Pharmacology & Therapeutics 99 (2003) 261 – 282
www.elsevier.com/locate/pharmthera
Associate editor: M. Endoh
Transgenic rabbits as therapeutic protein bioreactors and
human disease models
Jianglin Fana,*, Teruo Watanabeb
a
Laboratory of Cardiovascular Disease, Department of Pathology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8575, Japan
b
Saga Medical School, Saga, Japan
Abstract
Genetically modified laboratory animals provide a powerful approach for studying gene expression and regulation and allow one to
directly examine structure-function and cause-and-effect relationships in pathophysiological processes. Today, transgenic mice are available
as a research tool in almost every research institution. On the other hand, the development of a relatively large mammalian transgenic model,
transgenic rabbits, has provided unprecedented opportunities for investigators to study the mechanisms of human diseases and has also
provided an alternative way to produce therapeutic proteins to treat human diseases. Transgenic rabbits expressing human genes have been
used as a model for cardiovascular disease, AIDS, and cancer research. The recombinant proteins can be produced from the milk of
transgenic rabbits not only at lower cost but also on a relatively large scale. One of the most promising and attractive recombinant proteins
derived from transgenic rabbit milk, human a-glucosidase, has been successfully used to treat the patients who are genetically deficient in
this enzyme. Although the pronuclear microinjection is still the major and most popular method for the creation of transgenic rabbits, recent
progress in gene targeting and animal cloning has opened new avenues that should make it possible to produce transgenic rabbits by somatic
cell nuclear transfer in the future. Based on a computer-assisted search of the studies of transgenic rabbits published in the English literature
here, we introduce to the reader the achievements made thus far with transgenic rabbits, with emphasis on the application of these rabbits as
human disease models and live bioreactors for producing human therapeutic proteins and on the recent progress in cloned rabbits.
D 2003 Elsevier Inc. All rights reserved.
Keywords: Transgenic; Rabbit; Atherosclerosis; Animal model; Bioreactor; Gene targeting
Abbreviations: apo, apolipoprotein; CETP, cholesteryl ester transfer protein; ES, embryonic stem; FHC, familial hypertrophic cardiomyopathies; HDL, highdensity lipoprotein; HL, hepatic lipase; IDL, intermediate-density lipoprotein; LCAT, lecithin:cholesterol acyltransferase; LDL, low-density lipoprotein; Lp(a),
lipoprotein(a); LPL, lipoprotein lipase; mAb, monoclonal antibody; MMP-12, matrix metalloproteinase-12; MyHC, myosin heavy chain; VLDL, very low
density lipoprotein; WAP, whey acidic protein; YAC, yeast artificial chromosome.
Contents
1.
2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . .
Applications of transgenic rabbits in biomedical studies . . .
2.1. Transgenic rabbits as bioreactors . . . . . . . . . . .
2.2. Mammary-specific promoters . . . . . . . . . . . . .
2.3. Recombinant proteins produced from transgenic rabbit
2.4. Antibodies produced from transgenic rabbits . . . . .
Transgenic rabbits as human disease models . . . . . . . . .
3.1. Transgenic rabbits for atherosclerosis studies . . . . .
3.1.1. Apolipoprotein A-I transgenic rabbits . . . .
3.1.2. Apolipoprotein B transgenic rabbits . . . . .
3.1.3. Apolipoprotein (a) transgenic rabbits . . . . .
* Corresponding author. Tel.: +81-298-53-3165; fax: +81-298-54-9039.
E-mail address: [email protected] (J. Fan).
0163-7258/03/$ – see front matter D 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0163-7258(03)00069-X
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3.1.4. Apolipoprotein E2 transgenic rabbits . . . . . . . . . . . . . . . . . . . . . . .
3.1.5. Apolipoprotein E3 transgenic rabbits . . . . . . . . . . . . . . . . . . . . . . .
3.1.6. Apolipoprotein B mRNA editing enzyme catalytic polypeptide 1 transgenic rabbits
3.1.7. Hepatic lipase transgenic rabbits . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.8. Lecithin:cholesterol acyltransferase transgenic rabbits . . . . . . . . . . . . . .
3.1.9. Lipoprotein lipase transgenic rabbits . . . . . . . . . . . . . . . . . . . . . . .
3.1.10. 15-Lipoxygenase transgenic rabbits . . . . . . . . . . . . . . . . . . . . . . .
3.1.11. Matrix metalloproteinase-12 transgenic rabbits . . . . . . . . . . . . . . . . .
3.1.12. Double transgenic rabbits . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.13. Watanabe heritable hyperlipidemic transgenic rabbits . . . . . . . . . . . . . .
3.1.14. Transgenic rabbits versus transgenic mice. . . . . . . . . . . . . . . . . . . .
3.2. Transgenic rabbits as hypertrophic cardiomyopathy models . . . . . . . . . . . . . . . .
3.3. Transgenic rabbits as models for acromegaly and diabetes mellitus . . . . . . . . . . . .
3.4. Transgenic rabbits as models for AIDS and cancer study . . . . . . . . . . . . . . . . .
3.5. Other transgenic rabbits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. The methods for creating transgenic rabbits . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Pronuclear microinjection method . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.1. Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.2. Superovulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.3. Microinjection, embryo transfer, and detection of founders . . . . . . . . . . .
4.1.4. Variables affecting the success of transgenic rabbit generation . . . . . . . . . .
4.2. Nuclear transfer method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Other methods for transgenic rabbits . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Gordon et al. (1980) first reported the technology to
introduce a foreign gene into mice. Since then, tremendous
progress and achievements have been made in the transgenic research. Among these are the creation of transgenic
and knockout mice for research (Wight & Wagner, 1994),
transgenic livestock for the production of organs for xenotransplantation and for the production of therapeutic proteins from the milk (Houdebine, 2000), and more recently
advances in animal cloning (Colman, 2000). Generally
speaking, genetically modified animals can be classified
into two categories based on the gain of function or loss of
function of the genes: transgenic animals that bear a new
gene (called the transgene), which is integrated into the
genome, and knockout animals in which endogenous
gene(s) have been inactivated through homologous recombination (Doetschman et al., 1987; Thomas & Capecchi,
1987). Unfortunately, the production of knockout animals
has not been successful in species other than the mouse.
‘‘Transgenic animals’’ (in a broad sense) is sometimes used
to refer to both transgenic and knockout animals. Here, we
will use the term ‘‘transgenic’’ to designate all genetically
modified animals. Transgenic animals may also be divided
simply based on their size: small transgenics (mice and rats),
intermediate transgenics (rabbits), and large transgenic livestock (pigs and ruminants). The generation and applications
of each type of transgenic animal may depend entirely upon
the researcher’s interests. For example, transgenic mice may
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be used for the study of gene expression and regulation and
as a model of human pathophysiology, whereas large transgenic livestock are instead exclusively used for the production of organs intended for xenotransplantation or for the
production of therapeutic proteins. As an intermediate
between transgenic laboratory animals and farm animals,
transgenic rabbits can be used as both bioreactors and
models for some specific human diseases for which mice
are generally not suitable, as discussed later in Section
3.1.14. In this article, we specifically address the progress
in transgenic rabbits, including applications of transgenic
rabbits as bioreactors and human disease models and as
other research tools.
Rabbits are one of the most recently domesticated
species. Originating from Spain, wild rabbits were kept in
rabbit gardens or hunting grounds in ancient Rome. Further
domestication took place in late antiquity and during the
Middle Ages in monasteries in France. Rabbit breeds and
hybrid strains were developed during the 19th century based
on different mutations of coat color and other visible traits.
For many centuries, rabbits have been used for both
livestock production and animal experiments. Classical
experimental uses of rabbits include antibody production,
development of new surgical techniques, studies of physiology (e.g., circulation [atherosclerosis] and hypertension),
and toxicity tests of new drugs (Manning et al., 1994). On
the other hand, rabbits are also important in livestock
production, especially in the Mediterranean region and some
developing countries for meat, fur, and angora wool pro-
J. Fan, T. Watanabe / Pharmacology & Therapeutics 99 (2003) 261–282
duction. Thus, gene transfer into rabbits is an attractive
technique for improving the performance and applications
of rabbits in research and livestock production.
The first reports on the application of transgenic technology to the rabbit were by Brem et al. (1985) and Hammer
et al. (1985) as an initial trial to express human growth
hormone under the control of the mouse metallothionein
promoter. Although these transgenic rabbits did not turn out
to be functional or useful since the transgenic protein levels
were too low, these studies definitely paved the way for
subsequent work to exploit the rabbit model as an alternative model for investigating human diseases such as
atherosclerosis, cancer, AIDS, hypertrophic cardiomyopathy, and diabetes mellitus. In addition, because of their
appropriate size and short lactation period, transgenic rabbits have been used subsequently to produce biologically
active recombinant proteins in the milk. There have been 90
English language publications on transgenic rabbits based
on a Medline search using the keywords ‘‘transgenic rabbits’’ or ‘‘transgenic and rabbits’’ as of September 2002.
More than 50% of these reported studies of transgenic
rabbits have been for cardiovascular research, including
research on atherosclerosis, dyslipidemias, hypertrophic
cardiomyopathy, and diabetes mellitus, while about 30%
of the reported studies aimed at producing bioreactive
proteins from rabbit milk. We will discuss the potential
use of these models in biomedical fields and describe the
basic procedures for making transgenic rabbits.
