Expression Systems and Species Used For Transgenic Animal Bioreactors

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Expression Systems and Species Used for Transgenic Animal Bioreactors

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BioMed Research International
Volume 2013, Article ID 580463, 9 pages
http://dx.doi.org/10.1155/2013/580463

Review Article
Expression Systems and Species Used for
Transgenic Animal Bioreactors

Yanli Wang,1,2 Sihai Zhao,1,2 Liang Bai,1,2 Jianglin Fan,3 and Enqi Liu1,2
1
Laboratory Animal Center, Xi’an Jiaotong University School of Medicine, Xi’an, Shaanxi 710061, China
2
Xi’an Jiaotong University Cardiovascular Research Center, Xi’an, Shaanxi 710061, China
3
Department of Molecular Pathology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi,
Yamanashi 409-3898, Japan

Correspondence should be addressed to Enqi Liu; [email protected]

Received 2 November 2012; Revised 15 January 2013; Accepted 17 February 2013

Academic Editor: James D. Murray

Copyright © 2013 Yanli Wang et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Transgenic animal bioreactors can produce therapeutic proteins with high value for pharmaceutical use. In this paper, we compared
different systems capable of producing therapeutic proteins (bacteria, mammalian cells, transgenic plants, and transgenic animals)
and found that transgenic animals were potentially ideal bioreactors for the synthesis of pharmaceutical protein complexes.
Compared with other transgenic animal expression systems (egg white, blood, urine, seminal plasma, and silkworm cocoon), the
mammary glands of transgenic animals have enormous potential. Compared with other mammalian species (pig, goat, sheep, and
cow) that are currently being studied as bioreactors, rabbits offer many advantages: high fertility, easy generation of transgenic
founders and offspring, insensitivity to prion diseases, relatively high milk production, and no transmission of severe diseases to
humans. Noticeably, for a small- or medium-sized facility, the rabbit system is ideal to produce up to 50 kg of protein per year,
considering both economical and hygienic aspects; rabbits are attractive candidates for the mammary-gland-specific expression of
recombinant proteins. We also reviewed recombinant proteins that have been produced by targeted expression in the mammary
glands of rabbits and discussed the limitations of transgenic animal bioreactors.

1. Introduction bacteria, mammalian cells, transgenic plants, and insects [1].


As we know, bacteria are limited in their ability to perform
The term bioreactor, which originally meant a tank in which the posttranslational protein modifications necessary for
cells, cell extracts, or enzymes carried out a biological many targets [2, 3], and transgenic plant [4–6] and insect
reaction, now often refers to a growth chamber (fermenter production systems involve relatively slow production set-
or fermentation vessel) for cells or microorganisms used in ups and have yet to cross many regulatory hurdles [7–9]. A
the production of recombinant proteins. Transgenic animal comparison of the different systems used to produce recom-
bioreactors used to produce therapeutic proteins have existed binant pharmaceutical proteins is summarized in Table 1.
for decades; several proteins produced in these systems Another important consideration is cost. Although direct
are now in clinical trials, and one has been approved for comparison of the production costs associated with these
marketing. different systems is rather difficult, a previous study suggested
Compared with other systems of production for recom- that building a large-scale (10,000 liter bioreactor) manu-
binant proteins, transgenic animal bioreactors are, overall, facturing facility for mammalian cells takes 3–5 years and
an attractive platform because transgenic animal bioreactors costs US$ 250–500 million, whereas a transgenic farm with
represent powerful tools to address the growing need for a single purification facility should not cost more than US$
therapeutic recombinant proteins. The ability of transgenic 80 million and would most likely cost less [7]. Establishing
animals to produce complex, biologically active proteins in a commercial production herd of a company’s transgenic
an efficient and economic manner is superior to those of goats could be accomplished at approximately a tenth of the
2 BioMed Research International

