Development of an HSV-tk transgenic mouse model
for study of liver damage
Yan Zhang, Shu-Zhen Huang, Shu Wang and Yi-Tao Zeng
Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai Jiao Tong University, People’s Republic of China
The morbidity of severe liver disease is usually very
high, seriously threatening the patient’s health. Availab-
ility of animal models expressing related hepatic disor-
ders should provide a means of studying the pathogenic
mechanism of such diseases. Among these are the gen-
etically engineered animal models. Unfortunately, cur-
rently available transgenic models are unsatisfactory for
experimental use, because those transgenic mice expres-
sing toxic protein often die too early due to the over-
expression of toxic protein in such a vital organ. Others,
for example the alb-uPA transgenic mouse and FAH
–
knockout mouse developed in the 1990s can be main-
tained only with constant medical treatment [1,2].
In 1989, Heyman et al. [3] developed a new trans-
genic mouse in which ablation of a specific cell type is
TK-dependent. In such a transgenic mouse, the inser-
ted herpes simplex virus thymidine kinase (HSV-tk)
gene products can phosphorylate certain nucleoside
analogues such as ganciclovir (GCV) that are not
metabolized by conventional cellular enzymes. Phos-
phorylated nucleoside analogues such as GCV triphos-
phate are potent toxic metabolites for cells.
Nevertheless, neither GCV nor the HSV-tk alone is
harmful to cells. Hence, this conditional cell-depleting
effect is achieved by expressing HSV-tk with a cell-
specific promoter. It has been used for depletion of

with inflammatory infiltration in the liver, and diffuse proliferation of
hepatocytes. In addition, the blood test demonstrates a significant increase
of serum alanine aminotransferase, aspartate aminotransferase and total
bilirubin. In conclusion, the transgenic mouse model with hepatocyte-speci-
fic expressed HSV-tk developed hepatitis with administration of GCV, had
morphological and clinical chemical characteristics indicative of hepato-
cellular disease and should be useful for the the study of inducible liver-
specific diseases.
Abbreviations
ALT, alanine aminotransferase; AST, aspartate aminotransferase; FISH, fluorescence in situ hybridization; GCV, ganciclovir; HSV-tk, herpes
simplex virus thymidine kinase; RT, reverse transcription.
FEBS Journal 272 (2005) 2207–2215 ª 2005 FEBS 2207
lymphoid cells, growth hormone-secreting cells, inter-
leukin-2 and interleukin-4-expressing cells, dendritic
cells or fibroblasts under the control of a cell-specific
promoter [4–9]. Such a system is used in the transgenic
rats of Kawasaki et al. [10], in which the rats develop
experimental hepatitis on administration of GCV. The
genome of the mouse is much better characterized than
that of the rat and the cost of producing and maintain-
ing transgenic mice is less than for rats. The high
conservation and strong liver-specific regulatory
machinery of the mouse serum albumin cluster makes
it appropriate for use as a promoter for hepatic-speci-
fic expression [11,12].
In this study, HSV-tk transgenic mice were produced,
in which the inserted gene is regulated by an albumin
enhancer ⁄ promoter; liver injury is readily induced in this
model. Among five founder transgenic mice generated,
only one (TK5$) transmitted the transgene to progeny

A total of 182 eggs were microinjected and subse-
quently reimplanted into eight pseudopregnant foster
mothers, of which five became pregnant and gave birth
to 36 mice. Among them, six mice showed the inser-
tion of the HSV-tk gene as detected by PCR. The
integration rate was 16.7% (6 ⁄ 36). One line of trans-
genic mice is female; the transgene is transmitted to
the offspring at a rate of about 50% according to
Mendel’s laws (Fig. 2A).
Chromosomal localization of transgene
integration as demonstrated by fluorescence
in situ hybridization (FISH)
More than 50 metaphases were analysed for each
transgenic mouse. All of the metaphase cells showed
one positive hybrid signal. According to the standard
idiograms of mouse chromosomes, the integration site
is located at 2F1-G3 in TK5-F1-455 (Fig. 2B). The
A
B
Fig. 1. Comparison of different HSV-tk transfected Hep-G2 cells
post-treatment with GCV and the negative control group. (A) Mor-
phology of the Hep-G2 cells transfected with pLLTK after adminis-
tration of GCV. Left, 40 lmolÆL
)1
GCV; right, no GCV (original
magnification, · 200). (B) Comparison of cellular survival rates
among HSV-tk transfected Hep-G2 cells post-treatment with GCV
and the negative control group. *P < 0.05. Results are expressed
as mean ± SD of three separate experiments. Bar1, cells trans-
fected with pCMV-TK (positive control group); Bar2, cells trans-