2. Applications of transgenic rabbits in biomedical
studies
2.1. Transgenic rabbits as bioreactors
Hammer et al. (1985) established the first transgenic
livestock animals, including sheep, rabbits, and pigs, in an
attempt to develop a way to produce recombinant proteins
from these animals. Since then, production of a number of
recombinant proteins from transgenic animals has been
reported. A good example is human factor IX, which is
now used to treat human hemophilia B (Lubon & Palmer,
2000). Although transgenic mice may serve as a predictive
model to evaluate the usefulness of expression constructs
and to study the properties of expressed proteins, they are
not at present useful as bioreactors for producing large
quantities of recombinant proteins that can satisfy commercial demands. Often, researchers pretest constructs in mice
prior to microinjecting into the more expensive livestock
species. Large transgenic animals, such as cows, pigs,
sheep, goats, and rabbits, have been used as bioreactors,
and many pharmaceutical companies have made efforts to
produce different valuable therapeutic proteins (Houdebine,
1995, 2000). The chief objective of using bioreactors is the
economical production of valuable complex human therapeutic proteins in easily accessible fluids. By using con-
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structs with the tissue-specific expression, it is now possible
to express and produce large amounts of human recombinant proteins in the extracellular space, urine, seminal
plasma, milk, and blood from large transgenic animals.
Proteins obtained from bioreactors have several advantages
compared with proteins from other sources. First, using
transgenic bioreactors to produce the proteins may reduce
the contamination in the products of contaminants such as
HIV and virus hepatitis compared with the levels of contamination in proteins isolated directly from human blood,
thereby avoiding tragedies such as the infection of many
hemophilia patients in Japan with viruses present in human
blood products. The second major advantage of producing
foreign proteins in transgenic animals is the superior preservation of the native protein activity compared with that of
bacteria- and yeast-derived recombinant proteins. This is
due to the fact that bacteria do not add carbohydrates to
polypeptide chains and cannot necessarily generate all
proteins in their mature native structures. The mammary
glands of transgenic bioreactors appear to accomplish protein postsynthesis modifications such as carboxylation,
glycosylation, and amidation, all of which are essential for
full biological activity of many proteins (Houdebine, 1995,
2000). The use of eukaryotic cells (cultured mammalian
cells) can overcome these problems in some cases, but the
culturing of animal cells on an industrial scale remains an
expensive technique. In addition, the production of therapeutic active peptides as fusion peptides in the milk of
transgenic animals also has several advantages over chemical synthesis. The scale on which peptides can be synthesized chemically is limited by considerations of reactor size,
reagent handling and disposal, and cost of purification
(McKee et al., 1998). Therefore, bioreactors can be an ideal
source of recombinant proteins and can be used to produce
physiologically active substances at relatively low cost. The
criteria for selecting the most suitable animal species for
gene farming are totally based on the quantity of proteins
needed per year, the capacity of the facility, and the potential
commercial value of the recombinant proteins in addition to
other factors such as time until milk production and milk
volume, etc., as summarized in Table 1 (Ziomek, 1998). A
simplified rule for choosing transgenic bioreactors is: the
production of a protein (such as albumin) in tons should be
Table 1
Comparison of transgenic milk expression system between different species
Animal
Gestation
(months)
Maturation
(months)
Milk yield
per lactation
(L)
Elapsed months
from microinjection
to milk
Mouse
Rabbit
Pig
Sheep
Goat
Cow
0.75
1
4
5
5
9
1
5–6
7–8
6–8
6–8
15
0.0015
1 – 1.5
200 – 400
200 – 400
600 – 800
8000
3–6
7–8
15 – 16
16 – 18
16 – 18
30 – 33
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carried out using transgenic cows, in hundreds of kilograms
using sheep or goats, and in kilograms per year using rabbits
(Castro et al., 1999). Transgenic rabbits are highly suitable
as an intermediate animal for the production of recombinant
proteins on a relatively large scale. Compared with other
large farm animals, rabbits have other unique features.
Rabbit husbandry can be done under specific pathogen-free
barrier facilities, rabbits have shorter reproductive interval,
and transgenic rabbit founders can be generated with a
reasonable efficiency (Table 2) (Buhler et al., 1990). The
protein content of rabbit milk is 14% compared with 5%
in cow’s milk and a lactating female rabbit can produce
170 –220 g of milk per day and yield up to 10 kg of milk per
year under semiautomatic hygienic milking conditions
(Duby et al., 1993). Thus, considering both economical
and hygienic aspects, rabbits are attractive for the mammary
gland-specific expression of recombinant proteins. Researchers and pharmaceutical companies are focusing their
attention on achieving relatively large-scale production of
proteins using transgenic rabbits.
2.2. Mammary-specific promoters
Several promoters have been successfully used to direct
tissue-specific expression of recombinant proteins. In rabbits, caseins are the major protein constituents of milk and
their concentration in rabbit’s milk is above 60 mg/mL,
whereas the concentration of whey acidic proteins (WAP) is
15 mg/mL in the milk. Therefore, the aS1- and b-casein
promoter and the WAP promoter, along with the b-lactoglobulin promoter, have been extensively used to direct the
tissue-specific expression of recombinant proteins in transgenic rabbits (Castro et al., 1999). These promoters can be
from different species such as mouse, cattle, or sheep in
addition to the rabbit endogenous promoters. Common
problems using heterologous DNA (typically consisting of
promoter and cDNA) are the ectopic or nonspecific expression of the transgene (Massoud et al., 1996). As discussed in
Section 4.1.4, such problems need to take into account
whenever one is using this model for the production of
recombinant proteins. Most researchers have found that
genomic sequences direct higher levels of expression than
cDNA sequences. It is generally recommended that large
genomic DNA such as yeast artificial chromosome (YAC)
Table 2
Reproductive performance of rabbits
Reproductive parameter
Value
Age at sexual maturity
Conception rate
Gestation time
Litter size
Lactation period
Litter interval (mean)
Litters per year
4 – 5 months
65%
30 – 33 days
5 – 12
40 – 50 days
44 days
4–7
or BAC should be used to generate transgenic rabbits (Brem
et al., 1996; Giraldo & Montoliu, 2001). A specific mammary-specific expression cassette, designated the pBC1
milk expression vector kit (cat. no. K270-01, Invitrogen),
originally created by Genzyme Transgenic, is now commercially available. This vector uses the goat b-casein promoter
to drive high-level expression of a variety of cDNA-based
constructs. For example, using this construct, high levels of
transgenic proteins have been obtained in transgenic goat
milk: 6 g/L for human tissue plasminogen activator (tPA),
14 g/L for antithrombin III, 20 g/L for a1-proteinase
inhibitor, and 10 g/L for an anticancer monoclonal antibody
(mAb) (Ziomek, 1998).
2.3. Recombinant proteins produced from transgenic rabbit
milk
Using an appropriate promoter, a number of recombinant
proteins have been produced from transgenic rabbit milk or
blood, as summarized in Table 3. Recombinant human
proteins produced by transgenic rabbits include human
a1-antitrypsin (Massoud et al., 1990, 1991), interleukin-2
(Buhler et al., 1990), tPA (Reigo et al., 1993), erythropoietin
(Rodriguez et al., 1995; Massoud et al., 1996; Korhonen et
al., 1997), insulin-like growth factor-1 (Brem et al., 1994;
Wolf et al., 1997; Zinovieva et al., 1998), extracellular
superoxide dismutase (Stromqvist et al., 1997), growth
hormone (Hammer et al., 1985; Limonta et al., 1995), aglucosidase (Bijvoet et al., 1998), salmon calcitonin
(McKee et al., 1998), equine chorionic gonadotropin (Galet
et al., 2001), nerve growth factor-b (Coulibaly et al., 1999),
protein C (Chrenek et al., 1999), and chymosin (Brem et al.,
1995). It must be admitted that not all these transgenic
rabbits as bioreactors or the recombinant proteins produced
are functional or practical (some studies have not yet
progressed beyond the research stage) due to low levels of
expression; however, these studies have opened the door for
possible technical advances that will permit the production
of large quantities of these human therapeutic proteins and
their use in the future. One of the best examples of such
proteins reported until now is human a-glucosidase from
rabbit milk, which is the first transgenic product from rabbit
milk used to treat patients (Van den Hout et al., 2000, 2001).
Pompe’s disease (also called glycogen storage disorder type
II) is a fatal muscular disorder caused by lysosomal aglucosidase deficiency; patients with this disease have a
rapidly fatal or slowly progressive impairment of muscle
functions due to concomitant storage of lysosomal glycogen
in the muscles and massive cardiomegaly. Hitherto, these
patients have been treated with human acid a-glucosidase
produced from genetically modified Chinese hamster ovary
cells. In 1998, a group of scientists in the Netherlands
generated transgenic rabbits using a fusion between the
human acid a-glucosidase gene in its genomic context and
the bovine aS1-casein promoter. This protein isolated from
transgenic rabbit milk was shown to exert therapeutic effects
J. Fan, T. Watanabe / Pharmacology & Therapeutics 99 (2003) 261–282
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Table 3
Therapeutic recombinant proteins produced from transgenic rabbits
Proteins
Potential use
Protein levels
Promoter
References
Human a1-antitrypsin
Emphysema
1 mg/mL plasma
Human a1-antitrypsin
Human IL-2
Human tPA
Human erythropoietin
?
Thrombosis
Anemia
Human insulin-like
growth factor-1
GH deficiency/resistance,
DM, osteoporosis,
cardiomyopathy
Osteoarthritis, ischemia and
post-ischemic reperfusion
GH deficiency
0.43 mg/mL
50 ng/mL
0.3 ng/mL
0.5 mg/mL
50 mg/mL
1 mg/mL
678 mg/mL
300 mg/mL
3 mg/mL
Rabbit b-casein
Bovine aS1-casein
Rabbit WAP
Bovine b-lactoglobulin
Rabbit WAP
Bovine aS1- casein
Bovine aS1- casein
Bovine aS1- casein
Mouse WAP
Massoud et al.,
1990, 1991
Buhler et al., 1990
Reigo et al., 1993
Rodriguez et al., 1995;
Massoud et al., 1996;
Korhonen et al., 1997
Brem et al., 1994;
Wolf et al., 1997;
Zinovieva et al., 1998
Stromqvist et al., 1997
80 ng/mL plasma
>4 mg/mL
50 mg/mL
8 mg/mL
2.1 mg/mL
Mouse metallothionein-I
Mouse WAP
Mouse WAP
Bovine aS1-casein
Ovine b-lactoglobulin
Hammer et al., 1985;
Brem et al., 1985;
Limonta et al., 1995
Bijvoet et al., 1999
McKee et al., 1998
27.1 mg/mL
Rabbit WAP
Galet, 2000
Neuropathy
50 – 250 mg/mL
Bovine aS1-casein
Coulibaly et al., 1999
hPC deficiency
Cheese production
?
