Original Article

Yonago Acta medica 2016;59:174–182

Evaluation of an Hprt-Luciferase Reporter Gene on a Mammalian Artificial
Chromosome in Response to Cytotoxicity
Takeshi Endo,* Natsumi Noda,*† Yasushi Kuromi,*‡ Kenji Kokura,§ Yasuhiro Kazuki,§||¶ Mitsuo Oshimura§and Tetsuya Ohbayashi‡*Tottori Industrial Promotion Organization, Tottori 689-1112, Japan, ‡Division of Laboratory Animal Science, Research Center for Bioscience and Technology, Tottori University, Yonago 683-8503, Japan, §Chromosome Engineering Research Center, Tottori University,
Yonago 683-8503, Japan, ||Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Tottori University,
Yonago 683-8503, Japan and ¶Division of Molecular and Cell Genetics, Department of Molecular and Cellular Biology, School of Life
Sciences, Tottori University Faculty of Medicine, Yonago 683-8503, Japan

ABSTRACT

is such a house-keeping gene, and has been utilized inmany studies of gene expression as a reference standard.
As we compared the gene expression inductions by thecompounds (mostly drugs) of internal control genes, using the Open TG-GATEs (Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System), HPRTgene presented the least variation.4 Although it has been
widely used as a standard in real-time quantitative reverse transcription PCR (RT-qPCR), the Hprt promoter/enhancer has not been extensively analysed.5, 6
Luciferase assay systems enable the real-time monitoring of gene expression in living cells. We used thegreen-emitting luciferase (SLG) from Rhagophthalmus
ohbai as a reporter gene in this study.7 Reliable expression systems are needed for the evaluation of in vitro
gene analysis, but transgene expression in various celllines established by random genomic integration using
conventional methods can be unstable or non-uniform
due to gene silencing. Mammalian artificial chromosome technology has been developed to overcome thisproblem. It has been demonstrated that human artificial
chromosomes (HACs) and mouse artificial chromosomes (MACs) are independently retained in host cells
and provide stable expression of transgenes.8, 9 In addition to these characteristics, features that allow cell-tocell transfer of HACs and MACs by microcell-mediatedchromosome transfer (MMCT) has shown potential for

Hypoxanthine guanine phosphoribosyltransferase (Hprt) is known as a house-keeping gene,and has been used as an internal control for real-timequantitative RT-PCR and various other methods of gene
expression analysis. To evaluate the Hprt mRNA levels
as a reference standard, we engineered a luciferase reporter driven by a long Hprt promoter and measured its
response to cytotoxicity.
Methods We constructed a reporter vector that harbored a phiC31 integrase recognition site and a mouse
Hprt promoter fused with green-emitting luciferase(SLG) coding sequence. The Hprt-SLG vector was
loaded onto a mouse artificial chromosome containing a multi-integrase platform using phiC31 integrase
in mouse A9 cells. We established three independent
clones.
Results The established cell lines had similar levelsof expression of the Hprt-SLG reporter gene. Hprt-SLG
activity increased proportionately under growth conditions and decreased under cytotoxic conditions after
blasticidin or cisplatin administration. Similar increases
and decreases in the SLG luminescent were observed
under growth and cytotoxic conditions, respectively, tothose in the fluorescent obtained using the commerciallyavailable reagent, alamarBlue.
Conclusion By employing a reliable and stable expression system in a mammalian artificial chromosome,the activity of an Hprt-SLG reporter can reflect cellnumbers under cell growth condition and cell viability
in the evaluation of cytotoxic conditions.
Background†Present address: Department of Chemistry, School of Science, The
University of Tokyo, Tokyo 113-0033, Japan
Corresponding author: Tetsuya Ohbayashi, PhD
ohbayashi@med.tottori-u.ac.jp
Received 2016 March 18
Accepted 2016 May 17
Abbreviations: BAC, bacterial artificial chromosome; CAG, cytomegalovirus IE enhancer and chicken beta-actin promoter; HAC,
human artificial chromosome; Hprt, hypoxanthine guanine phosphoribosyltransferase; HS4, DNase I hypersensitive site 4; MAC,
mouse artificial chromosome; MI, multiple integrases; MI-MAC,
multiple integration site-containing mouse artificial chromosome;
MMCT, microcell-mediated chromosome transfer; PAC, P1 artificial chromosome; PGK, phosphoglycerate kinase; SLG, green-emitting luciferase.