cells account for 20–30% of the total hepatocytes of
TK5-F1-455 (Fig. 4A), whereas in mouse TK3, there
were approximately 60–70% HSV-tk staining hepato-
cytes, with visible slight yellowish-brown signals in the
focal necrosis, but several regenerative foci (regener-
ating parenchyma hepatocytes) displayed reduced or no
staining (Fig. 4D). The percentage of HSV-tk-positive
hepatocytes in the F2 mice (TK5-F2-327) of TK5 was
similar to those of TK5-F1-455 (data not shown).
Hematoxylin and eosin staining for histological
analysis
Microscopic analysis of the livers of GCV-treated
HSV-tk mice (F1 and F2) showed that the diseased liv-
ers display a number of abnormalities, including the
appearance of apoptosis bodies, hepatocyte vaculation,
lymphocyte infiltration, hepatocyte megalocytosis, and
diffused proliferation of hepatocytes (Fig. 5A). In
transgenic mouse TK3, mutifocal coagulation necrosis
was evident in the liver (Fig. 5B). Histological analysis
of the kidney showed no apparent abnormity in the
GCV-treated transgenic mice and wild-type mice (data
not shown).
Biochemical analysis of the blood
Twenty-one days after the injection of GCV, the values
of alanine aminotransferase (ALT), aspartate amino-
transferase (AST) and total bilirubin were significantly
increased in the TK5-F1 transgenic mice (P<0.05),
whereas there were no significant increase in the wild-
type control mice. The value of creatinine was not
altered significantly in either group (Fig. 6). The chan-

infiltration, diffuse proliferation of hepatocytes as well
as a significant increase of serum ALT, AST and total
bilirubin, can be easily recognized. However, renal
function is not affected by GCV treatment. This indi-
cates that GCV at the dosage used in this study is
associated with toxin-mediated hepatocyte damage in
our HSV-tk mice.
We used FISH and RT-PCR to investigate trans-
gene integration and HSV-tk expression in various
tissues of the transgenic mice. FISH indicated that the
Fig. 5. Histology of liver tissue (hematoxylin
and eosin stain). (A) GCV-treated TK5-F1 tra-
nsgenic mouse showed several apoptotic
bodies, variably severe cytoplasmic vacuoli-
zation, lymphocyte infiltration, hepatocyte
megalocytosis, and proliferation of hepato-
cytes. (B) GCV-treated TK3 mouse, showed
apoptosis bodies, mutifocal coagulation
necrosis with lymphocyte infiltration,
hepatocyte megalocytosis, and proliferation
of hepatocytes. (C) GCV-treated nontrans-
genic mouse. (D) Untreated transgenic
mouse. (Original magnification, · 200.)
Fig. 4. Immunohistochemical staining
observation of the HSV-tk expression.
(A) Liver of transgenic mouse TK5-F1-455.
HSV-tk-positive cells are clustered around
the central vein, scattered in the liver lobule
tissue, or clustered in the periportal areas.
(B) Liver of the wild-type mouse showed no

can cause chain termination and single-strand breakage
upon incorporation into DNA. Although blocking of
DNA synthesis of GCV is especially toxic for dividing
cells, it can also cause damage of nondividing cells,
such as hepatocytes, and liver toxicity of HSV-tk
[13,14]. This provides the basis for selected hepatocyte
killing using a hepatocyte-specific promoter in vivo.
Hepatocyte replication was not a prerequisite for
this effect, indicating that interference with DNA syn-
thesis during S phase of the cell cycle is not the only
mechanism of toxicity of phosphorylated GCV.
Furthermore, although the exact mechanism by which
suicide genes kill the HSV-tk-expressing cells is not yet
clear, apoptosis has been considered to be a major
contributor to GCV killing [15–18]. Song et al. [19]
have shown that GCV induced HSV-tk expressing cells
into apoptosis, thus inhibiting the growth of ovarian
cancer cells. Shibata et al. [20] injected the HSV-tk vec-
tor into rats with bladder cancer and observed apopto-
sis of bladder cancer cells. Kawasaki et al. [10] created
an AL-HSV-tk transgenic rat that expressed HSV-tk in
hepatocytes, in which apoptosis was demonstrated
after treatment with GCV. The administration of GCV
elicited leukocyte infiltration and induced chronic
hepatitis [21,22]. Although in the hepatitis model the
precise role of Kupffer cells is unclear, it is possible
that they are involved in inflammation [10]. Activated
Kupffer cells release cytokines and chemokines that
activate and transport T cells [23–25]. It seems that
hepatitis in the rat is primed by hepatocyte apoptosis