0.5 – 2 mg/mL
Mouse WAP
Bovine aS1-casein
Chrenek et al., 1999
Brem et al., 1995
Human extracellular
superoxide dismutase
Human GH
Human a-glucosidase
Salmon calcitonin
Equine chorionic
gonadotropin (eCG)
Human nerve growth
factor (hNGF-b)
Human protein C
Bochymosin
Glycogen storage disease
Osteoporosis, Paget’s disease,
and hypercalcemic shock
?
GH, growth hormone; IL-2, interleukin-2; tPA, tissue plasminogen activator.
in the treatment of mice with glycogen storage deficiency
and later on in the treatment of human a-glucosidase
deficiency (Bijvoet et al., 1998, 1999). In one of those
studies, the authors administered recombinant human aglucosidase from rabbit milk to four human babies who
were genetically deficient in a-glucosidase, at starting doses
of 15 or 20 mg/kg and later at 40 mg/kg (Van den Hout et
al., 2000). The activity of human a-glucosidase was shown
to be normalized in the muscles of these patients, and the
tissue morphology and motor and cardiac functions were
dramatically improved (Van den Hout et al., 2000). That
successful study provided convincing evidence that the milk
of transgenic rabbits is a safe source of therapeutic proteins
and has opened the way for further exploration of this
production method. Now, the Pharming Pharmaceutical in
the Netherlands has undertaken a transgenic rabbit program
to make human a-glucosidase to treat Pompe’s disease and
human C1 inhibitor to treat hereditary angioedema (http://
www.pharming.com/Technology/technology.html). It is
reasonable to hope that many more biopharmaceutical
proteins will soon be produced via transgenic rabbit milk.
2.4. Antibodies produced from transgenic rabbits
Since rabbits have a large quantity of blood (on average
50 – 60 mL/kg BW), it is possible to produce sufficient
amounts of Ab for diagnostic and therapeutic purposes.
Weidle et al. (1991) introduced the genes for the light and
heavy chains of a mouse mAb into transgenic rabbits. The
titers of mAb were about 200 mg/mL in the transgenic rabbit
serum, suggesting that transgenic rabbits can be used as a
tool to produce Ab. One critical obstacle, which should be
tackled before this technique can be applied for this purpose,
is that the endogenous immunoglobulin loci of the rabbit
must be rendered inactive, which requires homologous
recombination in totipotential cells (such as embryonic stem
[ES] cells). Unfortunately, as mentioned above and discussed in Section 4.2, the use of ES cell has not been
successful in rabbits. Therefore, there is a need for an
alternative means to overcome this problem, such as the
nuclear transfer technique. For many years, researchers have
attempted to generate rabbit mAb because rabbits recognize
antigens and epitopes that are not immunogenic in mice or
rats, two species from which mAb are usually generated.
However, rabbit mAb have not been obtained successfully
because no plasmacytoma fusion partner was available. In
this respect, Spieker-Polet et al. (1995) generated transgenic
rabbits carrying two oncogenes, c-myc and v-abl. These
transgenic rabbits developed plasmacytomas from which a
plasmacytoma cell line was isolated. This cell line was fused
with spleen cells of immunized rabbits, resulting stable
hybridomas that secreted Ab specific for the immunogen.
Interestingly, the rabbit hybridomas can be cloned and
propagated in nude mice and can be frozen without a change
in their ability to secrete specific mAb (Spieker-Polet et al.,
1995). These studies demonstrated that transgenic rabbits
can be used to produce mAb for the diagnosis of diseases
and for the treatment of patients in the future.
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3. Transgenic rabbits as human disease models
3.1. Transgenic rabbits for atherosclerosis studies
The ideal animal model of human atherosclerosis should
possess several important characteristics (Fan & Watanabe,
2000). It should be easy to acquire and maintain at a
reasonable cost, easy to handle, and of the proper size to
allow for all anticipated experimental manipulations.
Ideally, the animal should reproduce in a laboratory setting
and have well-defined genetic characteristics. Finally, the
animal model should share with man the most important
aspects of the disease process. Lesions should develop
naturally when the animal consumes a reasonable diet,
and lesions should develop slowly over the animal’s lifetime
with clinical sequelae in later middle to old age. The natural
history of lesion pathogenesis should range from fatty
streaks to atheromatous plaques with complications such
as calcification, ulceration, hemorrhage, and superimposed
thrombosis with luminal stenosis. Although there is no
species that satisfies all these requirements, cholesterol-fed
rabbits are the first developed and most generally used
model for the study of atherosclerosis (Fan & Watanabe,
2000). The first experiments on rabbits with the aim of
studying atherosclerosis study were performed a century ago
(Ignatowski, 1908). The rabbit has been extensively utilized
as an ideal model of atherosclerosis because of its size, easy
manipulation, and extraordinary response to dietary cholesterol. With the advent of genetically engineered rabbits,
transgenic rabbits have become a novel means to explore a
number of proteins that are associated hyperlipidemia and
atherosclerosis (Taylor & Fan, 1997; Fan et al., 1999b).
Compared with the most widely used transgenic model,
the mouse, rabbits have different lipoprotein metabolism
features, as summarized in Table 4. For example, (1) rabbit
lipoprotein profiles (low-density lipoprotein [LDL] rich) are
Table 4
Comparison of lipoprotein metabolism characteristics between mouse,
rabbit, and human
Lipoprotein
profile
CETP
Hepatic apoB
editing
apoB48
Hepatic lipase
activity
Hepatic LDL
receptor
apoA-II
Dietary
cholesterol
Atherosclerosis
Mouse
Rabbit
Human
HDL-rich
LDL-rich
LDL-rich
No
Yes
Yes
No
Yes
No
Chylomicron
VLDL
High, 70% in
circulation
Usually high
Chylomicron
Chylomicron
Low, liver-bound
High, liver-bound
Down-regulated
Down-regulated
Yes
Resistant
(most strains)
Resistant
No
Sensitive
Yes
–
Susceptible
–
CETP, cholesteryl ester transfer protein.
similar to those of humans but unlike those of mice (highdensity lipoprotein [HDL] rich); (2) rabbit liver does not edit
apolipoprotein (apo) B mRNA and thus produces apoB-100
only as does the human liver, but mouse liver also produces
apoB48; therefore, apoB48 is present in both hepatically
derived very low density lipoprotein (VLDL) and intestinally derived chylomicrons; (3) rabbits have abundant
cholesteryl ester transfer protein (CETP) in their plasma as
do humans whereas mice are deficient in CETP; and (4) as
mentioned above, rabbits are susceptible to cholesterol-rich
diet-induced atherosclerosis, whereas most strains of mice
are resistant to cholesterol diet-induced atherosclerosis. In
addition, the rabbit lacks an analogue of human apoA
(hapoA)-II and has relatively lower hepatic lipase (HL)
activity compared with mice and thus provides a unique
system to assess the effects of these genes on plasma
lipoproteins and atherosclerosis susceptibility (Brousseau
& Hoeg, 1999). Rabbit strains have a more diverse genetic
background than inbred and outbred mouse strains. This
might be favorable when studying complex disease models
such as atherosclerosis, obesity, and diabetes mellitus or
developing therapeutic strategies since it resembles more
accurately the diverse situation in humans. However, this
may also hamper its use in defining the effects of gain of
function or loss of function of the target gene and elucidate
the mechanism(s) of single gene related diseases. Despite
this limitation, transgenic rabbits have become a unique tool
in demonstrating a number of gene functions in physiological and pathological processes.
To date, transgenes for human apo(a), apoA-I, apoB,
apoE2, apoE3, HL, lecithin:cholesterol acyltransferase
(LCAT), lipoprotein lipase (LPL), 15-lipoxygenase (15LO), and matrix metalloproteinase-12 (MMP-12) as well
as for rabbit apoB mRNA editing enzyme catalytic polypeptide 1 (APOBEC-1) have been expressed in rabbits
(Table 5). In addition, human apoA-I, LCAT, apo(a), and
LPL have been introduced into Watanabe heritable hyperlipidemic (WHHL) rabbits, which are deficient for LDL
receptor function. All of these transgenes have been found
to have significant effects on plasma lipoprotein metabolism
and/or atherosclerosis. These studies have provided new
insights into the mechanisms responsible for the development of atherosclerosis. We will briefly describe the
features of each of these transgenic rabbits and discuss the
findings from these models.
3.1.1. Apolipoprotein A-I transgenic rabbits
Duverger et al. (1996a, 1996b) reported the generation of
five lines of transgenic New Zealand white rabbits expressing human apoA-I in the liver. The plasma levels of human
apoA-I in transgenic rabbits ranged from 8 to 100 mg/dL.
When these transgenic rabbits were fed a cholesterol diet
(0.48 g cholesterol per 120 g of diet) for 14 weeks, the
atherosclerotic lesions in the thoracic aorta were reduced by
50% compared with those in control rabbits (15 ± 12% vs.