gene reporter; hypoxanthine phosphoribosyltransferase; luciferase; mouse artificial chromosome;reference standards
Key words

House-keeping genes have been routinely used as internal controls for normalization in gene expression
analysis.1–3 Hypoxanthine guanine phosphoribosyltransferase (Hprt), a nucleotide metabolizing enzyme,
174

Cell viability monitoring system

integrase optimized for mammalian codon usage and isdriven by the CAG promoter.

gene therapy application as well as for gene analyses invarious situations.10–13 A mouse A9 cell line, derivativeof mouse fibroblast L cells used for toxicity testing, wasuseful for a donor cell of MMCT.14
The construction of larger promoter/reporter vectors
requires the handling of large DNA regions, which isdifficult with common cloning approaches. The bacterial
artificial chromosome (BAC) recombineering method is
a powerful tool for the manipulation of long DNA fragments.15, 16 Because recombination takes place in bacteria by employing intrinsic bacterial/phage machinery,large vector construction can be achieved without complicated cloning steps. It has been reported that a mouse
CD40L gene vector constructed using BAC recombination showed functional expression from a HAC.17
Phage integrases, enzymes that integrate DNA intoa bacterial host genome, have been reported to work inmammalian cells.18, 19 PhiC31, R4, TP901 and Bxb1 integrases mediate efficient site-specific recombination in
mammalian cells, and transgenesis in mice was also reported by pronuclear injection of phiC31 integrase.20–24
The multiple integrases (MI) system on an artificial
chromosome (MI-HAC/MI-MAC), an application of
mammalian artificial chromosome technology, wasdeveloped for loading gene(s) onto HACs and MACs.
The MI platform has five gene loading sites for distinct
recombinase/phage integrases.25 By using this recombinase-mediated MI system, targeted recombinant cells
can be obtained at high efficiency and these recombinants retain stable transgene expression compared withthe random integration method.25 Recently, it has beenshown that transchromosomic mice were generated in
fewer steps by direct use of mouse embryonic stem cells
harboring MI-MAC.26
Here, we constructed a long Hprt-promoter/luciferase reporter vector using a BAC recombineering method
and loaded it onto the MI-MAC system. We confirmed
luciferase activity that was proportionate with cell numbers in established Hprt-luciferase cell lines.

Hprt promoter cloning by BAC recombination

A detailed f low diagram for the construction ofphiC31neoHprt-SLG is described in Fig. 1 and Table 1.
Briefly, the locus-specific homology arm (white boxes
in Fig. 2A) of the Hprt promoter region was synthesized
for the retrieving 20 kb BAC fragment. The arm wasligated into the inspB4ins3 vector, and then the coding
sequences of the Rhagophthalmus ohbai luciferase gene,
SLG (pSLG-test vector, Toyobo, Osaka, Japan) was
inserted. Additionally, a phiC31neo module (the phiC31
integrase attB site and a neomycin resistance gene cassette) was also ligated into the inspB4ins3 vector. The
vector was digested by AscI and AvrII and then ligatedinto PPAC ori km. The Hprt gene promoter region was
retrieved by gap-repair from a BAC clone (B6Ng01126E09; Riken, Tokyo, Japan) into the P1 artificial
chromosome (PAC) vector using E. coli strain DY380.13,
14
Clones were selected at 32 °C on LB agar containing
kanamycin. To check whether the clones were precisely
retrieved by gap-repair of the promoter arms, the clones
were amplified by PCR, and the vector was confirmedby restriction enzyme digestion.
Cell culture and compounds

Mouse A9 (MI-MAC) cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 10% FBS at 37 ºC.26 Blasticidin (InvivoGen, San Diego, CA) and cisplatin (Wako,
Osaka, Japan) were diluted with culture medium at thetime of use.
Establishment of Hprt-SLG cells

