 1960s, it at-
tracted attention after its discovery in adults during carci-
nogenesis , with approximately 80% of HCC patients
showing an increase in the AFP level [7,8]. Therefore, AFP
has been used for many years as a diagnostic and prognos-
tic serum marker for HCC [9,10] and a transgenic system
for AFP was proposed to be a valuable tool for examining
the mechanism of the transcriptional regulation during li-
ver development and hepatocarcinogenesis .
N-nitrosodiethylamine (DEN) was reported to induce
hepatocelluar carcinoma after a single injection in neonatal
infant male mice . Hepatocelluar foci and microcarci-
noma induced by DEN have alpha-fetoprotein positive
characteristics . Naugler et al. demonstrated that the
administration of DEN in male mice caused larger in-
creases in the IL-6 concentration than in females, and the
estrogen-mediated inhibition of IL-6 production reduces
the HCC risk in females .
Owing to the recently developed in vivo imaging sys-
tems equipped with a sensitive cooled charge coupled de-
vice (CCD) camera , an optical imaging method using
fluorescence and bioluminescence is useful for monitoring
the progression or intervention of diseases in live small
0304-3835/$ - see front matter ? 2011 Elsevier Ireland Ltd. All rights reserved.
⇑Corresponding author at: Molecular Imaging Research Center and
Department of Nuclear Medicine, Korea Institute of Radiological and
Medical Sciences (KIRAMS), 75 Nowon-gil, Gongneung-Dong, Nowon-Gu,
Seoul 139-706, Republic of Korea. Tel.: +82 2 970 1339; fax: +82 2 970
E-mail address: firstname.lastname@example.org (J.H. Kang).
Cancer Letters 310 (2011) 53–60
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/canlet
animals. Among them, bioluminescence imaging (BLI) can
be applied to the quantitative and real-time analysis of
gene expression or the tracking of target cells, such as stem
cells and immune cells in living animals . This tech-
nique is more convenient and cheaper than other imaging
modalities, such as positron emission tomography (PET)
and MRI, and has less harmful effects from radiation or
magnetic fields . Luciferase from firefly Photinus pyralis
(fLuc) is used most widely as a BLI reporter gene .
Molecular imaging based on reporter gene expression al-
lows tissue-specific events or processes to be measured
using the BLI reporter gene expression vector controlled
by specific enhancer/promoters [18–20]. Maggi et al. 
reported that a reporter gene expressing transgenic animal
model can support the rapid monitoring of the entire body
in which a given compound is active, measure the drug re-
sponse in a range of tissues according to the administration
routes, evaluate the minimum concentration of drug
needed to obtain the desired pharmacologic response inde-
pendently of its plasma levels, and discover the active
metabolites and their action profiles.
In the present study, fLuc expressing transgenic mice
controlled by the AFP enhancer/promoter (enh/promoter)
were produced to screen for the development of AFP-
producing HCC. These models are expected to be useful
for monitoring the agents or drugs that modulate the AFP
level and measuring the specific signaling events impor-
tant for HCC development.
2. Materials and methods
2.1. Cell lines and transient transfection
The AFP-producing human HCC cell line, HuH-7 (JCRB
0403) and the AFP-nonproducing human embryonic kid-
ney cell line, 293A (ATCC CRL-1573), were chosen for this
comparative study of reporter gene expression according
to the levels of AFP expression. The HuH-7 and 293A cells
were cultured in Dulbecco’s modified eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum
(FBS) and 1% antibiotics in an atmosphere containing 5%
CO2at 37 ?C. All transient transfections were carried out
according to the manufacture’s instructions. To determine
the level of AFP secreted from the cultured cells, 2 ? 105
cells were plated in the wells of a 6-well plate and incu-
bated for 48 h. The media was exchanged with fresh media
without FBS. After further incubation for 48 h, the number
of cells was counted using the trypan blue exclusion meth-
od, and the AFP content in the medium was measured
using an AFP IRMA kit (Immunotech, Murmanská, Czech).
Each value represents the mean ± SD of three wells.