30 ± 8%). This study showed that the protective effects of
J. Fan, T. Watanabe / Pharmacology & Therapeutics 99 (2003) 261–282
267
Table 5
Transgenic rabbits for atherosclerosis and dyslipidemias
Transgenes expressed
Expression
tissue
Effects on lipoproteins
Effects on atherosclerosis
Human hepatic lipase
Human lipoprotein lipase
Liver
Multiple
VLDL#, IDL#, HDL#
VLDL#, IDL#, LDL", HDL#
Human lecithin:cholesterol acyltransferase
Rabbit apoB mRNA editing protein
Human apo(a)
Human apoA-I
Human apoB-100
Human apoC-III
Human apoE2
Human apoE3
15-Lipoxygenase
Macrophage metalloelastase
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Macrophage
Macrophage
LDL#, HDL"
LDL#
Lp(a) formation
HDL"
LDL" and HDL#
ND
VLDL", IDL", HDL" (,)
VLDL#, LDL", HDL"
ND
ND
Anti-atherogenic
Anti- or pro-atherogenic
dependent on cholesterol levels
Anti-atherogenic
ND
Pro-atherogenic
Anti-atherogenic
ND
ND
Atherogenic
Atherogenic (high expressor)
Pro-atherogenic
ND
VLDL, very low density lipoprotein; LDL, low-density lipoprotein; IDL, intermediate-density lipoproteins; HDL, high-density lipoprotein; ND, not
determined.
human apoA-I on diet-induced atherosclerosis were associated with the HDL levels via the mechanism of reverse
cholesterol transport (Duverger et al., 1996a). Unfortunately, these human apoA-I rabbits suffered from impairment of signal transduction of the endothelial nitric oxide
(NO) system and showed impaired endothelium-derived
vasorelaxation (Lebuffe et al., 1997). Also, the anti-atherosclerotic effect of human apoA-I was not confirmed in later
studies of these authors using either same or different lines
of the human apoA-I transgenic rabbits (Mackness et al.,
2000; Boullier et al., 2001).
3.1.2. Apolipoprotein B transgenic rabbits
The development of transgenic rabbits expressing human
apoB-100 by using an 80-kb human apoB genomic DNA
was described by Fan et al. (1995). Four lines of transgenic
rabbits were generated, with plasma levels of human apoB100 ranging from 12 to 94 mg/dL. Expression of human
apoB-100 in these transgenic rabbits resulted in a 2- to 3fold increase of total cholesterol and triglycerides (TG)
compared with those in age- and sex-matched control
rabbits. Nearly all of the cholesterol and human apoB-100
was in the LDL fraction, with striking enrichment of the TG
content. Transgenic rabbit LDL was further found to contain
large amounts of apoC-III and apoE. The atherosclerosis
susceptibility was not determined in these animals. To study
the effect of HL on LDL modulation, these transgenic
rabbits were crossbred with HL transgenic rabbits (Rizzo
et al., 1999). Rouy et al. (1998) produced a line of human
apoB transgenic rabbits using a 90-kb P1 phagemid clone
and found that the plasma level of human apoB was 17.6
mg/dL. It is envisioned that human apoB transgenic rabbits
may be useful for investigating some lipid-lowering agents
(such as antisense inhibitor for inhibiting apoB synthesis)
with the aim of treating hypercholesterolemia in humans
(Isis Pharmaceuticals) (http://www.isispharmaceuticals.com/
press/press02/052102-Cardio.htm).
3.1.3. Apolipoprotein (a) transgenic rabbits
Elevated plasma levels of lipoprotein(a) [Lp(a)] constitute an independent risk factor for coronary heart disease,
stroke, and restenosis (Ishibashi, 2001). However, apo(a), a
unique component of Lp(a), is naturally present exclusively
in Old World monkeys, humans, and hedgehog. Therefore,
there are no convenient experimental animal models of
Lp(a). Studies on transgenic mice expressing human apo(a)
revealed that murine apoB cannot bind to human apo(a) to
form Lp(a) particles (Chiesa et al., 1992). To investigate the
Lp(a) assembly and its possible role in atherosclerosis, our
laboratory along with others have reported the generation of
transgenic rabbits expressing human apo(a) (Fan et al.,
1998a, 1999a; Rouy et al., 1998). The human apo(a) levels
of transgenic rabbits from those studies were 2.5 mg/dL in
transgenic rabbits generated with YAC vector and 1.8 – 4.5
mg/dL in transgenic rabbits generated with apo(a) cDNA.
Those studies showed that transgenic rabbits expressing
human apo(a) exhibited efficient assembly of human
Lp(a)-like particles, suggesting that such rabbits may be
useful as a model for the study of Lp(a) (Fan et al., 1999a).
To examine the effect of Lp(a) on the development of
atherosclerosis, we studied transgenic rabbits expressing
human apo(a) on both chow and cholesterol diets. We did
not find any atherosclerotic lesions in transgenic rabbits on a
regular chow diet, suggesting that lower plasma apo(a) is
not atherogenic. On a 0.3% cholesterol diet for 16 weeks,
human apo(a) transgenic rabbits had more extensive atherosclerotic lesions than nontransgenic rabbits although the
cholesterol levels in the plasma of both groups of rabbits
were similarly elevated. Compared with the lesions in nontransgenic control rabbits, the areas of atherosclerotic
lesions in human apo(a) transgenic rabbits were increased
in the aorta, iliac artery, and carotid artery (Fan et al., 2001a)
(Fig. 1). Furthermore, we found that human apo(a) transgenic rabbits on a cholesterol-rich diet had a greater degree
of coronary atherosclerosis than control rabbits (Fig. 1).
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delayed fibrinolytic activity and increased plasma plasminogen activator inhibitor-1 in transgenic rabbits and that
Lp(a) promotes smooth muscle cell proliferation in atherosclerotic lesions of transgenic rabbits (Ichikawa et al.,
2002).
3.1.4. Apolipoprotein E2 transgenic rabbits
Transgenic rabbits expressing high levels of human
apoE2 (Cys 112 and Cys 158), an apoE variant, were
generated by Huang et al. (1997). The apoE2 homozygous
patients manifest type III hyperlipoproteinemia and are
predisposed to premature atherosclerosis. The study of
Huang et al. demonstrated that overexpression of human
apoE2 (30 – 70 mg/dL) resulted in a marked accumulation of
b-VLDL (intestinal and hepatic remnant lipoproteins), a
hallmark of type III hyperlipoproteinemia. Even on a chow
diet, these rabbits developed spontaneous atherosclerosis in
the aortic arch and proximal abdominal aorta. A more
intriguing finding from their study was that male transgenic
rabbits showed more extensive atherosclerosis than transgenic females, suggesting that sex hormones play an
important role in modulating type III hyperlipoproteinemia
(Huang et al., 1997).
Fig. 1. Lp(a) enhances the development of diet-induced atherosclerosis in
transgenic rabbits expressing human apo(a). Rabbits were fed a 0.3%
cholesterol diet for 16 weeks and the atherosclerotic lesions of their aorta
(upper panel) and coronary arteries (lower panel) were analyzed. The aortas
were stained by Sudan IV to visualize lipid-stained lesion areas (red in
color); apparently, transgenic rabbits (upper panel, right) have more
extensive lesions than do control rabbits. Coronary arteries were
investigated by histology and immunohistochemistry using Ab against
apo(a) and apoB. Compared with control rabbits, transgenic rabbits have
larger plaque in coronary artery with reduced lumen (see hematoxylin-eosin
[HE] staining). Both apo(a) and apoB are present in the lesions of
transgenic rabbit coronary artery when stained with Ab against apo(a) and
apoB, suggesting that Lp(a) may participate in the lesion development (Fan
et al., 2001a).
That study is currently being extended to clarify the mechanism(s) responsible for atherogenicity of apo(a) in transgenic rabbits. Recently, we reported that Lp(a) causes
3.1.5. Apolipoprotein E3 transgenic rabbits
Fan et al. (1998b) generated transgenic rabbits expressing human apoE3 (Cys112 and Arg158) using human apoE3
genomic DNA together with the hepatic control region.
Three lines of transgenic rabbits were established, and their
human apoE3 levels were 6, 11, and 13 mg/dL, respectively.
Analysis of these transgenic rabbits revealed that increased
expression of human apoE3 results in reduced VLDL and
increased accumulation of LDL, which is apparently different from the effects in transgenic mice expressing the same
transgene. The mechanism(s) responsible for this phenomenon were investigated, and the results showed that apoErich particles have a greater affinity for cell surface receptors, thereby increasing remnant clearance from the plasma.
In addition, these particles appear to compete more effectively than LDL for receptor-mediated binding and clearance, resulting in delayed clearance and the accumulation of
LDL in the plasma. The effects of human apoE3 expression
on diet-induced atherosclerosis were briefly described in a
preliminary report (Taylor, 1997). Further studies will be
required to more fully elucidate the role of apoE3 in
atherosclerosis susceptibility. Another important finding
obtained from apoE3 transgenic rabbits is that overexpression of apoE3 causes combined hyperlipidemia by stimulating hepatic VLDL production and reducing VLDL
lipolysis. This study using transgenic rabbits expressing
different levels of human apoE also revealed that the
differential expression of apoE may, within a narrow range
of concentrations, play a critical role in modulating the
plasma cholesterol and TG levels and may represent an
important determinant of specific types of hyperlipoproteinemia (Huang et al., 1999).
J. Fan, T. Watanabe / Pharmacology & Therapeutics 99 (2003) 261–282
3.1.6. Apolipoprotein B mRNA editing enzyme catalytic
polypeptide 1 transgenic rabbits
Transgenic rabbits expressing rabbit APOBEC-1 were
generated by Yamanaka et al. (1995) to determine whether
hepatic expression of this enzyme would reduce plasma
LDL cholesterol concentrations in an attempt to use this
enzyme for treating hyperlipidemias. Two transgenic founders were created: one founder had a single copy of the
APOBEC-1 transgene and the other had 17 copies. Analysis of these two transgenic and control rabbits showed
that the lipoprotein profiles of transgenic rabbits were
characterized by reductions of plasma VLDL, intermediate-density lipoproteins (IDL), and LDL accompanied by
increased HDL cholesterol. Although the lipoprotein profiles are favorable in transgenic rabbits, the transgenic
rabbit with high copy number of transgene suffered from
liver dysplasia, which may compromise the potential use of
APOBEC-1 for gene therapy to treat hyperlipidemias
(Yamanaka et al., 1995).