Table 1: Comparison of the different systems used to produce from these animals [11]. Since then, production of a number
recombinant pharmaceutical proteins. of recombinant proteins from transgenic animals has been
reported. Many laboratories and pharmaceutical companies
Mammalian Transgenic
Bacteria have made efforts to produce a variety of valuable therapeutic
cells animals
proteins from transgenic animals, such as cows, pigs, sheep,
Production level ++ + ++++ goats, and rabbits [7, 8, 12, 13].
Investment cost +++++ + +++ Selection of a suitable method for expressing a recombi-
Production cost +++++ ++ ++++ nant protein is dependent on the characteristics and intended
Scaling-up ability +++++ + ++++ application of the recombinant protein [14]. Presently, milk
Collection +++++ +++++ ++++ is the most mature system for producing recombinant pro-
teins from transgenic organisms. Blood, egg white, seminal
Purification +++ ++++ +++
plasma, silk gland, and urine are other theoretically possi-
Posttranslational
+ ++++ ++++ ble systems. The advantages and disadvantages of different
modifications
expression systems are shown in Table 3.
Glycosylation + ++++ ++++
Stability of product +++++ +++ ++++
2.1. Mammary Gland, the Best Bioreactor Available. The
Contaminant pathogens +++++ ++++ ++++
mammary gland has generally been considered the organ of
Products on the market ++++ +++++ +++ choice to express valuable recombinant proteins in transgenic
Table adapted from [1]. animal bioreactors because milk is easily collected in large
volumes. Milk is currently the best available bioreactor.
Table 2: Comparative estimated production cost between cell Foreign proteins are commonly reported to be produced in
culture and transgenics. transgenic milk at rates of several grams per liter. Based on the
assumption of average expression levels, daily milk volumes,
Production scale Cost
(Kg/year)
System
(dollars/gram product)
and purification efficiencies, 5.400 cows would be needed to
produce the 100.000 kg of human serum albumin that are
Cell culture 147 required per year worldwide, 4.500 sheep would be required
50
Transgenics 20 for the production of 5000 kg 𝛼-antitrypsin (𝛼-AT), 100 goats
100
Cell culture 48 for 100 kg of monoclonal antibodies (mAbs), 75 goats for
Transgenics 6 75 kg of antithrombin III, and two pigs to produce 2 kg of
Table adapted from [7]. human clotting factor IX [15]. As a result, a great deal of effort
has been made to produce transgenic bioreactors not only
with the traditional “dairy” species, such as sheep, goats, and
cost of building a commercial cell-culture facility [10]. As cows, but also with rabbits and pigs.
shown in Table 2, protein production costs are substantially A number of examples leave no doubt about the capacity
lower for transgenic animals than for cell culture. Therefore, of the mammary gland to synthesize, mature, and secrete
transgenic animal bioreactors show a financial advantage foreign proteins. Apart from these successes, a certain num-
over cell culture or other systems, even when all costs are ber of failures in animal mammary gland systems have
taken into account. occurred for various reasons: (1) purely technical issues such
Recombinant proteins are produced from transgenic as problems in the generation of transgenic mammals, (2)
animal body fluids. Milk, egg white, blood, urine, seminal the need for more fundamental knowledge in areas such as
plasma, and silkworm cocoon from transgenic animals are protein maturation or secretion, and (3) the fact that certain
candidate sources of recombinant proteins produced at an bioactive proteins produced in milk can have adverse affects
industrial scale. on an animal’s health; this is particularly true when they
The technology for using the mammary glands of trans- are produced at high concentrations and the protein can be
genic animals as primary bioreactors has been developed reabsorbed.
for large transgenic animals, such as cows, goats, sheep,
and pigs. Instead, many laboratories and pharmaceutical 2.2. Blood and Egg: Alternative Recombinant Protein Secretion
companies have made efforts to produce a variety of valuable Medium. The mammary gland of a transgenic animal is
therapeutic proteins using transgenic rabbits. In this paper, the most popular protein bioreactor. However, there are
we compared different systems and species of transgenic alternative systems based on production of useful pharma-
animal bioreactors. ceutical proteins in blood and eggs [16]. Animal blood, which
collects secretions from many tissues, may be used as a
2. Protein Production Platforms Using source of recombinant proteins. For example, human 𝛼-AT
Transgenic Animals was obtained at a high level from the serum of transgenic
rabbits [17], and human hemoglobin has been produced in a
In 1985, Hammer and colleagues established the first trans- transgenic swine circulatory system. This protein seemed to
genic livestock animals, including sheep, rabbits, and pigs, in have been matured appropriately for its functionality [18, 19].
an attempt to develop a way to produce recombinant proteins In principle, the human component of the pigs’ blood was
BioMed Research International 3

Table 3: Comparison of the different transgenic animal species used to produce recombinant pharmaceutical proteins.