2.2. Reporter vector construction
The pGL-Basic vector (Promega, Madison, WI) was used
to make a construct containing 2.1 Kb of the alpha-fetopro-
tein enh/promoter. The AFP enh/promoter gene was ampli-
fied from the plasmid, pDRIVE-AFP-hAFP (Invivogen, San
Diego, CA), using a polymerase chain reaction and inserted
into the Mlu I and Xho I sites of pGL-Basic. The primer con-
50-GAT CAC GCG TGC TTA GAA ATA TGG GGG TAG-30and 50-
GAT CCT CGA GGT TGC TAG TTA TTT TGT TAT TG-30, respec-
tively. The resulting plasmid was designated pAFP-fLuc.
pCMV-fLuc was also constructed using the pcDNA3.1/
Hygro(+) vector (Invitrogen) as a backbone. The fLuc gene
was amplified from the plasmid pGL-Basic by PCR and in-
serted into the HindIII and XhoI sites.
2.3. Transient transfection analysis of plasmid DNA
To normalize the transfection efficiency, the fLuc activ-
ity of pCMV-fLuc transfected into HuH-7 and 293A cells
was calculated as 100%. After 24 h transfection, the cells
were washed with phosphate buffered saline (PBS) and
harvested using a lysis solution (Tropix, Bedford, MA).
The cell lysates with luciferase substrate A (ATP) and B
(D-luciferin) were placed on an opaque 96 well plate and
measured using a Luminometer (Spectramax, Molecular
devices, Sunnyvale, CA). The analysis program used was
Softmax pro5.2 (Molecular devices).
2.4. Generation of transgenic mice and RT-PCR
Transgenic mice were generated at Macrogen (Seoul,
Korea). The transgenic mice were generated by the micro-
injection of a linear transgene containing the AFP enh/pro-
moter-fLuc sequence, in which pAFP-fLuc was digested
with MluI and SalI into super-ovulated fertilized eggs of
C57BL/6 mice. The presence of the transgene in the foun-
ders was confirmed by PCR using the genomic DNA ex-
tracted from the tail. The primers for genotyping were as
follows: forward primer, 50-AATAGGCATAGAGCCAGGACT
and reverse primer, 50-AACGCGCCCAAC ACCGGCATA. The
total RNA was extracted from the liver using TRI-Reagent
(Molecular Research Center, Cincinnati, OH), precipitated
with isopropanol, and dissolved in DEPC-treated distilled
water. cDNA synthesis was performed according to the
manufacturer’s instructions using Superscript III reverse
transcriptase (Invitrogen). The following primers were
used to detect the fLuc mRNA by RT-PCR: forward primer,
50-GGCCTTTATGAG GATCTCTCT and reverse primer, 50-
CGCCTTGATTGACAAGGATGG. Beta-actin mRNA was used
as the internal standard.
2.5. In vivo bioluminescence imaging
All BLIs were acquired using an IVIS imaging system
series 200 (Xenogen, Hopkinton, MA) to detect the biolu-
minescence signals in the transgenic mice. The BLIs of
the neonates of the positive founders were acquired to
confirm the fLuc activity. Each neonate was injected intra-
peritoneally with 3 mg of D-luciferin (Molecular Imaging
Products Company, Bend, OR) in PBS and the BLIs were ac-
quired immediately over a 1 min period. Ex vivo imaging of
the excised organs was performed after a D-luciferin injec-
tion to confirm the signal location. The intestines, stomach,
liver, heart and lungs were imaged using an IVIS imaging
system. To measure the intensity of emitted light, a region
of interest (ROI) was drawn over the emitted region and
J.H. Park et al./Cancer Letters 310 (2011) 53–60
the total photon efflux was determined. The biolumines-
cent signals are expressed in units of photons per cm2
per second per steradian (p/cm2/s/sr).
2.6. AFP expression in neonatal liver
After the BLIs of 10 neonates from two positive lines
(founder number 39 and 67) under 10 days after birth
were acquired, the mice were sacrificed and the AFP pro-
duction level was determined using the total RNA and total
cell protein from a neonatal liver. The AFP expression level
was detected from the band intensity of the RT-PCR prod-
ucts on agarose gel (Gel-pro Analyzer 3.1, Media Cybernet-
ics, Bethesda, MD) and is presented as the relative band
intensity of the AFP PCR product to the internal control.