3.1.7. Hepatic lipase transgenic rabbits
Fan et al. (1994a) established transgenic rabbits overexpressing human HL as the first transgenic rabbits expressing an enzyme for lipoprotein metabolism. The rationale for
preferentially using rabbits is that rabbits have a lower level
of activity of HL, which has been considered to be responsible for their susceptibility to diet-induced atherosclerosis.
The construct used for producing the transgenic rabbits was
composed of human HL cDNA and the human apoE/CI
hepatic control region. HL expression in transgenic rabbits
had a significant effect on plasma lipid and lipoprotein
levels. Total cholesterol and TG levels were reduced by
42% and 58% in transgenic rabbits compared with nontransgenic controls. Lipoprotein analysis revealed that overexpression of HL led to a remarkable reduction of HDL,
VLDL, and IDL. When HL transgenic rabbits were fed a
diet containing 0.3% cholesterol and 3% soybean oil, they
showed attenuated hypercholesterolemia compared with
control rabbits (Fan et al., 1994b). A preliminary study
showed that reduced hypercholesterolemia in HL transgenic
rabbits was associated with a diminished extent of aortic
atherosclerosis (Taylor, 1997). It should be noted that
transfer of HL into mice caused a pro-atherogenic effect
(Amar et al., 2000), again suggesting that rabbits and mice
have intrinsic differences even when the same transgene is
expressed.
3.1.8. Lecithin:cholesterol acyltransferase transgenic
rabbits
Hoeg et al. (1993, 1996a, 1996b) expressed human
LCAT in both WHHL and wild-type New Zealand white
rabbits using the human LCAT genomic DNA construct.
Several reports about the effects of human LCAT on
lipoprotein metabolism using this model have been published (for a review, see Brousseau & Hoeg, 1999). Human
LCAT overexpression in transgenic rabbits resulted in a
269
substantial change in plasma lipid and lipoprotein profiles:
plasma total, free, and esterified cholesterol concentrations
as well as the phospholipid concentration were significantly
increased in both low and high expressor F1 progeny
compared with control rabbits (Hoeg et al., 1996b). The
elevation of plasma total cholesterol content was due to a
marked increase in HDL cholesterol concentration. On a
0.3% cholesterol diet for 17 weeks, LCAT transgenic
rabbits had significantly reduced atherosclerosis compared
with control littermates (Hoeg et al., 1996a). In contrast to
transgenic rabbits, transgenic mice expressing human
LCAT showed enhanced atherosclerosis (Mehlum et al.,
1997).
3.1.9. Lipoprotein lipase transgenic rabbits
Transgenic rabbits expressing human LPL were recently
generated in our laboratory using a human LPL cDNA
construct with a chicken b-actin promoter (Araki et al.,
2000). LPL is the rate-limiting enzyme involved in the
hydrolysis of TG-rich lipoproteins. LPL transgenic rabbits
have 650 ng/mL of human LPL in their postheparin
plasma, and their LPL activity is 4 times higher than that
of littermate rabbits. In LPL transgenic rabbits, plasma TG
was decreased by 80% and HDL by 59%. A conspicuous
reduction in VLDL and IDL was observed in the plasma
lipoprotein fraction (Fan et al., 2001b). With LPL transgenic
rabbits, we initially tested the hypothesis that increased LPL
activity would influence diet-induced hypercholesterolemia
and subsequent atherosclerosis. Transgenic rabbits showed
marked protection against diet-induced hypercholesterolemia and subsequently showed attenuation of atherosclerosis
(Fan et al., 2001b). Since the cholesterol levels in LPL
transgenic rabbits are markedly lower than those of control
rabbits, we could not answer our question as to whether LPL
per se is anti-atherogenic or whether the anti-atherogenic
effect of LPL is dependent only upon its lipid-lowering
effect. In the second experiment, we fed transgenic rabbits a
diet containing a high content of cholesterol to make them to
have equally high hypercholesterolemia with control rabbits. Under circumstances in which both transgenic and
control rabbits had similar hypercholesterolemia, transgenic
rabbits showed increased aortic atherosclerosis. Preliminary
studies revealed that LPL transgenic rabbits had lower bVLDL levels accompanied by increased small dense LDL
levels, suggesting that small LDL are more atherogenic than
large remnant lipoproteins when the total cholesterol levels
are the same (Fan et al., 2002). We are currently investigating whether LPL may enhance the lesion development by
increasing the retention of the apoB-containing particles in
the lesions.
3.1.10. 15-Lipoxygenase transgenic rabbits
Shen et al. (1995) reported the generation of transgenic
rabbits expressing the human 15-LO gene driven by a
lysozyme macrophage-specific promoter. When fed a diet
containing 10% corn oil and 0.25% cholesterol for 13.5
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weeks, transgenic rabbits had significantly smaller lesion
areas than their littermates even when both groups of rabbits
had similar levels of hypercholesterolemia (Shen et al.,
1996). This anti-atherogenic effect of 15-LO found in
rabbits was unexpected and rather surprising since it is
contrary to the general notion that oxidative modification
of LDL increases LDL atherogenecity (Cyrus et al., 1999;
Steinberg, 1999). It was speculated that LO may exert
protective effects by regulating the expression of redoxsensitive genes and/or that the effects of LO at different
stages of lesion development may differ considerably (Kuhn
& Chan, 1997).
3.1.11. Matrix metalloproteinase-12 transgenic rabbits
MMP-12 has been implicated in atherosclerosis and
inflammatory processes (Matsumoto et al., 1998). MMP12 derived from macrophages and foam cells in plaques
may influence the plaque stability and the rupture of
atherosclerotic lesions. To clarify MMP-12 functions in
vivo, we recently generated transgenic rabbits that
expressed human MMP-12 under the control of a macrophage-specific promoter, human scavenger receptor promoter (Horvai et al., 1995). Two transgenic founder rabbits
were shown by Southern blot analysis to have human
MMP-12 transgene integration (Wang et al., 2002a).
Human MMP-12 mRNA was expressed in peritoneal
macrophages, alveolar macrophages, and tissues that contain significant numbers of macrophages, including the
spleen, lung, and bone marrow in transgenic rabbits. High
levels of human MMP-12 protein were detected in the
conditioned media of cultured peritoneal and alveolar
macrophages from transgenic rabbits. We believe that this
transgenic rabbit model with increased expression of
human MMP-12 may become a useful model for further
mechanistic studies of atherosclerosis, many other inflammatory diseases, and cancer invasion; these rabbits are also
an ideal model for testing the in vivo actions of MMP-12
inhibitors.
3.1.12. Double transgenic rabbits
Double transgenic rabbits expressing both human apoE
and HL were made by crossbreeding apoE and HL transgenic rabbits (Barbagallo et al., 1999). These double transgenic rabbits make it possible to compare the functional
roles of these proteins in remnant metabolism and whether
there is a combined or synergistic effect of apoE and HL in
response to dietary cholesterol consumption. This study
showed that coexpression of apoE and HL led to dramatic
reductions of total cholesterol and of total VLDL, IDL, and
LDL, suggesting that apoE and HL have complementary
and synergistic functions in plasma cholesterol and lipoprotein metabolism. Rouy et al. (1998) generated double
transgenic rabbits expressing human apo(a) and apoB. Their
study revealed that rabbit apoB is more weakly associated
with human apo(a) through a disulfate bond than is human
apoB, suggesting that there is an intrinsic difference
between rabbit and human apoB in terms of the cysteine
site, which is required for Lp(a) formation.
3.1.13. Watanabe heritable hyperlipidemic transgenic
rabbits
Three human transgenes (LCAT, apo(a), and LPL) have
been introduced into WHHL rabbits to study the relationship between LDL receptor activity and these genes.
WHHL rabbits are genetically deficient in LDL receptor
and develop spontaneous hypercholesterolemia and atherosclerosis on a chow diet (Watanabe, 1980). This model has
been used as a model of human familial hypercholesterolemia. The advantages of using WHHL rabbits are 2-fold:
they allow the study of these protein functions in the
setting of LDL receptor defects and also make it possible
to study their relationship with hypercholesterolemia and
atherosclerosis without consumption of a cholesterol diet.
In LCAT transgenic WHHL rabbits, it has been found that
LCAT modulates LDL metabolism via the LDL receptor
pathway, ultimately influencing atherosclerosis susceptibility (Brousseau et al., 2000). To study Lp(a) atherogenicity,
we generated WHHL transgenic rabbits expressing human
apo(a) (Fan et al., 2000). With this model, we were able to
test the hypothesis that increased plasma levels of Lp(a)
may enhance the development of atherosclerosis in the
setting of hypercholesterolemia and to examine whether
the LDL receptor is involved in Lp(a) catabolism. Recently, we reported that transgenic WHHL rabbits developed more extensive advanced atherosclerotic lesions
than did nontransgenic WHHL rabbits (Sun et al., 2002).
In particular, the advanced atherosclerotic lesions in transgenic WHHL rabbits were frequently associated with calcification, which was barely evident in nontransgenic
WHHL rabbits. These results demonstrate for the first time
that Lp(a) accelerates advanced atherosclerotic lesion
formation and may play an important role in vascular calcification (Sun et al., 2002).