Principles for recombinase-mediated integration using
the MI-MAC system are described elsewhere.25, 26 The
phiC31neoHprt-SLG PAC vector was purified usinga Large Construction Kit (Qiagen, Hilden, Germany). A9 MI-MAC cells were co-transfected withphiC31neoHprt-SLG and pCAG-phiC31 using Lipofectamine 2000 (Invitrogen) for 6 h (Fig. 2B). Twenty-fourhours after transfection, cells were expanded for 24 hand then selected with 600 μg/mL G418 (Invitrogen).
Surviving colonies were picked and recombination
checked by genomic PCR analyses.

EXPERIMENTAL PROCEDURES
Vectors

The inspB4ins2 vector is described elsewhere.26 The
vector has two insulator cassettes consisting of repetitive 5′-DNaseI hypersensitive site 4 (HS4) elementsfrom chicken beta-globin to prevent promoter interference from neighboring regions. The PPAC ori km vector
was modified from the pPAC4 vector (Children’s Hospital Oakland Research Institute, Oakland, CA). The
BstEII/AscI region was replaced by a linker sequence
containing a multiple cloning site (McaTI, FseI, PmeIand AvrII). pCAG-phiC31 expresses a bacterial phiC31

PCR analyses

Amplified regions and primer sequences for genomic
PCR are described in Fig. 2C and Table 2, respectively.
All PCR reactions were performed with KOD FX neo
polymerase (Toyobo) under the following conditions.
175

T. Endo et al.

For junction PCR (Fig. 2C; JP-5, JP-3); 95 °C for 2 minfor 1 cycle, 98 °C for 10 sec and 68 °C for 1–1.5 min for
35 cycles. For long PCR (Fig. 2C; LP-6, LP-14, LP-10
and LP-15); 95 °C for 2 min for 1 cycle, 98 °C for 10 sec
and 74 °C for 6–15 min for 5 cycles, 98 °C for 10 sec
and 72 °C for 6–15 min for 5 cycles, 98 °C for 10 sec
and 70 °C for 6–15 min for 5 cycles, 98 °C for 10 sec
and 68 °C for 6–15 min for 20 cycles, 68 °C for 10 min
for 1 cycle. Extension time was modulated according
to target size. Long PCR products were digested withappropriate restriction enzymes to confirm amplificationof the target.

are known to be deleterious for subsequent BAC retrieving (Fig. 2A white boxes). The module containing all components was transferred to a PAC vector
backbone (Fig. 1, steps 6 to 8), and the resulting PAC
vector was used as a retrieving vector. We successfully
retrieved 20 kb of Hprt promoter from the BAC clone togive phiC31neoHprt-SLG (Fig. 1 step 9). The large 20kb promoter/enhancer region of Hprt was successfully
recloned into an expression vector for Hprt gene expression using BAC recombineering method.
Vector transfection into A9 MI-MAC cells
Figure 2A and B represent a genomic map of the mouse
Hprt gene and schematic map of the MI-MAC andphiC31neoHprt-SLG vector array, respectively. The
retrieved phiC31neoHprt-SLG vector was co-transfected into A9 MI-MAC cells with a phiC31 integrase
expression vector (Fig. 2B), and 32 candidate colonies
were isolated. We performed sequential PCR analysesof these clones to clarify the relationship between total
isolated colony numbers and accurate integration ineach step. Figure 2C shows a post-integration map at
the phiC31 site on the MI-MAC and the regions amplified by PCR using the primer sets in Table 2. All PCR
analysis results are summarized in Table 3. First, 26 of
32 clones were 5′-junction-PCR-positive (#2–11, #13–15,#17, #19–23 and #25–31). Among 16 of these clones (#2,
5, 6, 9, 10, 13, 15, 17, 19, 21, 22, 26–28, 30 and #31) 10
were positive for the 3′-junction PCR (#2, 10, 13, 15, 17,
21, 22, 26, 27 and #30). Subsequently, only four clones(#2, 17, 21 and #30) were positive for the 3′-6 kb PCR
that included the SLG reporter element, and then #21
clone was excluded by failure to amplify the 3′-15 kb
fragment (LP-15 in Table 3 and Fig. 2C). We, therefore,
obtained three cell lines (#2, #17 and #30; Hprt-SLG
cells) that integrated the entire vector region from the
16 clones examined. These clones produced almost thesame levels of luciferase activity (Fig. 3A), and FISH
analysis showed Hprt-SLG signals on the MAC (Fig.
3B). Thus, we presumed that these lines had uniform reporter gene expression from the MI-MAC.