The primers were used to detect mouse AFP mRNA: for-
ward primer, 50-GCTGTCACTGCAGATTTCTC and reverse
primer, 50-CTCACATGGACA TCTTCACC. Mouse beta-actin
was used as the internal standard. For western blot analy-
sis, the total protein was extracted from a frozen liver
using CelLytic™ MT Mammalian Tissue Lysis/Extraction
Reagent (Sigma–Aldrich, Inc., Louis, MO) and measured
according to the Bradford method. The proteins were
transferred to an Immune-Blot™ PVDF membrane (Bio-
rad, Hercules, CA). The primary antibody, AFP antibody
and beta-actin antibody, were purchased from Cell signal-
ing Technology, Inc. (Danvers, MA), and the secondary
antibody, anti-rabbit IgG-HRP, was purchased from Sig-
ma–Aldrich, Inc. The immunoreactive bands were detected
using ECL reagent (SuperSignal?West Pico Chemilumines-
cent Substrate, Thermo Scientific, Barrington, IL).
2.7. DEN induced HCC in transgenic mouse
The DEN (Sigma–Aldrich)-induced HCC mouse model
was produced by an intraperitoneal injection. Twelve
3 week old male transgenic mice from a single positive line
(founder number 39) were injected intraperitoneally once
with 20 mg/kg body weight of DEN. HCC was determined
at32 weeksofageby3TMRI.MRscanningusinga3 TMAG-
NETOM trio (Siemens AG, Munich, Germany) with a wrist
coil. Before scanning, the animals were anesthetized with
2% isoflurane in oxygen. Gadolinium-based contrast media
(Primovist, Bayer HealthCare, Germany, 1.25 mmol/kg)
izer images were obtained (TR = 8.6 ms, TE = 4.00 ms, FoV
read 180 mm, 3.0 mmslice thickness)and after positioning,
all MR images were acquired using a T2-weighted VIBE se-
quence:TR = 1620 ms, TE = 37 ms,
256 ? 256 matrices, 1.0 mm slice thickness and number of
excitations = 2. After the MRI scan, the BLIs were acquired
for approximately 6 months. The care, maintenance and
treatment of animals in these studies followed protocols
FoV read60 mm,
2.8. Histological examination
The HCC bearing transgenic mice were sacrificed after
imaging and a histological assessment of liver specimen
was performed. Immunohistochemistry was processed after
deparaffinization and rehydration from specimens fixed in
10% buffered paraformaldehyde. Antigen retrieval was per-
formed with sodium citrate (pH, 6.0) in blocking solution
(PBS added 5% (v/v) horse serum) for 2 h and the AFP IHC
antibody (goat anti-mouse antibody, IHC world) as the pri-
mary antibody was incubated overnight at 4 ?C. The primary
the second antibody, anti-mouse IgG/biotinylated (Vector
Laboratory, Burlingame, CA). Color development was per-
formed using an AP substrate kit (Vector Laboratory).
3.1. In vitro studies
The fLuc activities of the transient transfected cells were measured to
confirm the constructed reporter vector. The HuH-7 and 293A cells were
selected as the AFP-producing and AFP-nonproducing cells, and the level
of AFP secretion was determined using a radioimmunoassay. The amount
of AFP produced from the HuH-7 and 293A cells was 2133.95 ± 92.80 and
0.27 ± 0.14 ng/day/105cells, respectively. The HuH-7 and 293A cells
transfected with pAFP-fLuc or pCMV-fLuc showed high fLuc activities
compared to the untransfected control group. When the transfection effi-
ciency was normalized to pCMV-fLuc, the fLuc activity of the pAFP-fLuc
transfected HuH-7 cells was found to be approximately two times higher
than that of the pAFP-fLuc transfected 293A (Fig. 1). Therefore, the fLuc
activities of the pAFP-fLuc transfected HuH-7 exhibited specific reporter
gene expression in the AFP-producing cells.
3.2. Production of transgenic mice
After injecting the fertilized eggs with a linear transgene containing
the AFP enh/promoter-fLuc sequence, they were implanted into the uter-
ine of foster mothers. The presence of the inherited transgene was con-
firmed by the genomic PCR, and 11 transgenic mice were finally
selected among 110 founders. There were eight lines of male transgenic
mice (founder numbers: 39, 42, 57, 59, 74, 78, 81 and 82) and three lines
of female transgenic mice (founder numbers: 37, 55 and 67). One positive
founder of the 11 transgenic mice (female founder number 37) did not
transmit the transgene to its progenies.
3.3. Confirmation of reporter gene expression in transgenic mice
After 10 positive founders were mated with the wild type mice, the
neonates (under 1 day after birth) of three male lines (founder 39, 59
and 78) and 1 female line (founder 67) expressed fLuc in their liver using
IVIS-200 (Fig. 2). On the other hand, the neonates of the wild type mouse
Fig. 1. Relative fLuc activities of HuH-7 and 293A transfected with pAFP-
fLuc compared to those of transfection with pCMV-fLuc.