Production systems
Points to consider
Milk Blood Egg white Seminal fluid Urine Silk cocoon Others
Production level +++++ +++++ +++++ +++ ++ ++ ++
Investment cost +++ +++ +++ + + +++ +++
Production cost ++++ ++++ ++++ ++ + +++++ ++++
Scaling-up ++++ ++++ ++++ ++ + ++++ +++
Collection +++++ ++++ +++++ +++ +++ +++++ +++++
Purification +++ ++ +++ ++ ++ +++ ++
Effect on organism +++ ++ +++ +++ +++ ++++ ++++
Posttranslational modifications ++++ +++++ +++ +++ +++ ++ ++
Glycosylation ++++ ++++ +++ +++ +++ ++ ++
Contaminant pathogens +++ ++ +++ +++ ++ ++++ ++++
Products on the market ++++ + ++ + + ++ +
Table adapted from [1].

intended to be used as a blood substitute, but similarities transgenic animals [30]. Seminal fluid is a relatively abundant
between the porcine and human blood components made biological fluid in some species and it can be easily collected.
isolation of the human hemoglobin arduous. Additionally, This is the case for pig. The boar’s male accessory sex glands
recombinant antibodies were found in the blood of transgenic possess many characteristics that make them appropriate for
goats, pigs, and rabbits [20, 21]. Blood is a less-than-ideal the production of recombinant proteins. Pig semen contains
fluid for protein production because its harvest is invasive and 30 mg of protein per mL and boars can produce 200–300 mL
bioactive proteins could affect an animal’s health to the point of semen for a total of 6–9 g of protein per ejaculate [7]. The
of making the system impractical. collection and handling of boar semen is a well-established
The use of transgenic eggs for large-scale production of process, performed on a large scale at swine artificial insemi-
recombinant proteins is another method being contemplated. nation units worldwide. Also of interest is the fact that protein
Interest in this system is driven by the fact that a single secretion by these tissues is uniquely exocrine, minimizing
hen can produce an impressive number of eggs (up to 330 the risk of a biologically active recombinant protein upsetting
eggs/year) and egg white naturally contains approximately the host’s own physiology. The limitation of this system is
4 g of protein [22–24]. Transgenic chicken stably produced a that we do not know how complex proteins are matured and
human erythropoietin fusion protein not only in their serum secreted in semen [26].
and egg white but also in the egg yolk, as was expected [25].
However, the egg system has been hampered by the lack of an 2.4. Silkworm Cocoon, a Good Candidate. The silkworm
efficient transgenesis system in poultry [7]. has acquired the ability to synthesize bulk amounts of silk
proteins in its silk glands. To utilize this capacity for mass
2.3. Urine- or Seminal-Fluid-Specific Expression Systems. production of useful proteins, transgenic silkworms have
Urine is an abundant biological fluid already used to prepare been generated that synthesize recombinant proteins in the
proteins such as gonadotropins for pharmaceutical use. If silk gland and secrete them into the silk cocoon; many
it happens that a foreign protein is matured in a more recombinant proteins have been produced using this system,
appropriate manner in the urothelium than in the mammary particularly over the last decade [31–33]. The transgenic
gland, or if the resulting side effects of the protein are silkworm is not only suitable for the production of genetically
less deleterious for the animals, a system using expression modified scaffolds for fibrous proteins such as collagens,
in the urothelium may be useful [26]. Work has indicated elastin, and silk which can be used to produce fabrics and
that expression of the human growth hormone gene driven biomaterials for medical purposes, but also suitable for the
by the mouse uroplakin II gene promoter was expressed production of recombinant proteins that can be used for
specifically in the urothelium, and up to 100–500 ng/mL of pharmaceutical purposes. More specifically can yield up to
human growth hormone was found in the resulting urine 4 mg of recombinant protein per silkworm. This yield is
[27]. Other work has also been explored [28, 29]. Compared quite high compared with other systems, and production
with milk, one advantage of using the bladder as a bioreactor using transgenic silkworms is much cheaper and faster than
is that animals can urinate earlier than they can lactate. The production using transgenic livestock [34]. The advantages of
limiting factor for bladder production of proteins has been transgenic silkworms are convenience and cost effectiveness
yield. Although the bladder epithelium does secrete proteins, with increased product yields in most cases ranging from
the rates are minimal, and thus protein production rates with 20- to 10 000-fold compared with laboratory methods. The
this system are extremely low. disadvantages are that it is costly and time consuming to
The seminal fluid of the male ejaculate has also been maintain nondiapause transgenic silkworms and that the use
considered as a site for recombinant protein secretion in of denaturing chemicals to extract the recombinant proteins
4 BioMed Research International