Fluorescence in situ hybridization (FISH) mapping

FISH analysis was performed using a standard protocol.11, 26 Metaphase nuclei from established cell lines (A9Hprt-SLG cells) were spread on slides. Biotin-labeled
Hprt-SLG vector and digoxigenin-labeled mouse minor
satellite DNA were prepared as hybridization probes.
To suppress background signals, a fifty-fold amount of
non-labeled mouse Cot-1 DNA was added during hybridization. Chromosomal DNA was counter-stainedwith DAPI-Fluoromount-G (Southern Biotechnology
Associates, Birmingham, AL). Fluorescence images
were captured by Metafer, and analyzed with ISIS (Carl
Zeiss, Oberkochen, Germany).
Luciferase and cell viability assays

A9 Hprt-SLG cells were seeded at 5 × 104 cells per wellin a 96-well micro-clear bottom black plate (Greiner,
Kremsmünster, Austria) 24 h prior to compound addition. After culture for 72 h with blasticidin (0–10 μg/mL)or cisplatin (0–20 μM), cells were washed twice with
PBS and then subjected to the following analyses. Luciferase activity was measured with a Phelios luminometer(Atto, Tokyo, Japan) using Tripluc assay reagent (Toyobo).
A cell viability assay was performed using alamarBlue(AbDSerotec, Oxford, UK) according to the manufacturer’s instructions and an Infinite F500 fluorescent plate
reader (Tecan, Männedorf, Switzerland). Three independent wells were used to determine the Luciferase activity.

Evaluation of Hprt-SLG luminescence comparedwith cell numbers

RESULTS
Construction of the Hprt-SLG reporter vector

To verify luminescence linearity of Hprt-SLG cells,
we seeded clone #17 at various densities from 1,000 to
40,000 cells in a 96-well black plate and performed aluciferase assay after 2 h. Luciferase activity showed linear luminescence (Fig. 4A, upper panel, green line). Thisluminescence profile was mostly consistent with cell viability measured with an alamarBlue cell viability assay(Fig. 4A upper panel, blue line). Similar to the MTT as-

To develop a retrieving vector, the following components were sequentially ligated into inspB4ins2: insulator sequence (HS4), retrieving arm, SLG luciferase
and phiC31neo module (Fig. 1, steps 1 to 5). Detailed
operations for each step are described in Table 1. The
locations of the Hprt retrieving arms were determined
by RepeatMasker to avoid repetitive elements, which
176

Cell viability monitoring systemsay, the Alamar Blue reagent detects reduced substrates
in response to metabolism in living cells. 27 We also
seeded 10,000 cells, and measured luciferase activity every 24 h up to 72 h. The correlation of luminescence andliving cells showed an almost linear ratio through the
incubation period (Fig. 4A lower panel). According tothese results, we considered that luminescence intensityof Hprt-SLG reflected living cell numbers under conditions of growth. To assess the reporter response under
Step

NheI

1

toxic conditions, we administrated cytotoxic compounds
to clone #17. Treatment with blasticidin (Fig. 4B upper
panel) or cisplatin (Fig. 4B lower panel) for 72 h decreased SLG activity in a dose-dependent manner. Next,
we estimated cell viability using the alamarBlue cell
viability assay. We found a positive correlation between
SLG activity and cell viability using both blasticidin
Fig. and1
cisplatin. We also obtained similar results using clones#2 and #30 (data not shown).