revealed moderate hepatocyte vacuolation and an
increased number of inflammatory cells. In this study,
we found that there was more severe focal necrosis of
the liver tissue in TK3 than in TK5 mice. Moreover,
the liver regenerating focus was more evident in TK3,
in which clones of transgene expression-deficient cells
were formed as detected by immunohistochemistry.
The reasons for the different pathological changes
between these two mouse strains are not clear. We sug-
gest that these differences may be associated with the
quantities of HSV-tk expressing cells. In addition, the
patchy focal necrosis and regeneration in the TK3 liver
could be explained by the possibility that this founder
mouse may be a mosaic as approximately 5–10% of
founder mice showed mosaicism of some sort (either
multiple integration sites or patchy cellular distribu-
tion). Boucher et al. [27] compared the efficacy of the
HSV-tk ⁄ GCV system in two human carcinoma cell
lines after exposure to GCV and found that the killing
effect depended on the concentration of the tk enzyme,
the number of cells expressing HSV-tk, different cell
types and the overall confluence of the HSV-tk expres-
sing-cells. These results emphasise the importance of
cell-specific metabolism in HSV-tk ⁄ GCV-mediated
cytotoxicity. In conclusion, the killing of cells with
HSV-tk ⁄ GCV is a complex interactive sequence of
biochemical and cellular events involving incorporation
and accumulation of the monophosphorylate deriv-
ative of GCV into DNA, disruption and inhibition of
the cell cycle, gap junction metabolite transfer, and

of the pathogenesis of liver diseases and potential
therapies.
Experimental procedures
Plasmid construction and generation of
transgenic mice
Total DNA was extracted from KM mouse blood for PCR
amplification of murine albumin promoter ()310 bp to
+25 bp) and enhancer ()9192 bp to )11 250 bp). The
primers for albumin promoter and enhancer are: pro1,
5¢-CTTAGGTACCTCCATGCCAAGGCCCACA-3¢; pro2,
5¢-CTTGCTCACCATGGTGGCGACCGGTAGTGGGGT
TGATAGGAAAGG-3¢; en1, 5¢-ACGAGTCTAGAGTG
GAGCTTACTTCTTTGATTTGA-3¢; en2, 5¢-CCGCGTC
GACGGAAAAGCGCCTCCCCTAC-3¢; The 1800 bp of
the HSV-tk coding sequence were also amplified by PCR
from the pTK-neo plasmid and the consensus Kozak
sequence GCCACC was introduced in front of the transla-
tion start codon ATG by the primers tk1, 5¢-CGTA
TACCGGTGCCACCATGGCTTCGTACCCCGGC-3 ¢ and
tk2, 5¢-CCGCGTCGACGGAAAAGCGCCTCCCCTAC-3¢)
[32]. Recombinant plasmid pLLTK was obtained by insert-
ing all three of the amplified fragments into the multiple
cloning sites of pcDNA3.1(+) ⁄ zeo (Invitrogen, Carlsbad,
CA, USA) by cohesive-blunt end ligation. Then pLLTK
was digested with HindIII and the 4200 bp fragment of
LLTK (Fig. 7) was obtained with the QIAquick gel extrac-
tion kit (Qiagen, Valencia, CA, USA). After purification
with S & S Elutip minicolumns (Schleicher & Schuell,
Keen, NH, USA), the DNA fragment was microinjected
into the male pronuclei of the KM mouse fertilized eggs