may result in extracted proteins that cannot retain their expressed in mice and goats and for heterotrimeric fibrinogen
original structures. produced in sheep.
Proteins that require posttranslational modification (e.g.,
glycosylation) have been expressed successfully, including
2.5. Others. Fish may present other specific, unique, and anti-thrombin-III in goats [68] and extracellular superoxide
unexplored opportunities for use of transgenic animals as dismutase, a complex N-glycosylated homotetramer that
bioreactors for production of important proteins. Some bio- carries copper and zinc atoms and is sensitive to proteolysis,
pharmaceutical companies have ongoing projects to express produced in physiologically active form in transgenic rabbits
factor VII, insulin, collagen, human calcitonin, pleurocidin, [47]. In recent years, several bioactive proteins have been
and human defensins in mucus produced by tilapia and expressed successfully in different animal mammary glands
salmon [35]. However, at present, we have little knowledge [69], such as lactoferrin [70, 71], human parathyroid hor-
about transgenic fish bioreactors. mone [72], 𝛼-fetoprotein [73], lysozyme [74], and butyryl-
The use of transgenic plants to produce novel products cholinesterase [75].
has great biotechnological potential, as they are relatively ATryn, produced by GTC Biotherapeutics, is made from
inexpensive, safe sources for potentially valuable bioactive the milk of transgenic goats that produce human antithrom-
metabolites, diagnostic proteins, and vaccines [4–6]. For bin, a plasma protein with anticoagulant properties [76].
example, nicotiana hybrids provide an advantageous pro- Early in 2006, the European Medicines Agency approved
duction platform for partially purified, plant-made vaccines ATryn for use in European Union countries; the drug was
that may be particularly well suited for use in veterinary also approved by the FDA in 2009 for treatment of patients
immunization programs [36]. Transgenic plants are superior with hereditary antithrombin deficiency. It was stated that
in terms of storage and distribution issues. one transgenic goat could produce the same amount of
antithrombin in a year as 90.000 blood donations [77]. ATryn
is the first medicine produced using transgenic animals. Its
3. Recent Successes: Different Animals Used as value is in the proof and acceptance of an intransgenic-animal
Mammary Gland Bioreactors platform to produce therapeutic proteins. Undoubtedly, an
increasing number of drugs produced by transgenic animal
As what we discussed in Section 2.1, transgenic animal mammary glands will be approved in the near future.
mammary glands are the best available bioreactors. They can
express a variety of interesting recombinant proteins—large 4. Rabbits versus Livestock: Bigger Is Not
and small, simple and complex—with high efficiency and full Always Better
bioactivity and have been extensively and successfully used in
different animals. Several animal species have been successfully used as trans-
Transgenic mice may only serve as a predictive model genic bioreactors. However, the criteria for selecting the most
to evaluate the usefulness of expression constructs and to suitable animal species for molecular pharming are based
study the properties of expressed proteins. However, they on the quantity of proteins needed per year, the capacity
are not at present useful as bioreactors for producing the of a facility, and the potential commercial value of the
large quantities of recombinant proteins that can satisfy recombinant proteins in addition to other factors such as
commercial demands. time until milk production and milk volume. The features
The largest and most complex protein successfully pro- of milk secretion in livestock are summarized in Table 4. A
duced to date is the human clotting protein factor VIII, simplified rule for choosing transgenic bioreactors is that the
a large heterodimer that was correctly processed into a production of a protein (such as albumin) in tons should be
bioactive protein in pigs [67]. The gene contains 26 exons carried out using transgenic cows, in hundreds of kilograms
and the cDNA alone is 7.6 kb long. At the other extreme, using sheep or goats, and in kilograms per year using rabbits
small peptides are unstable in biological systems, but they [78]. For example, in goats, a lactating female can produce up
can be expressed in transgenic animals if fused to carrier to 600–800 L of milk per year that can contain approximately
proteins. For example, the salmon calcitonin peptide was 5 g/L recombinant protein to yield approximately 4 kg of
fused to a small milk protein (human a-lactalbumin) and suc- protein per year.
cessfully expressed and amidated in rabbits [61]. Calcitonin In comparison with other large domestic livestock spe-
was cleaved from the fusion precursor in vitro during the cies, the rabbit is a relatively small animal with a short
purification process to yield a peptide with potent bioactivity gestation time, sexual maturity period (only four months for
in vivo. This is one approach to expressing physiologically females and five months for males) (Table 5), and optimal
active polypeptides without compromising the physiology or size. Handling in reproduction favors the application of
health of the transgenic production animal. Even multimeric transgenic technology in rabbits; gene transfer into rabbits
proteins have been expressed and assembled in vivo by the is an attractive technique for improving their performance,
coinjection of separate transgene constructs containing the and applications have been developed that use rabbits as fast
individual protein chains. Correct assembly into bioactive bioreactors for the production of therapeutic proteins used
proteins requires coexpression of the individual proteins at in biomedical research [78]. Rabbits are efficient breeders
the same time in the same cell with the correct stoichiometry. and will produce milk containing a desired protein within 8
This has been accomplished in vivo for heterodimeric mAbs months after the start of a project (Table 4).
BioMed Research International 5

Table 4: Comparison of transgenic milk expression systems in different species.