BamHI
HS4

HS4

inspB4ins2

BamHI fill-in/self ligation

AvrII
HS4

2

HS4

inspB4ins2-B
BamHI
HS4

3

NheI

AvrII

HS4

BglII

NcoI BamHI AvrII
HS4

HS4

arm

HS4

arm

SLG

NcoI

HS4

arm-SLG-inspB4ins3

AscI

7

arm

SLG

HS4

arm

BamHI

AvrII

HS4

φC31attB neo

arm-SLG-inspB4ins3-φC31neo

AscI
AvrII

PmeI
HS4

SLG

NheI
AvrII
φC31attB neo

AvrII
HS4

BamHI

HS4

AscI

6

arm

HS4

arm-inspB4ins3

AvrII

5

AvrII

HS4

inspB4ins3

4

HS4

SLG

HS4

φC31attB neo

SLG

HS4

φC31attB neo

PPAC ori km

PPAC arm-SLG-φC31neo
Km

8

HS4

Linearize

PPAC arm-SLG-φC31neo
Km

BAC

9

hprt 20 kb
HS4

SLG

Retrieving

Cm

HS4

φC31attB neo

PPAC arm-SLG-φC31neo
Km

Fig. 1. Construction of phiC31neoHprt-SLG.
Flow chart of PAC vector construction for BAC retrieving. Arrows indicate the sequential steps via the operations described in Table 1.
The retrieving vector is composed of an Hprt homologous arm-SLG sequence flanked by HS4 insulators and a phiC31neo cassette.
BAC, bacterial artificial chromosome; Hprt, hypoxanthine guanine phosphoribosyltransferase; HS4, DNase I hypersensitive site 4; PAC,
P1 artificial chromosome; SLG, green-emitting luciferase.

177

T. Endo et al.
Table 1. Operations for constructing the phiC31neoHprt-SLG PAC vector
Steps in Fig. 1

Operation

Fragment/digestion (origin)

Insert site

1 to 2Fill-in/self ligation

BamHI

2 to 3

Add HS4 fragment

NheI-AvrII (inspB4ins2)

AvrII

3 to 4

Add arm fragment

BglII-BamHI (synthetic gene arm)

BamHI

4 to 5

Add SLG fragment

NcoI-BamHI (pSLG-test)

NcoI-BamHI

5 to 6

Add phiC31neo fragment

NheI-AvrII (phiC31neo inspB4ins2)

Fig. 2
Fig. 2
AvrII

6 to 7

Transfer to PAC vector

AscI-AvrII (arm-SLG-inspB4ins3-phi C31neo)

AscI-AvrII

8

Linearization

PmeI

9

Retrieving of BAC

Exon 1

Fig. 2

mouse Hprt

BAC, bacterial artificial chromosome; HS4, DNase I hypersensitive site 4; PAC, P1 artificial chromosome; SLG, green-emitting luciferExon 1
mouse Hprt
ase.
–20,608–20,174–322 +146–20,608–20,174–322 +146
Exon 1–20,174–322 +146

AA–20,608

10 kb
mouse Hprt
10 kb

MI-MAC
MI-MACA

10 kb

MI-MAC

2xHS4
2xHS4

B
B
B

2xHS4

FRT

phiC31attPR4attP

TP901attP

Bxb1attP

PGK

FRT

phiC31attPR4attP
TP901attP
phiC31 integrase

Bxb1attP

mHPRT promoter (20kb)

SLG 2xHS4 phiC31attB neo

Hprt-SLGmHPRTphiC31neo
promoter(20kb)
phiC31attB neo
PGK
FRTSLG 2xHS4 phiC31attP(39kb)
phiC31neo Hprt-SLG(39kb)
mHPRT promoter (20kb)

phiC31 integrase
R4attP

TP901attP

Bxb1attP

phiC31 integrase

SLG 2xHS4 phiC31attB neo

phiC31neo Hprt-SLG(39kb)