analysed statistically using Student’s t-test (SAS Software).
A P-value < 0.05 was considered significant.
PCR analysis for the integration of the transgene
The transgene in the founder animals and their progeny
was identified by PCR analysis of genomic DNA obtained
from tail biopsies. PCR analysis was performed in 25 lL
reaction mixtures. The primers (stk1, 5¢-GTATACCGG
TATGCCCACGCTACTGCGG-3¢; SH552: 5¢-GCACTC
GAGACCCGTGCGTTTTATTCTGTCT-3¢) for HSV-tk
were designed to amplify a 390 bp region. Amplification
was performed on a thermocycler for 30 cycles of: 1 min at
94 °C, 1 min at 59 °C and 30 s at 72 °C. PCR products
were then separated electrophoretically on 2% agarose gel
and visualized after ethidium bromide staining.
Chromosomal localization of transgene integration
sites by using FISH following G-banding
Chromosome preparation was performed following the
reported methods with modifications [33–35]. FISH was
carried out according to the previous study with some
modifications [36,37]. The DNA fragment LLTK was used
as probe and labelled with the DIG-Nick translation mix
(Roche) according to the manufacturer’s protocol. Finally,
slides were counter-stained with propidium iodide anti-
fading solution (Sigma-Aldrich), and examined on a
fluorescent microscope (Leica DM RXA2, Wetzlar,
Germany). The nuclei were red and the hybridization sig-
nals were yellow-green. Previously photographed G-ban-
ded metaphases were relocated, and re-photographed.
Chromosomal localization of the transgene integration site
was determined by combining FISH hybridization signals

The slides were then washed three times and incubated with
biotinylated goat anti-rabbit immunoglobulins (DAKO) at
a dilution of 1 : 300 in NaCl ⁄ Tris for 1 h at room tempera-
ture with protection against light. After washing three
times with NaCl ⁄ Tris, peroxidase-conjugated streptavidin
(DAKO) diluted 1 : 300 was added for 1 h at room tem-
perature and washed three times with NaCl ⁄ Tris. Finally,
the signal was visualized by incubating the slides with 3–3¢-
diaminobenzidine (DAKO). The examined mice included
TK5-F1-455, TK5-F2-325 and TK3.
Induction of liver damage
For the present study, mice were housed individually at
22 °C using a 12 h light ⁄ 12 h dark photoperiod. Three trans-
genic mice of TK5-F1 (including three #), TK5-F2 (including
two # and one $) and TK3 aged 8–14 weeks, received tail
vein injections of sodium GCV (10 mgÆkg
)1
) at 48 h intervals
through a 29-gauge needle on 10 occasions, meanwhile three
nontransgenic mice served as the control. GCV was dissolved
in NaCl ⁄ P
i
and filter-sterilized before administration.
Y. Zhang et al. Development of an HSV-tk transgenic mouse model
FEBS Journal 272 (2005) 2207–2215 ª 2005 FEBS 2213
Pathological examination
Twenty-one days after GCV treatment the mice were killed
under pentobarbital anaesthesia. The tissues were fixed with
4% (v ⁄ v) phosphate-buffered formalin and paraffin-embed-
ded sections were stained using hematoxylin and eosin as

hai Jiao Tong University, China) for their performing
the FISH and microinjections. The study was supported
by: the Chinese National Program for High Technology
Research and Development (No.2002AA216091).
References
1 Grompe M, Lindstedt S, al-Dhalimy M, Kennaway NG,
Papaconstantinou J, Torres-Ramos CA, Ou CN &
Finegold M (1995) Pharmacological correction of
neonatal lethal hepatic dysfunction in a murine model of
hereditary tyrosinaemia type I. Nat Genet 10, 453–460.
2 Braun KM, Thompson AW & Sandgren EP (2003)
Hepatic microenvironment affects oval cell localization
in albumin-urokinase-type plasminogen activator trans-
genic mice. Am J Pathol 162, 195–202.
3 Heyman RA, Borrelli E, Lesley J, Anderson D, Rich-
man DD, Baird SM, Hyman R & Evans RM (1989)
Thymidine kinase obliteration: creation of transgenic
mice with controlled immune deficiency. Proc Natl Acad
Sci USA 86, 2698–2702.
4 Borrelli E, Heyman RA, Arias C, Sawchenko PE &
Evans RM (1989) Transgenic mice with inducible dwarf-
ism. Nature 339, 538–541.
5 Dzierzak E, Daly B, Fraser P, Larsson L & Muller A
(1993) Thy-1 tk transgenic mice with a conditional lym-
phocyte deficiency. Int Immunol 5, 975–984.
6 Kamogawa Y, Minasi LA, Carding SR, Bottomly K &
Flavell RA (1993) The relationship of IL-4- and IFN
gamma-producing T cells studied by lineage ablation of
IL-4-producing cells. Cell 75, 985–995.
7 Salomon B, Lores P, Pioche C, Racz P, Jami J & Klatz-