Species Elapsed months from


Gestation (months) Maturation (months) Milk yield per lactation (L)
microinjection to milk
Mouse 0.75 1 0.0015 3–6
Rabbit 1 5-6 1–1.5 7-8
Pig 4 7-8 200–400 15-16
Sheep 5 6–8 200–400 16–18
Goat 5 6–8 600–800 16–18
Cow 9 15 6000–8000 30–33

Table 5: Reproductive performance of rabbits. dismutase [47], growth hormone [48–50], 𝛼-glucosidase [37,
38], salmon calcitonin [61], equine chorionic gonadotropin
Reproductive parameter Value
[60], nerve growth factor-𝛽 [55, 56], chymosin [58], C1
Sexual maturity 4-5 months inhibitor [39], clotting factor VIII [40, 41], tissue nonspecific
Conception rate 65% alkaline phosphatase [63], bovine follicle-stimulating hor-
Gestation time 30–33 days mone [59], protein C [62], lactoferrin [64], interferon beta
Litter size 5–12 [65], and antithrombin [66], as summarized in Table 6.
Lactation period 40–50 days One of the best examples of recombinant proteins
Litter interval 44 days reported from rabbit milk is human 𝛼-glucosidase, which was
Litters per year 4–7 the first transgenic product from rabbit milk used to treat
Pompe’s disease (also called glycogen storage disorder type
II) [37, 82, 83]. Pompe’s disease is a fatal muscular disorder
caused by lysosomal 𝛼-glucosidase deficiency; patients with
Rabbit milk naturally contains 2.5-fold as much protein as
this disease have a rapidly fatal or slowly progressive impair-
sheep milk and 4.8-fold that of goat milk. A lactating female
ment of muscle functions due to concomitant storage of
rabbit can produce 170–220 g of milk per day and yield up to
lysosomal glycogen in the muscles and massive cardiomegaly.
10 kg of milk per year under semiautomatic hygienic milking
In 1998, a group of scientists in The Netherlands generated
conditions [79]. Expression levels of transgenic protein can
transgenic rabbits using a fusion between the human acid
be as high as 20 grams per liter. For small- and medium-sized
𝛼-glucosidase gene in its genomic context and the bovine
facilities, the rabbit system is ideal to produce up to 50 kg of
𝛼-S1-casein promoter. This protein isolated from transgenic
protein per year. Thus, the transgenic rabbit system is a lower
rabbit milk was shown to exert therapeutic effects in the
cost alternative primarily because rabbits are smaller and less
treatment of mice with glycogen storage deficiency and
expensive to maintain than livestock.
later in the treatment of human 𝛼-glucosidase deficiency
Specific pathogen-free rabbits are available and free of
[37]. Subsequently, they administered recombinant human
infectious agents. Although for conventional rabbits there
𝛼-glucosidase from rabbit milk to four human babies who
are no known prion diseases (similar to scrapie of the
were genetically deficient in 𝛼-glucosidase, at starting doses
sheep, human Creutzfeldt-Jacob disease and kuru, bovine
of 15 or 20 mg/kg and later at 40 mg/kg. The activity of
spongiform encephalopathy of the bovines) [80], humans
human 𝛼-glucosidase was shown to be normalized in the
can be infected with avian influenza, a virus from poultry
muscles of these patients, and their tissue morphology and
[81], though rabbit. There has been no known serious disease
motor and cardiac functions were dramatically improved
transmission to humans from the rabbit, which makes the
[82]. That successful study provided convincing evidence that
rabbit safer than other dairy livestock and poultry. Therefore,
the milk of transgenic rabbits is a safe source of therapeutic
the system of transgenic rabbits is safe to produce therapeutic
proteins and has opened the way for further exploration of
proteins.
this production method.
It must be admitted that not all transgenic rabbit bioreac-
5. Transgenic Rabbit Mammary tors or the recombinant proteins they produce are functional
Gland Bioreactor or practical due to low levels of expression; however, these
studies have opened the door for possible technical advances
Considering both economical and hygienic aspects, rabbits that will permit the production of large quantities of human
are attractive for the mammary-gland-specific expression therapeutic proteins and their use in the future.
of recombinant proteins. Using an appropriate promoter, a
number of recombinant proteins have been produced from
rabbit milk, including hormones, bioactive peptides, and 6. Conclusions
therapeutic proteins. Recombinant human proteins produced
by transgenic rabbits include 𝛼-AT [17], interleukin-2 [51], The various mammals used as bioreactors are rabbits, pigs,
tissue plasminogen activator [57], erythropoietin [42–46], sheep, goats, and cows. Each of these species offers advantages
insulin-like growth factor-1 [52–54], extracellular superoxide and drawbacks. Rabbits are sufficient to produce several
6 BioMed Research International