MI-MAC
MI-MACPGK phiC31attR

PGK phiC31attR
JP-5 PGK5
1.2 kbJP-5 PGK5
1.2 kbPGK phiC31attR

LP-14 PGK5
JP-5 PGK5
LP-14 PGK5
LP-14 PGK5

C
C

PGK

1.2 kb

PAC

2xHS4

MI-MAC
Hprt

neoG418 3AS
PAC

2xHS4

Hprt

neo

G418 3AS
neo

PAC

2xHS4

G418
14 kb3AS
14 kbLP-10

PAC#17

LP-10

PAC#17

14 kb

LP-10

LP-6

HPRT #40
Hprt
LP-6 HPRT #40
HPRT #36
HPRT #36

JP-3

LP-15

LP-15

HPRT#33
10 kb

2xHS4 phiC31attL PGK
phiC31attL-B Fw2

0.7 kb

R4 attP
R4R4attP
attP Rv

R4attP Rv
0.7 kb
R4attP Rv
6
kb
SLG 2xHS4 phiC31attL PGK R4 attP
R4attP Rv
6 kbFw2
R4attP Rv
JP-3 phiC31attL-B
0.7 kbHPRT #31

kb#40
LP-6 10 HPRT
10 kb
HPRT#33
HPRT#36

PAC#17

SLG

Fw2
JP-3
2xHS4 phiC31attL
SLG phiC31attL-B
PGK

HPRT #31

6 kb
15 kb

HPRT #31

15 kb

R4attP Rv
R4 attP Rv
R4 attP Rv

Fig. 2. Mouse Hprt promoter vector and MI-MAC integration.
HPRT#33
R4 attP
RvA) Genomic map of the mouse Hprt gene. Vertical blackLP-15
lines indicate
exons. Numbers besidewhite boxes represent
nucleotide
positions
15 kb
C arms.
of Hprt retrievingB) MI-MAC integration and the retrieved Hprt-SLG PAC vector. The MI platform consists of five phosphoglycerate kinase (PGK) promoter-attP arrays. FRT, phiC31attP, R4attP, TP901attP and Bxb1attP indicate yeast recombinase FLP, phage integrase phiC31, R4, TP901
and Bxb1 recognition sequence, respectively. Recombination between phiC31attB and attP by phiC31 integrase is represented.
C) Map of PAC vector integrated into the MI-MAC at the phiC31 site. Double-headed arrows with fragment length indicate regions amplified by PCR. Primer sequences are described in Table 2. Names of PCR-amplified regions are indicated to the left of the double-headed arrows.
Hprt, hypoxanthine guanine phosphoribosyltransferase; HS4, DNase I hypersensitive site 4; MI-MAC, multiple integration site-containing mouse artificial chromosome; PAC, P1 artificial chromosome; PGK, phosphoglycerate kinase; SLG, green-emitting luciferase.