3.1.14. Transgenic rabbits versus transgenic mice
As discussed above, rabbits (LDL-mammals like
humans) have different features of lipoprotein metabolism
from mice (HDL-mammals); therefore, these two species
show different phenotypes even when the same gene is
introduced. Table 6 summarizes the differences of the
characteristic phenotypes of lipoproteins and the development of atherosclerosis between mice and rabbits after
gene transfer. Thus, it is likely that expression of the
same transgenes in two different species results in different phenotype changes, thereby affecting the interpretation
of experimental findings. For example, the expression of
either human HL or LCAT in rabbits induced protection
against atherosclerosis (Hoeg et al., 1996a; Taylor, 1997)
but led to enhanced lesion formation in transgenic mice
(Mehlum et al., 1997; Amar et al., 2000). Overexpression
of apoE in mice caused inhibition of atherosclerosis
(Shimano et al., 1992) but led to increased plasma LDL
J. Fan, T. Watanabe / Pharmacology & Therapeutics 99 (2003) 261–282
Table 6
Different phenotypes manifested in mouse and rabbit after the same gene
transfer
Genes
Mouse1
transferred
Rabbit
LCAT
Anti-atherogenic
HL
apo(a)
apoE3
15-LO
LPL
Pro-atherogenic
References
Hoeg et al., 1996a;
Mehlum et al., 1997
Anti-atherogenic Pro-atherogenic
Taylor, 1997;
Amar et al., 2000
Unbound with
Bound with
Chiesa et al., 1992;
apoB
apoB
Fan, 1999
Anti-atherogenic Atherogenic
Shimano et al.,
(high expression) 1992; Fan et al., 1998b
Pro-atherogenic Anti-atherogenic Shen et al., 1996;
Cyrus et al., 1999
Myopathies
No abnormalities Levak-Frank et al.,
in muscle
1995; Koike et al.,
2002
LCAT, lecithin:cholesterol acyltransferase; HL, hepatic lipase; LPL,
lipoprotein lipase; 15-LO, 15-lipoxygenase.
1
Refers to both transgenic and knockout.
and spontaneous atherosclerosis in transgenic rabbits (Fan
et al., 1998b). High expression of human LPL results in
myopathies in transgenic mice but not in transgenic
rabbits (Levak-Frank et al., 1995; Koike et al., 2002).
This further indicates that the species (rabbit vs. mouse)
may affect the interpretation of results from transgenic
studies.
3.2. Transgenic rabbits as hypertrophic cardiomyopathy
models
In addition to their wide use in lipid metabolism and
atherosclerosis research, transgenic rabbits are also used to
study other human familial hypertrophic cardiomyopathies
(FHC). FHC is a common disease that is diagnosed by the
presence of left ventricular hypertrophy in the absence of an
increased external load. The pathology is characterized by
myocyte hypertrophy, disarray, and increased interstitial
collagen. In human FHC, mutations in eight genes, all
encoding sarcomeric proteins, have been identified. The
most common gene responsible for human FHC is b-MyHC,
which accounts for 35 –50% of FHC cases. Rabbit hearts are
similar to human hearts but differ from those of mice. For
example, in the hearts of mice, the most commonly used
transgenic model, the most abundant component of the
cardiac sarcomere, the myosin heavy chain (MyHC), consists of the ‘‘fast’’ MyHC isoform (a-MyHC), whereas the
‘‘slow MyHC (b-MyHC) is the major isoform in the healthy
human adult (Kavinsky et al., 1984). In this respect, the
rabbit atrium expresses a-MyHC at all developmental
stages, whereas the ventricles express both a- and b-MyHC
isoforms, with b-MyHC as the predominant adult isoform
(James et al., 2000). Thus, MyHC expression in rabbits is
highly similar to that of the human heart. Marian et al.
(1999) successfully established transgenic rabbits express-
271
ing human mutant b-MyHC-Q403 as a model for human
FHC. Transgenic rabbits carrying the mutant transgene bMyHC-Q403 showed substantial myocyte disarray and a 3fold increase in interstitial collagen expression in their
myocardia. The mean septal thickness was significantly
increased in the mutant transgenic rabbits compared with
wild-type transgenic and nontransgenic rabbits. Thus, transgenic rabbits expressing mutant human b-MyHC-Q403 may
provide a good human FHC model. Recently, these authors
reported that myocardial contraction and relaxation were
reduced in these transgenic rabbits, as demonstrated by
tissue Doppler imaging (Nagueh et al., 2000), and showed
that treatment with simvastatin resulted in regression of
cardiac hypertrophy and fibrosis and improvement of cardiac function (Patel et al., 2001). James et al. (2000, 2002)
developed murine a- and b-cardiac MyHC promoters for
transgenic rabbits and recently created transgenic rabbits
expressing rabbit mutant essential light chain (M149V)
under the control of the b-MyHC promoter in order to
investigate whether this mutation causes FHC in rabbits.
Their study showed no apparent abnormalities in the transgenic rabbits from young to adult stages. They concluded
that the M149V mutation is not causative for FHC (James et
al., 2002).
3.3. Transgenic rabbits as models for acromegaly and
diabetes mellitus
Costa et al. (1998) produced transgenic rabbits expressing the bovine growth hormone gene in liver and
kidney. These rabbits showed enlargement of the head
and limbs and reduction of visceral fat. They also
showed marked hyperinsulinemia, hypertriglyceridemia,
and hyperglycemia, suggesting that these transgenic rabbits may have insulin resistance (although the authors did
not perform any glucose tolerance experiments). Although this model may be potentially useful for studying
acromegaly or diabetes mellitus, these rabbits suffered
from sterility (Costa et al., 1998), which limits their
usefulness. It may be impossible to expand such a transgenic
line.
3.4. Transgenic rabbits as models for AIDS and cancer
study
In addition to their cardiovascular studies, transgenic
rabbits have also been used in AIDS and tumorigenesis
studies. HIV-1 has been shown to be the causative agent of
AIDS in humans; however, very little is known about the
infection process and induction mechanisms underlying
AIDS, partly due to the lack of small laboratory animal
models for studying disease progression and testing diagnostic, therapeutic, and preventive measures. In vitro
studies showed that rabbit T-lymphocytes expressing human
CD4 become highly permissive for HIV-1 infection (Yamamura et al., 1991), which led researchers to generate trans-
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genic rabbits expressing human CD4 as a possible model for
the study of AIDS (Speck et al., 1998). Two groups have
successfully generated transgenic rabbits expressing human
CD4 (Dunn et al., 1995; Snyder et al., 1995) and they found
that lymphocytes from CD4 transgenic rabbits are susceptible to HIV-1 infection associated with rapid apoptosis
(Leno et al., 1995), suggesting that these models may be
useful for the development of therapeutic agents for AIDS
in the future.
Other human disease models include transgenic rabbits
expressing oncogenes, which develop lymphoma and leukemia (Knight et al., 1988; Sethupathi et al., 1994) and skin
carcinoma (Peng et al., 1993, 1995, 1999, 2001). These
models may be valuable for studying oncogenes and tumorigenesis and may provide unique models for evaluating
antitumor therapies in the future.
3.5. Other transgenic rabbits
In addition to the two uses of transgenic rabbits
described above (Tables 3, 5, and 7), namely as bioreactors
and disease models, several other uses for transgenic
rabbits have been reported. For example, tyrosinase is
known to be essential for melanization, and the expression
of murine tyrosinase results in the rescue of rabbit albinism
(Aigner & Brem, 1993; Aigner et al., 1996; Brem et al.,
1996; Jeffery et al., 1997); therefore, it may be possible to
use this enzyme as a marker for screening transgenic
animals. Taboit-Dameron et al. (1999) developed transgenic
rabbits expressing human CD55 and CD59 molecules in
order to control hyperacute rejection at the time of xenotransplantation. Previously, only a low expression rate was
observed in gene transfer studies using transgenic animals.
Those authors improved the DNA construct, making it
possible to achieve high molecular expressions in transgenic rabbits. These rabbits may become an effective model
for studying xenotransplantation. Some researchers also
attempted to use gene transfer in rabbits to improve
efficiency and quality in rabbit production since rabbits
are widely used for meat marked in Mediterranean region
and some developing countries (Brem et al., 1998). How-
ever, such research is still in immature stage and challenged
by public acceptance.
4. The methods for creating transgenic rabbits
4.1. Pronuclear microinjection method
The generation of transgenic rabbits is not only time
consuming because rabbits have a longer gestation period
than mice but is also expensive. Some important parameters
that may influence the frequency of transgene integration in
mice have been described (Brinster et al., 1985), but those in
other species, including rabbits, have not been systematically investigated. Therefore, the success rate (defined as the
rate of integration of transgenes in pups screened) in rabbits
is still lower than that in mice, and factors affecting the
success of transgenic rabbit production require more study.
There are four methods for generating transgenic animals:
(1) pronuclear microinjection, (2) injection of genetically
modified ES cells into blastocysts, (3) gene transfer into
sperms and oocytes, and (4) nuclear transfer of transfected
somatic cells. For transgenic rabbits, the most successful
commonly used method is pronuclear microinjection, while
use of the other methods is still restricted to mice and has
not been fully established for practical use in rabbits. Here,
we describe the protocol currently used in our laboratory.
