Transgenic rabbits as therapeutic protein bioreactors and human disease models

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-

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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

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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

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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).

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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

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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-

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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

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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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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

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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

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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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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.

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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

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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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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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