J.H. Park et al./Cancer Letters 310 (2011) 53–60
did not show fLuc expression (Fig. 2A). Six to seven neonates could be ob-
tained from the transgenic mouse and 2 (Fig. 2D), 3 (Fig. 2B and E) or 4
(Fig. 2C) neonates showed BLI in the trunk of the body. This result corre-
sponded to Mendelism. The D-luciferin injected transgenic neonates were
sacrificed and their organs were isolated to determine the origin of the
bioluminescence (BL) signal of the transgenic mice. Ex vivo images of
the excised organs (the intestine, stomach, liver, heart and lung) showed
fLuc expression (Fig. 3). BLI indicated that the fLuc activity in the trans-
genic neonates of the positive founder was located exclusively in the liver
and not expressed in the other organs (lower panel of Fig. 3A). The BLIs of
the non-transgenic neonates of the positive founder did not appear, even
in the liver (upper panel of Fig. 3A). The expression of fLuc in the trans-
genic mice was confirmed at the mRNA level. As shown in Fig. 3B, fLuc
mRNA was expressed in the livers from the neonates of the transgenic
mice (founder 39, 58, and 78) but not in the wild type mouse.
3.4. Correlation with the AFP expression level and fLuc activity in neonatal
The BLI of two neonates from one positive line (founder 39) was per-
formed as a function of time after birth to determine if fLuc expression is
reduced by a decrease in the AFP level after birth (Fig. 4). BLIs in the neo-
nates of the positive founder were obtained at 1 day, 5 days and 10 days
after birth. At 5 days after birth, the fLuc activity was less intense than
that 1 day after birth and was barely observed at 10 days. By quantifying
the light intensity, a rapid decrease in fLuc activity was observed in the
liver region (Fig. 4B). The extracted RNA and protein from the liver exhib-
ited a pattern of decrease in the AFP amounts after birth, which showed
the same pattern in the expressed fLuc activity by the BLI system
(Fig. 5). The relative AFP expression level to b-actin correlated with the
fLuc activity (RNA: R2= 0.8182, protein: R2= 0.9494).
3.5. BLIs of DEN induced HCC in transgenic mouse
The aim of this study was to monitor HCC in transgenic mice induced
by DEN as a chemical carcinogen with BLIs. The HCC model was evaluated
using a 3T MRI scan (Fig. 7A). Seven of the DEN-treated twelve transgenic
mice did not develop tumors until 32 weeks after the DEN treatment. The
BLI of the DEN induced HCC in the transgenic mice showed distinct activ-
ity in the abdominal region, which increased with time for 8–14 months
after the DEN treatment (Fig. 6). To confirm where the BL signal origi-
nated in the DEN treated transgenic mouse, the transgenic mouse was in-
jected with D-luciferin, sacrificed and BLI were acquired with IVIS-200. Ex
vivo BLI showed that the fLuc activity was located exclusively in the nod-
ules of the liver (Fig. 7B).
The chemical carcinogen-induced HCC regions manifested as multiple
nodules of various sizes and color (Fig. 7C). Not all nodules could be de-
tected on BLI. The margin of the tumor and abnormal nuclei were signif-
icant according to H&E staining (Fig. 7D). The induced-tumor regions
were determined to be HCC or hepatocellular adenoma. Immunohisto-
chemistry was performed against AFP to confirm AFP expression in BL sig-
nal-positive nodules. As shown in Fig. 7E, the BL signal positive nodules
expressed AFP protein and indicated by black arrows.
Non-invasive molecular imaging techniques provide
valuable temporal and spatial information on diseases in
small animal models. In vivo molecular imaging is an
essential basic tool for evaluating the effect of gene ther-
apy and physical function in medicine and can obtain a
quantifiable image of the targeted organs or tissues. There-
fore, molecular imaging with the BL reporter genes has
been applied extensively to monitoring the disease pro-
gression and the development of new therapeutics.