178

Cell viability monitoring system

DISCUSSION
In this report, we used BAC recombination and mammalian artificial chromosome technology to engineer an
Hprt-reporter whose activity reflected living/dead cell
viability in response to cytotoxic compounds.
Although the house-keeping Hprt gene has beenused and verified as a standard for gene expression experiments, few reports have analyzed the Hprt gene promoter. Therefore, we examined a 20 kb long region of
the Hprt promoter with the expectation of achieving reliable expression. Without using PCR cloning or a standard “cut and ligation” approach, we employed a BAC
recombination method to acquire the long promoter
region of the Hprt gene. In this Hprt promoter retrieving
experiment, the BAC recombination efficiency (successfully retrieved bacterial clones) was 1%–6%. Retrieving
rates we achieved for other genes were similar (datanot shown). Recombination efficiency depends on the
retrieving sequence and its length, and we considered
the targeted Hprt promoter region within the limits ofthe procedure to achieve vector construction. Using along Hprt promoter, we constructed the phiC31neoHprtSLG reporter vector for integration at the phiC31 siteon the MI-MAC. The phiC31neoHprt-SLG and phiC31
integrase expression vectors were co-transfected into A9
MI-MAC cells, and we isolated G418 resistant colonies.
Transfected A9 MI-MAC clones were mostly positive
for the 5′-junction PCR assay, but we detected only threelines with full-length integration (#2, #17 and #30; Table3). These three lines produced nearly equivalent luciferase activity [clone #21 displayed low activity in spiteof containing the SLG element (data not shown)]. We
presume that low luciferase activity of clone #21 resulted from deletion of the Hprt promoter region during the
integration step into the MI-MAC. While we employeda 39 kb PAC vector in this study, it is considered that
intact integrations will occur at lower frequencies withlarger vector constructions.
Luciferase activity of the three correctly integrated
cell lines exhibited linearity with respect to cell numbers. We confirmed that this correlation was retained
under continuous growth conditions for up to 72 h (Fig.
4A lower panel). We also confirmed proportionate decreases in luciferase activity and cell numbers under the
toxic conditions of exposure to blasticidin or cisplatin(Fig. 4B). Blasticidin is a nucleoside antibiotic generally used in mammalian or bacterial cell selection andcisplatin is an anti-neoplastic drug widely used as achemotherapeutic drug for cancer patients. Considering
these results, the Hprt-SLG reporter can report on not
only living (growing) cells, but also on toxicity-induced
cell death. Although, cisplatin evoked a mild cytotoxic
response up to 20 μM, we confirmed similar tendencies
of luciferase activity and cell viability in response to
blasticidin and cisplatin, suggesting that the Hprt-SLG
reporter has the potential to evaluate cytotoxicity causedby different actions. We also note that employing a 20 kb
Hprt promoter contributed to reliable expression of the
Hprt-reporter gene on the MAC. Now, we are verifying
the usability of the Hprt-luciferase reporter in other celllines by MAC transfer (manuscript in preparation).
Stability and uniformity of reporter gene expression
are important factors and are required for reliable resultsin gene expression analyses. In addition, the transferrable feature of HACs and MACs by the microcell-mediated chromosome transfer method makes it possible
to establish various cell lines using different recipients,
including mouse embryonic stem cells.10 Transchromosomic mice generated from such HAC/MAC-transferred mouse embryonic stem cells can pave the way
for authentic in vivo analysis. Recently, a simultaneousgene-loading system for HACs has been developedand is a further multi-purpose tool for gene analysis.28
Furthermore, an evaluation system for osteogenic differ-

Table 2. Primers used for PCR analysis to verify the reporter construct in Fig. 2C
Primer name5′-sequence

Usage

PGK5

AATGGAAGTAGCACGTCTCACTAGTCTC

5′-junction/long PCR

G418 3AS

GGTAGCCAACGCTATGTCCTGATAGCGGTC

5′-junction PCR

phiC31attL-B Fw2

CTCGTCGGCCGGCTTGTCGACG

3′-junction PCR

R4attP Rv

AGTTGGGTGCACCCGCAGAGTGTA

3′-junction/long PCR

PAC#17

CTCTAGCGGGGGGATCTGCATGCACLong PCR

HPRT#31GTGTATGAGGCCTCTCTGGTCATAACCTG

Long PCR

HPRT#33

GTTACTATCGAGCCTGTGACAACCACGTGG

Long PCR

HPRT#36

CTGCAGGCCCAGGTTGGTAAGCTCTCTCLong PCR

HPRT#40GCGGAGTGATTATCTGGGAATCCTCTGGGLong PCR

HPRT, hypoxanthine guanine phosphoribosyltransferase; PAC, P1 artificial chromosome; PGK, phosphoglycerate kinase.

179T. Endo et al.
Table 3. Summary of PCR analysis of phiC31neoHprt-SLG vector-transfected MI-MAC A9 cells#Clone

JP-5

JP-3

PCR regions in Fig. 2C
LP-15

LP-6

LP-10

LP-14

1
–
2
+
+
+
+
+
+
3
+
NT
4
+
NT
5
+
–
6
+
–
7
+
NT
8
+
NT
9
+
–
10
+
+
–
11
+
NT
12
–
13
+
+
–
14
+
NT
15
+
+
–
16
–
17
+
+
+
+
+
+
18
–
19
+
–
20
+
NT
21
+
+
+
–
22
+
+
–
23
+
NT
24
–
25
+
NT
26
+
+
–
27
+
+
–
28
+
–
29
+
NT
30
+
+
+
+
+
+
31
+
–
32
–
Hprt, hypoxanthine guanine phosphoribosyltransferase; MI-MAC, multiple integration site-containing mouse artificial chromosome; NT,
not tested; SLG, green-emitting luciferase.