4.1.1. Animals
To produce most of the transgenic rabbits reported thus
far, the New Zealand white strain and Japanese white strain
have been most often used, although some researchers have
used ZIKA hybrid rabbits (Brem et al., 1998) or Dutch
Belted rabbits (Buhler et al., 1990). The procedure for
microinjection is illustrated in Fig. 2. To produce transgenic
rabbits, four types of specific pathogen-free rabbits are
required: donor females (4 – 5 months old) and fertile males
to provide zygotes, sterile (vasectomized) males (over 5
months old), and foster females (7– 9 months old) to serve
as zygote recipients. We found that New Zealand white and
Japanese white rabbits work equally well. Whenever it is
Table 7
Transgenic rabbits for other human disease models
Possible
disease models
Transgenes
expressed
Phenotype
References
Hypertrophic cardiomyopathy
Human mutant b-myosin
heavy chain
Bovine growth hormone
Human CD4
Myocyte disarray and
increased fibrosis
Acromegaly and diabetes mellitus
Increased susceptibility
to HIV infection
Lymphocytic leukemia
Lymphoid and non-lymphoid tumors
Papillomas and skin cancer
Marian et al., 1999
Costa et al., 1998
Dunn et al., 1995;
Snyder et al., 1995
Knight et al., 1988
Sethupathi et al., 1994
Peng et al., 1993
ND
Taboit-Dameron et al., 1999
Acromegaly
AIDS
Tumorigenesis
Xenotransplantation
Rabbit c-myc oncogene
Rabbit E k-myc oncogene
Rabbit EJ-ras DNA
and papilloma virus DNA
Human CD55/CD59
J. Fan, T. Watanabe / Pharmacology & Therapeutics 99 (2003) 261–282
273
Fig. 2. The procedure for the production of transgenic rabbits by pronuclear microinjection. Donor rabbits are superovulated by the injection of 150 U of PMS
followed 3 days later by 150 U of hCG. The donor rabbits are mated to fertile males, and single-cell embryos are flushed from the oviducts 19 hr later. A DNA
solution ( 8 ng/mL) of the construct of interest is microinjected into the male pronucleus of the embryo while it is immobilized by a holding pipette under a
gentle vacuum. Injected embryos are implanted through the fimbrial end of the oviduct (15 – 20 embryos per oviduct) of a pseudopregnant recipient rabbit that
was mated with a vasectomized male 24 hr earlier. Founder pups are identified 1 month after birth by DNA screening using Southern blotting analysis or
polymerase chain reaction. Modified from J.M. Taylor and J. Fan (1997). Transgenic rabbit models for the study of atherosclerosis. Frontiers in Bioscience 2:
d298 – d308. With copyright permission of publisher (no. 9206988).
possible, all rabbits should be kept in a barrier room where
temperature and humidity are maintained at 23 C and 55%,
respectively. The rabbits are maintained with 12 hr light/
dark cycle and given water and food ad libitum. The
requirement for specific pathogen-free condition in the
facility for the rabbits seems to be critical for the superovulation, pregnancy of recipients, and colony breeding, as
described in Section 4.1.4.
4.1.2. Superovulation
In order to collect as many embryos as possible from each
donor rabbit, superovulation is generally induced using
hormone treatment. In rabbits, two types of hormone injection, pregnant mare’s serum gonadotropin (PMS) (Fan et al.,
1999b) and follicular stimulating hormone (FSH) (Kauffman
et al., 1998), are commonly used. On average, one donor
rabbit can yield 20 –30 eggs after appropriate hormone
injection. The protocol for superovulation and the time course
of the production of transgenic rabbits are shown in Fig. 3. On
the first day, donor rabbits are injected intramuscularly with
150 U of PMS. On the fourth day, donor rabbits are mated
with two or more males to ensure that the eggs are fertilized.
In our laboratory (Fig. 2), one donor rabbit is mated with two
or three males. After mating, 100 –150 U of human chorionic
gonadotrophin (hCG) is injected intramuscularly to induce
ovulation. In the case of FSH, 0.5 AU of hormone is
administered subcutaneously at 12 hr intervals for 3 days
for a total of 6 times. On the fourth day, the donor rabbits are
mated with fertile males and then hCG is administered.
In our laboratory, we use Japanese white rabbits 16
weeks old or older as zygote donors. Although there is no
apparent difference between young and old rabbits in terms
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J. Fan, T. Watanabe / Pharmacology & Therapeutics 99 (2003) 261–282
Fig. 3. Schematic illustration of two protocols for hormone-induced superovulation and timed events of transgenic rabbit production.
of egg recovery, our experience has shown that younger
rabbits tend to have more zygotes. However, if the rabbit is
too young (younger than 16 weeks), the percentage of
morphologically abnormal eggs, such as a thick or ovalshaped zona pellucida, increases. Such abnormal eggs are
not suitable for microinjection (Fan et al., 1999b). By
comparing two types of hormones, we found that FSH
induction resulted in a more stable number of eggs than
did PMS induction. However, the FSH method requires
multiple hormone injections, while PMS injection results in
slightly unstable recovery and fewer eggs compared with
FSH. Also, we sometimes observe that rabbits show either
no response (no eggs) or an excessive response (more than
100 fertilized eggs per rabbit) to PMS injection. At any rate,
fertilized eggs obtained using either treatment are usable for
microinjection. In our experience, there is no apparent
difference between these two methods in terms of in vitro
zygote development, number of pups obtained after an
embryo transfer, or transgenic efficiency.
Fertilized eggs are recovered by flushing medium through
the oviducts. In rabbits, it is possible to perform oviduct
perfusion in vivo under appropriate anesthesia as well as
hormone injection of the same rabbits several times. However, at the second superovulation, the number of ovulations
dramatically decreases. It is possible that antibodies against
the hormone are produced, leading to a reduction in the
ovarian follicle density in the ovary.
Proven or foster does are used as recipients. They are
mated with sterile (vasectomized) males at the same time as
donors, so that the state of pseudopregnancy is synchronous
with the developmental stage of transferred embryos.
4.1.3. Microinjection, embryo transfer, and detection of
founders
The microinjection of rabbit embryos, like that of
mouse embryos, is performed under an inverted differential
interference contrast microscope as described in detail
previously (Fan et al., 1999b). A representative photograph
showing the microinjection of rabbit embryos is shown in
Fig. 4. The successful injection of several picoliters of
DNA solution, containing a few hundred copies of the
gene construct, can easily be evaluated by observing the
expansion of the pronuclei. After microinjection, the
embryos are incubated at 37 C for 2– 3 hr in the transfer
medium. The surviving embryos are transferred into the
oviduct of a recipient. One specific characteristic of a
rabbit embryo is a thick mucin layer that forms around
the zona pellucida in the oviduct. The presence of this
mucin layer greatly influences pregnancy. If there is no
mucin layer, the pregnancy rate drops dramatically. Thus,
it is desirable to transfer the embryo quickly into the foster
mother after the microinjection in order to assist the
formation of the mucin layer around the zona pellucida.
We routinely transfer 15 – 20 embryos into both oviducts of
a recipient. We collect a piece of tissue by ear biopsy,
isolate genomic DNA, and detect the transgene by polymerase chain reaction or Southern blot analysis. If the
transgenic proteins such as apolipoproteins are present in
the plasma, the plasma can be directly subjected to
Western blotting analysis or enzyme-linked immunoabsorbent assay (ELISA) using specific antibody (Fan et
al., 1999b). Once identified, founder transgenic rabbits
are usually bred to nontransgenics in order to (1) determine whether the founder is a germline transgenic and (2)
provide F1 animals for transgene expression analysis.
4.1.4. Variables affecting the success of transgenic rabbit
generation
A number of factors influence the success of the generation of transgenic rabbits. A number of problems with the
production of transgenic rabbits are commonly encountered:
a low pregnancy rate (less than 40%), small litter size (0– 2
J. Fan, T. Watanabe / Pharmacology & Therapeutics 99 (2003) 261–282
Fig. 4. Micrograph of the process of pronuclear microinjection of rabbit
embryos. (A) Microinjection under an inverted microscope stage. (B) The
embryo is held by a holder pipette on the left, and the injection pipette filled
with DNA solution is on the right. (C) Injection pipette is inserted into a
pronucleus and DNA solution is released. Note: Compared with (B), the
pronucleus is enlarged (swollen), indicating that the DNA injection is
successful.
per foster) and cannibalism, a low positive rate within the
pups, uncontrolled expression (ectopic expression), and
mosaic founders that are incapable of germline transmission.
In a previous report, we described the some parameters by
which the success of the production of transgenic rabbits
275
can be assessed (Fan et al., 1999b). Readers may also need
to refer to the book chapter published recently (Brem et al.,
1998). We recommend evaluating the following variables in
making transgenic rabbits. These variables include (1) the
embryo yield from each donor, (2) the survival rate of
embryos after injection, (3) the pregnancy rate after embryo
transfer, (4) the litter size of pups (pups per embryos
transferred), (5) the positive rate of transgenic pups (positive
pups per total pups or positive pups per embryos transferred), (6) the mosaic, no expression, and ectopic expression in transgenic founders. For convenience, we may
arbitrarily divide these factors into controllable and uncontrollable factors. Controllable factors refer to problems we
can overcome by refining our technique, modifying the
methods or materials, or enhancing performance proficiency. Uncontrollable factors are essentially of unknown
causes and their resolution awaits breakthroughs in technology and further investigations.
To improve factor 1, one needs to consider the procedure
for superovulation (materials and methods) and donor
rabbits (age and breeding conditions), as mentioned above.
For factor 2, the problem may be due to poor microinjection
(either technique or needles) and poorly prepared DNA
solution (agarose contamination, buffer, or high DNA concentration). It has been reported that the method of DNA
preparation influences the integration rate in transgenics
(Wall et al., 2000). Factors 3 and 4 may be basically
associated with factor 2, but it is more likely that recipients
are not in good condition due to age, state of pseudopregnancy, time and number of embryo transfers, breeding
conditions, etc. A lower positive rate (factor 5) of pups (if
there are a normal total number of pups) is usually attributed
to poor DNA (quality) or injection efficiency. Factor 6 is
more complicated than the other factors and can only be
solved by generating more transgenic founders. In our own
experience (25 transgenic rabbit founders expressing different levels of transgenes), the mosaicism rate within all
positive founders is less than 10%; therefore, if you are so
unfortunate as to have only one mosaic founder, you need to
generate several more founders. It is hard or impossible to
experimentally control or expect the expression level in
transgenic rabbits because the transgene is randomly
inserted into the host genome and the function of the
transgene is dependent upon the position where it is located
(so-called position effect) rather than on the copy number.