Some transgenic mouse models were developed by re-
porter gene expression controlled by AFP enh/promoter
[22,23]. Kwon et al.  reported a green fluorescent pro-
tein (GFP)-expressing transgenic mice model controlled by
AFP enh/promoter and could acquire bright GFP fluores-
cence images in their embryos, which was localized at york
sac and liver. On the other hand, they could invasively
acquire the fluorescent images in the embryos of model
Fig. 2. BLIs for the neonates of wild-type and four positive founders. Each positive founder was mated with the wild-type female or male partner. One day
after birth, their neonates were anesthetized and intraperitonial injected with D-luciferin. The neonatal fLuc expression images were obtained using an IVIS-
200 system. (A) Neonates from wild type mouse, (B) neonates from founder number 39, (C) neonates from founder number 59, (D) neonates from founder
number 67, (E) neonates from founder number 78.
J.H. Park et al./Cancer Letters 310 (2011) 53–60
animal. Cany et al.  also produced transgenic mice
detected b-galactosidase expression in fetal hepatocytes
invasively. These transgenic mouse models could not
Fig. 3. Confirmation of the BL signal and fLuc expression in the mRNA level of the liver from transgenic mice. (A) After the neonates injected with D-luciferin
were sacrificed, ex vivo images of the excised organs were obtained. Ex vivo BLI of neonates of the founder. (upper) Non-transgene neonate from founder,
(lower) transgenic neonate from founder. Int: intestine, St: stomach, Li: liver, H: Heart, Lu: lung. (B) fLuc expression of transgenic neonates (founder
numbers 39, 59 and 78) was confirmed by RT-PCR. b-actin was used as an internal control. W indicate a non-transgene neonate.
Fig. 4. BLIs of the neonates according to time after birth. BLIs of the neonates from positive founder 39 were obtained 1 day, 5 days and 10 days after birth.
(A) Longitudinal BLI of the neonate after birth (B) the quantified BL signal on the emitted region. The fLuc activities in region of interest (ROI) decreased with
time after birth.
J.H. Park et al./Cancer Letters 310 (2011) 53–60
demonstrate relationships between the AFP level and re-
porter gene activity using an imaging method and might
have restrictive use as an imaging model for monitoring
hepatocarcinogenesis owing to its invasiveness. The trans-
genic mouse expressing fLuc by the AFP enh/promoter
exhibited a high BLI in HCC induced by DEN as well as a
gradual increase in BL activity in the liver region with time
(Fig. 6). Because the biotransformation processes of some
chemical carcinogens is necessary for carcinogenesis to in-
duce HCC, reporter gene expressing transgenic mice mod-
els will be needed to monitor the chemical carcinogens
rather than an in vitro assay in a culture plate. Therefore,
transgenic mouse model expressing fLuc by the AFP enh/
promoter will allow researchers to assess the tumor–host
interactions, find the carcinogens that can lead to a malig-
nant transformation of hepatocytes, and perform drug
screening for HCC treatments using noninvasive and sensi-
tive BLIs. A transgenic mouse expressing the fLuc reporter
gene controlled by prostate specific antigen (PSA) enhan-
cer/promoter was produced for prostate cancer. The results
Fig. 5. Relationship between the AFP expression level and fLuc activity in neonatal liver. After BLIs of the neonates from 1 to 10 days after birth were
acquired, RT-PCR for AFP transcripts (A) and western blot for AFP protein level (B) was performed using a neonatal liver. The AFP expression level was
calculated as a relative band intensity of AFP PCR product and AFP protein band to internal control. Mouse b-actin was used as an internal standard.
Fig. 6. BLIs of DEN induced HCC in transgenic mouse. Eight months after treatment with DEN, the BLIs of the transgenic mouse were acquired for 6 months.
The BL activities (A) exhibited in the abdominal region of DEN treated transgenic mouse and showed an increasing pattern according to time progression
J.H. Park et al./Cancer Letters 310 (2011) 53–60
demonstrated that androgen withdrawal could induce a
decrease in fLuc expression in the prostate, which is sensi-
tive to the availability of an androgen .
In conclusion, a transgenic mouse model for the imag-
ing of AFP-producing cells or organs by BLI was developed
using an AFP enh/promoter and fLuc. The BL signals mea-
sured by BLIs in the liver correlated well with the AFP lev-
els. This transgenic mouse model may be used to detect
liver cancer induced by a chemical carcinogen without
the need for sacrifice as well as for subsequent continuous
monitoring of the hepatocarcinogenesis process in vivo.
The transgenic mice can be also used to screen modulating
agents by the AFP expression level, such as chemical agents
or natural extracts, using in vivo BLI system.
Conflicts of interest
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