Strauss M & Dorken B (1997) Liver-associated toxicity
of the HSV-tk ⁄ GCV approach and adenoviral vectors.
Cancer Gene Ther 4, 9–16.
15 Elshami AA, Saavedra A, Zhang H, Kucharczuk JC,
Spray DC, Fishman GI, Amin KM, Kaiser LR &
Albelda SM (1996) Gap junctions play a role in the
Development of an HSV-tk transgenic mouse model Y. Zhang et al.
2214 FEBS Journal 272 (2005) 2207–2215 ª 2005 FEBS
‘bystander effect’ of the herpes simplex virus thymidine
kinase ⁄ ganciclovir system in vitro. Gene Ther 3, 85–92.
16 Krohne TU, Shankara S, Geissler M, Roberts BL,
Wands JR, Blum HE & Mohr L (2001) Mechanisms of
cell death induced by suicide genes encoding purine
nucleoside phosphorylase and thymidine kinase in
human hepatocellular carcinoma cells in vitro. Hepatol-
ogy 34, 511–518.
17 Beltinger C, Fulda S, Kammertoens T, Meyer E, Uckert
W & Debatin KM (1999) Herpes simplex virus thymidine
kinase ⁄ ganciclovir-induced apoptosis involves ligand-
independent death receptor aggregation and activation
of caspases. Proc Natl Acad Sci USA 96, 8699–8704.
18 Freeman SM, Abboud CN, Whartenby KA, Packman
CH, Koeplin DS, Moolten FL & Abraham GN (1993)
The ‘bystander effect’: tumor regression when a fraction
of the tumor mass is genetically modified. Cancer Res
53, 5274–5283.
19 Song J, Kim H, Yoon W, Lee K, Kim M, Kim K, Kim
H & Kim Y (2003) Adenovirus-mediated suicide gene
therapy using the human telomerase catalytic subunit
(hTERT) gene promoter induced apoptosis of ovarian

noue H, Yoshiji H, Okamoto S, Fukui H & Ikenaka K
(1999) Cancer gene therapy with HSV-tk ⁄ GCV system
depends on T-cell-mediated immune responses and
causes apoptotic death of tumor cells in vivo. Int J
Cancer 83, 374–380.
27 Boucher PD, Ruch RJ & Shewach DS (1998) Differen-
tial ganciclovir-mediated cytotoxicity and bystander kill-
ing in human colon carcinoma cell lines expressing
herpes simplex virus thymidine kinase. Hum Gene Ther
9, 801–814.
28 Ellison AR, Wallace H, al-Shawi R & Bishop JO (1995)
Different transmission rates of herpesvirus thymidine
kinase reporter transgenes from founder male parents
and male parents of subsequent generations. Mol
Reprod Dev 41, 425–434.
29 Huttner KM, Pudney J, Milstone DS, Ladd D & Seid-
man JG (1993) Flagellar and acrosomal abnormalities
associated with testicular HSV-tk expression in the
mouse. Biol Reprod 49, 251–261.
30 Al-Shawi R, Burke J, Jones CT, Simons JP & Bishop
JO (1988) A Mup promoter-thymidine kinase reporter
gene shows relaxed tissue-specific expression and confers
male sterility upon transgenic mice. Mol Cell Biol 8,
4821–4828.
31 Braun RE, Lo D, Pinkert CA, Widera G, Flavell RA,
Palmiter RD & Brinster RL (1990) Infertility in male
transgenic mice: disruption of sperm development by
HSV-tk expression in postmeiotic germ cells. Biol
Reprod 43, 684–693.
32 Kozak M (1987) At least six nucleotides preceding the