One can imagine that the transgene expression may be
strongly influenced by the host genes in the vicinity,
resulting into either enhancement or inhibition of the transgene functions. One of the main hurdles of transgenesis
technology is the lack of appropriate models for testing
transgenic constructs before spending time and effort to
generate transgenic animals. Testing in transfected cells can
only tell one whether the transgenic construct is functional
(e.g., directs protein expression) but cannot predict whether
the construct will be faithfully expressed in transgenic
animals. The best method to test this is to generate (inex-
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J. Fan, T. Watanabe / Pharmacology & Therapeutics 99 (2003) 261–282
Fig. 5. Schematic illustration of the procedures for the generation of transgenic clone rabbits. Donor somatic cells from either adult or fetus are cultured in vitro,
during which time genetic manipulations such as transfection or gene targeting can be performed. Metaphase II recipient oocytes are collected from
superovulated female donors. Enucleation of recipient oocytes is performed by removing the chromosomes using a beveled pipette or piezo-drived pipette.
Nuclear transfer can be accomplished by two different methods. For the fusion method (A) on the left, the donor somatic cells (nuclei) are inserted under the
zona pellucida alongside the oocyte membrane. The cellular fusion is induced by a short high voltage pulse at right angles to the juxtaposition of the two cells.
For the piezo microinjection method (B) on the right, the donor cell is disrupted by suction into a piezoelectrically controlled microneedle before deposition in
the oocyte. Reconstructed embryos are activated by stimulation with strontium. After incubation in vitro for development, these reconstructed embryos are
transplanted into a surrogate pseudopregnant recipient rabbit and all pups born are homogeneous in genetic background as the cloned somatic cells.
J. Fan, T. Watanabe / Pharmacology & Therapeutics 99 (2003) 261–282
pensive) transgenic mice first before generating rabbits. It
should be mentioned in this regard that some specific
constructs (promoters) may show fidelity of expression in
transgenic mice but not in transgenic rabbits. To overcome
the problem of a lower expression in transgenic animals, one
can also consider using different promoters. Probably, the
best choice for this is to use genomic DNA such as YAC or
BAC to generate transgenic rabbits (Giraldo & Montoliu,
2001).
4.2. Nuclear transfer method
As mentioned above, pronuclear microinjection is the
most effective and practical method for the generation of
transgenic rabbits. However, several uncontrollable difficulties in this technique associated with high costs have
hampered the extensive use of this model. The problems of
low efficiency ( 5%), mosaicism, position effect, and
failure to establish ES cells are still not solved in transgenic
rabbits or in other transgenic livestock. Therefore, there is a
need to find an alternative means to generate genetically
modified transgenic or knockout rabbits. One promising
alternative method to pronuclear microinjection relies on the
somatic cell nuclear transfer method by which many cloned
animals have been created, the first being ‘‘Dolly’’ followed
by cloned cow, mouse, goat, and pig (Wilmut et al., 1997;
Kato et al., 1998; Wakayama et al., 1998; Baguisi et al.,
1999; Onishi et al., 2000).
Nuclear transfer of stably transfected somatic cells been
reported in sheep and cows. Schnieke et al. (1997b) reported
a transgenic cloned sheep that produced factor IX, a
therapeutic agent for hemophilia in the milk. They transfected sheep embryonic fibroblasts with the factor IX gene
and created the transgenic cloned sheep by nuclear transfer.
In 1997, Cibelli et al. transfected bovine embryonic fibroblasts with the pCMV/b-gal-neo gene, which is a fusion
gene between b-galactosidase and neomycin resistance gene
under the control of the cytomegalovirus promoter (Cibelli
et al., 1998b). They used these transfected cells and
obtained three heads of surviving transgenic cloned cows.
Nuclear transfer of transfected somatic cells for transgenesis
has several advantages over pronuclear microinjection in
addition to solving the problems noted above. First, it is
possible to determine the gender of transgenic animals in
advance. Thus, one can selectively produce either male or
female transgenic animals. If one uses transfected somatic
cells that are obtained from a female animal, all the newborns are female transgenic animals, whereas all the newborns are male if the transfected somatic cells are from a
male animal. Second, all transgenic animals produced by
nuclear transfer will have the same genetic background with
the same level of transgene expression since they are from
the cloned transfected cells. This feature is tremendously
important since some studies require that the animals have
homogeneous genetic backgrounds.
The basic method of nuclear transfer is shown in Fig. 5.
For transgenic cloning, somatic cells need to be transfected
277
with transgenes and cloned before nuclear transfer. Then,
these cloned cells are inserted into the perivitelline space of
the enucleated eggs using a micropipette. Immediately after
the insertion of somatic cells, the eggs are activated, fusion
is induced by electric stimulation, and the embryos are
reconstituted. Alternatively, somatic cells can be directly
inserted into the cytoplasm of enucleated eggs using a
piezo-drived micropipette and the reconstructed embryos
are activated by strontium. In an attempt to produce transgenic rabbits using cloning technology, we have tried to use
both electric fusion, which is used for cows and sheep, and
piezo injection method, which is used for mice and pigs.
Our preliminary data show that these reconstructed embryos
can develop to the morula or blastocyst phase in vitro (Wang
et al., 2002b). We have also performed implantation, but
thus far we have not obtained any ‘‘true’’ cloned transgenic
rabbits, which suggests there are a lot of issues regarding
this technique that still need to be resolved. While this work
was still under progress, transgenic nuclear transfer has been
proved possible in other species such as transgenic calves
(Cibelli et al., 1998a), sheep (Schnieke et al., 1997a), and
goats (Baguisi et al., 1999) via somatic cell nuclear transfer.
Recently, McCreath et al. (2000) successfully produced
gene-targeted sheep by nuclear transfer from cultured somatic cells. Our current goals are to generate transgenic
rabbits from recombinant somatic cells by nuclear transfer
to replace the current pronuclear microinjection, to increase
the efficiency of transgenic rabbit production to 100%, and
to overcome the problem of founder mosaicism. In addition,
in many experiments with rabbits as models of atherosclerosis, a genetically identical background is required because
of the great individual variations in the response to diet
manipulation and drug treatment. The birth of rabbits
derived from embryos in which the nuclei from donor 8or 16-cell embryos were transferred to recipient mature
oocytes suggests that the production of genetically identical
rabbits by nuclear transfer is possible (Stice & Robl, 1988;
Collas & Robl, 1990; Yang et al., 1992). Recently, Chesne
et al. (2002) produced the first cloned rabbits, although the
efficiency was low. That study is very encouraging because
rabbit cloning had once been thought impossible. We hope
that technical advances will speed the progress in this field.
4.3. Other methods for transgenic rabbits
In addition to pronuclear microinjection, transgenic rabbits were also generated by sperm-mediated gene transfer
described by two brief reports (Kuznetsov et al., 2000; Wang
et al., 2001). This method seems easy and efficient compared
with microinjection; however, the studies by this method are
still defined to feasibility of methodology and whether the
rabbits produced by this method are functional remains
questionable. Furthermore, establishment of rabbit ES cells
differentiating into a germ cell line has not been accomplished even though the preliminary studies were reported
almost 10 years ago (Graves & Moreadith, 1993), yet there
are no successful reports on knockout rabbits thus far.
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J. Fan, T. Watanabe / Pharmacology & Therapeutics 99 (2003) 261–282
5. Conclusions and perspectives
The advent of transgenic techniques for generating transgenic rabbits has allowed researchers to study human
diseases and produce foreign proteins. Unique transgenic
rabbit models for human diseases such as hyperlipidemia,
atherosclerosis, AIDS, and tumorigenesis have paved the
new way to enhance our understanding of molecular mechanisms of these diseases. Availability of genetically modified transgenic rabbits has also made it possible to produce
therapeutic proteins or antibodies on a relatively large scale
for different purposes. It has become clear that transgenic
rabbits have made a valuable contribution to the understanding of many human diseases and provided a unique
source for the production of recombinant proteins for
treatment and diagnosis of human disorders. In addition,
transgenic technology for rabbits may enhance our understanding of mammalian embryology (e.g., genetic elucidation of embryo development and abnormalities) since rabbit
eggs are larger and easy to handle.
In future, we need to find the right transgenes to be
expressed in rabbits for either research or bioreactors as both
human and rabbit genome are sequenced. Especially, it is
necessary to use large genomic sequences (such as YAC) to
solve the problems concerning the lack of regulatory elements and position effect in order to control expression
levels. Probably, we need to make more efforts in order to
establish ES cells in rabbits and eventually to be able to
generate knockout rabbits. It seems that there are many
obstacles that need to be worked out before the development
of gene targeting technology in the rabbits becomes available for biomedical research. Probably, nuclear transfer
technology coupled with transgenic technique may pave a
novel way to obtain transgenic animals in the future.
Compared with traditional microinjection methods, the
merits of somatic cell nuclear transfer for transgenic animal
production are unpredictable. Therefore, it is anticipated that
in the next few years transgenic rabbits generated by nuclear
transfer will be available for many research purposes.
Acknowledgments
The authors wish to thank all members (M. Araki, H.
Shimoyamada, L. Wu, M. Challah, H. Sun, H. Unoki, H.
Deng, N. Kojima, T. Ichikawa, X. Wang, J. Liang, T. Koike,
Y. Arai, and Y. Nakayama) in our laboratory who have
participated in this project, Drs. S. Kitajima and M.
Morimoto, Saga Medical School, and Drs. H. Shikama
and K. Honda, Yamanouchi Pharmaceutical, for their
generous support to this study. This work was supported
by Grants-in-Aid for Scientific Research from the Ministry
of Education, Science, and Culture of Japan, Ono Medical
Foundation, Japan, Uehara Memorial Foundation, Japan,
Japan Heart Foundation, Japan, Tokyo Biochemical Research Foundation, Ichiro Kanehara Foundation, Takeda
Medical Research Foundation, Mochida Memorial Foundation, and Japan Society for the Promotion of Sciences
(JSPS-RFTF96I00202), a grant of the Center for Tsukuba
Advanced Research Alliance (TARA) at the University of
Tsukuba.
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