Table 6: Recombinant proteins produced from transgenic rabbits.

Expressed proteins Promoter Expressed protein References


Human 𝛼1-antitrypsin Human 𝛼 1-antitrypsin DNA 1 g/L in plasma [17]

Bovine 𝛼s1-casein 8 g/L [37]


Human 𝛼-glucosidase
N-acetyl-𝛽-glucosaminyl NA [38]

Human C1 inhibitor NA NA [39]

Mouse WAP NA [40]


Human clotting factor VIII
Mouse WAP 0.005–0.161 g/L [41]

Rabbit WAP 0.0000003 g/L [42]


Rabbit WAP NA [43]
Human erythropoietin Bovine 𝛽-lactoglobulin 0.5 g/L [44]
Rabbit WAP NA [45]
Rabbit WAP 60–178 IU/L [46]

Human extracellular SOD Mouse WAP 3 g/L [47]

Mouse WAP 0.000012 g/L [48]


Human growth hormone Rat WAP 0.5–1.0 g/L [49]
Rat WAP 0.010 g/L [50]

Human IL-2 Rabbit 𝛽-casein 0.0005 g/L [51]

Bovine 𝛼s1-casein 1 g/L [52]


Human insulin-like growth factor Bovine 𝛼s1-casein 0.3 g/L [53]
Bovine 𝛼s1-casein 0.678 g/L [54]

Bovine 𝛼s1-casein 0.25 g/L [55]


Human nerve growth factor 𝛽
Adenoviral 0.346 g/L [56]

Human tPA Bovine 𝛼s1-casein 0.00005 g/L [57]


Bovine chymosin Bovine 𝛼s1-casein 1.5 g/L [58]
Bovine FSH Bovine 𝛼s1-casein 0.1 g/L [59]
Equine chorionic gonadotropin Rabbit WAP 0.022 g/L [60]
Salmon calcitonin Ovine 𝛽-lactoglobulin 2.1 g/L [61]
Human protein C Mouse WAP 0.0000001–0.0000003 g/L [62]
TNAP Human WAP NA [63]
Human lactoferrin Adenoviral 2.3 g/L [64]
Human interferon beta NA 2.2–7.2 × 107 IU/L [65]
Human antithrombin Adenoviral 4.8 g/L [66]
FSH: follicle stimulating hormone; IL-2: interleukin-2; NA: not available; SOD: superoxide dismutase; TNAP: tissue-nonspecific alkaline phosphatase; tPA:
tissue plasminogen activator; WAP: whey acidic protein.

kilograms of proteins per year. The rabbit is particularly the transgenic animals over several generations. This has been
flexible, allowing rapid generation of founders and scaling- taken into account in the development of gene pharming,
up. For very high protein production, larger animals are for example, by using only animals from prion disease-
needed. The recombinant proteins that have been prepared free countries (New Zealand) and keeping the animals in
in milk are mainly naturally secreted, which may facilitate very hygienic conditions. It may thus be considered that the
or complicate their purification. No matter what type of preparation of pharmaceutical proteins from milk is a safe
transgenic platform is used as a bioreactor, guidelines devel- process.
oped by the FDA require monitoring of the animals’ health, The raw potential for producing valuable proteins with
validation of the gene construct, and characterization of the transgenic animals seems apparent. The study of the proper-
isolated recombinant protein as well as the performance of ties of the recombinant proteins is of paramount importance
BioMed Research International 7

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Conflict of Interests
[17] M. Massoud, R. Bischoff, W. Dalemans et al., “Expression of
No Conflict of Interests is declared. active recombinant human 𝛼1-antitrypsin in transgenic rabbits,”
Journal of Biotechnology, vol. 18, no. 3, pp. 193–204, 1991.
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