Fig. 3

SLG luminescence(counts/sec)

70000
60000
50000
40000
30000
20000
10000

A

0

#2

#17

Clone

#30

B

Fig. 3. Luciferase activities and FISH analyses of Hprt-SLG cells.
A) Luciferase activities of A9 Hprt-SLG cells. Each clone was seeded 24 h prior to assay. Error bars represent standard deviation (n = 4).
B) Representative FISH image of A9 Hprt-SLG cells. Digoxigenin-labeled mouse minor satellite (red signal) and biotin-labeled Hprt-SLG
PAC vector (green signal) were used as detection probes. Arrow indicates MI-MAC and insert shows magnified image of Hprt-SLG and
MI-MAC signal. Hprt, hypoxanthine guanine phosphoribosyltransferase; MI-MAC, multiple integration site-containing mouse artificial
chromosome; PAC, P1 artificial chromosome; SLG, green-emitting luciferase.

180

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(40 x103 cells = 100%)

Relative luminescence (%)
Relative cell viability (%)

Relative luminescence (%)
Relative cell viability (%)

Cell viability monitoring system

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(0 µg/mL = 100%)
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Cell number (x 10 )
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18

20

Blasticidin (µg/mL)

AlamarBlue intensity

Relative luminescence (%)
Relative cell viability (%)(0 µM = 100%)
45000

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SLG luminescence (counts/s)

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Cisplatin (µM)

Fig. 4. Response curves of Hprt-SLG cells.
A) Hprt-SLG cells in normal culture conditions. Upper panel: Relative luciferase activity and cell viability were plotted against various cellnumbers. Luciferase activities and alamarBlue signals were measured at 2 h after cell seeding. Green rectangles and blue circles indicate
relative luminescence by SLG activity and relative cell viability by alamarBlue intensity, respectively. Lower panel: SLG luminescence andalamarBlue intensity were measured after continuous incubation for the indicated hours. Error bars represent standard deviation (n = 3).
B) Response of Hprt-SLG cells to cytotoxic compounds. Relative luciferase activity and cell viability were plotted against concentrationof blasticidin (upper panel) or cisplatin (lower panel). Hprt, hypoxanthine guanine phosphoribosyltransferase; SLG, green-emitting luciferase.
Acknowledgments: We wish to thank Dr. Masaharu Hiratsuka,
Tottori University for providing the inspB4ins2 vector. We appreciate Dr. Yoshihiro Nakajima for valuable discussions and Mr.
Naohiro Sunamura for technical advice. We also thank Ms. Miyuki Nomura for technical assistance.
This study was supported in part by the Ministry of Economy,
Trade and Industry (METI) (T.O. and M.O.), and the Regional
Innovation Strategy Support Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT)(T.O. and M.O.).

entiation has been established that utilizes a luciferase
reporter and a MAC.29 The combination of multiple
gene reporters, multiple gene loading and artificial chromosomes, will lead to diverse analyses of gene function
and to high throughput systems that will contribute to
drug development.
Noguchi et al. developed a dual-color luciferase assay
system in which the expression of multiple genes can betracked simultaneously using green- and red-emitting luciferases and this dual-color luciferase assay system wasused for an in vitro test to screen skin sensitizer.30, 31 By
using green- and red-emitting luciferases as the internal
control reporter and cytotoxicity specific reporter respectively, we can analysis quantitatively the cytotoxicity
of chemical compounds.
In the future, I would like to develop an in-vitro
nephrotoxicity test by using the dual-color luciferase
assay system of Hprt-SLG reporter and nephrotoxicity
marker gene- red-emitting luciferases.

The authors declare no conflict of interest.

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