Transgenic Research 9: 305–320, 2000.

© 2000 Kluwer Academic Publishers. Printed in the Netherlands.

305

Transgenic animal bioreactors

Louis Marie Houdebine

Unite de Biologie du Développement et Biotechnologie, Institut National de la Recherche Agronomique,
78352 Jouy-en-Josas Cedex, France, Tél.: 33 1 34 65 25 40; Fax: 33 1 34 65 22 41; E-mail: houdebine@biotec.
jouy.inra.fr

Key words: Recombinant proteins, transgenic animals, milk

Abstract
The production of recombinant proteins is one of the major successes of biotechnology. Animal cells are required
to synthesize proteins with the appropriate post-translational modifications. Transgenic animals are being used for
this purpose. Milk, egg white, blood, urine, seminal plasma and silk worm cocoon from transgenic animals are
candidates to be the source of recombinant proteins at an industrial scale. Although the first recombinant protein
produced by transgenic animals is expected to be in the market in 2000, a certain number of technical problems
remain to be solved before the various systems are optimized. Although the generation of transgenic farm animals
has become recently easier mainly with the technique of animal cloning using transfected somatic cells as nuclear
donor, this point remains a limitation as far as cost is concerned. Numerous experiments carried out for the last
15 years have shown that the expression of the transgene is predictable only to a limited extent. This is clearly
due to the fact that the expression vectors are not constructed in an appropriate manner. This undoubtedly comes
from the fact that all the signals contained in genes have not yet been identified. Gene constructions thus result
sometime in poorly functional expression vectors. One possibility consists in using long genomic DNA fragments
contained in YAC or BAC vectors. The other relies on the identification of the major important elements required to
obtain a satisfactory transgene expression. These elements include essentially gene insulators, chromatin openers,
matrix attached regions, enhancers and introns. A certain number of proteins having complex structures (formed
by several subunits, being glycosylated, cleaved, carboxylated...) have been obtained at levels sufficient for an
industrial exploitation. In other cases, the mammary cellular machinery seems insufficient to promote all the post-
translational modifications. The addition of genes coding for enzymes involved in protein maturation has been
envisaged and successfully performed in one case. Furin gene expressed specifically in the mammary gland proved
to able to cleave native human protein C with good efficiency. In a certain number of cases, the recombinant proteins
produced in milk have deleterious effects on the mammary gland function or in the animals themselves. This comes
independently from ectopic expression of the transgenes and from the transfer of the recombinant proteins from
milk to blood. One possibility to eliminate or reduce these side-effects may be to use systems inducible by an
exogenous molecule such as tetracycline allowing the transgene to be expressed only during lactation and strictly in
the mammary gland. The purification of recombinant proteins from milk is generally not particularly difficult. This
may not be the case, however, when the endogenous proteins such as serum albumin or antibodies are abundantly
present in milk. This problem may be still more crucial if proteins are produced in blood. Among the biological
contaminants potentially present in the recombinant proteins prepared from transgenic animals, prions are certainly
those raising the major concern. The selection of animals chosen to generate transgenics on one hand and the
elimination of the potentially contaminated animals, thanks to recently defined quite sensitive tests may reduce the
risk to an extremely low level. The available techniques to produce pharmaceutical proteins in milk can be used as
well to optimize milk composition of farm animals, to add nutriceuticals in milk and potentially to reduce or even
eliminate some mammary infectious diseases.
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Introduction A certain number of reviews have been written on

the subject during the last decade (Houdebine, 1994;
Proteins are the molecules, which have the most nu- Colman, 1996; Rosen et al., 1996; Clark, 1998; Wall,
merous biological activities in living organisms. Yet, 1999; Rudolph, 1999). The present paper aims at re-
they are traditionally only marginally used as thera- porting the major successes obtained so far and to ana-
peutic molecules. This is dearly due to the fact that lyze the hurdles which remain to generate transgenic
their administration must be done by injection but animals being more reliable bioreactors.
mainly because most of them are not available or not
known.
Genetic engineering offers now the possibility to The different animal systems to produce
know virtually all the proteins of a living organism but recombinant proteins
also to prepare them at an industrial scale as recom-
binant proteins. After the first success which allowed Blood
to prepare human insulin and growth hormone, it
appeared that many proteins could not be produced Serum, which collects secretion from many tissues,
by recombinant bacteria. In some cases, bacteria do may be the source of recombinant proteins. Human
not synthesize a foreign protein in large amount. In α1 antitrypsin synthesized essentially in liver was thus
other cases, the protein is in the form of aggregates, obtained at a high level from the serum of transgenic
which cannot be recovered easily. On the other hand, rabbits. This protein seemed matured in an appropri-
many proteins potentially utilizable as pharmaceut- ate manner (Massoud et al., 1991). One limitation in
icals cannot be synthesized in a satisfactory active this case was the difficulty to separate the recombin-
form by bacteria. In these cases, the proteins must be ant from the endogenous protein. The replacement of
obtained from recombinant animal cells. Fermentors the endogenous gene by the human genes using ho-
containing hybridoma, CHO cells, Sf9 insect cells in- mologous recombination would solve this problem.
fected by recombinant baculovirus or other cell lines Recombinant antibodies were also found in the blood
are extensively used to synthesize antibodies, human of transgenic pigs and rabbits (Lo et al., 1991; Weidle
erythropoietin, hepatitis B antigen, human factor VIII et al., 1991, Limonta et al., 1995). However, the
etc. Although efficient, this approach has intrinsic antibodies were present at a relatively low concentra-
limitations. The heaviest is probably the fact that, tion and they were hybrid molecules containing chains
despite quite significant improvement during the last from the endogenous antibodies. Replacement of loci
decade, animal cells in culture generally do not pro- harbouring the antibody genes by human loci may
duce large amount of recombinant proteins. This is allow mice to synthesize human antibodies (Mendes
obviously due to the fact that a fermentor contains a re- et al., 1997). The transfer of human chromosome 2
latively limited number of cells, which are maintained in mouse has been achieved using ES cells. This ad-
in non-optimal metabolic conditions. High capacity ditional chromosome was transmitted to progeny and
fermentors can produce relatively large quantities of was able to generate functional human antibodies after
recombinant proteins but not at a low cost. immunisation of the animals (Tomizuka et al., 1997).
This reality was perceived years ago and after the Although serum from transgenic animals is an
generation of the first transgenic mice, it was sug- abundant by-product of slaughterhouses it does not
gested that animals could become living fermentors appear presently as a potential source for many re-
(Palmiter et al., 1982). The demonstration was given combinant proteins. Indeed, many proteins are poorly
in 1987 when ovine β-lactoglobulin (Simons et al., stable in serum and some of them may hamper the
1987) and human tissue plasminogen activator (Gor- health of the animals.
don et al., 1987) were obtained in the milk of trans- Human hemoglobin has been obtained in the re-
genic mice. This pioneer work was confirmed since ticulocytes of transgenic swine (Sharma et al., 1994).
more than 100 foreign proteins have been produced This molecule was present at a relatively high level
experimentally from different organs and in several and it was functional. However, a large proportion was
animal species. The first protein obtained from trans- formed of hybrid molecules containing also the pig
genic goat milk is expected to be in the market in 2000. globins. The replacement of pig globin genes by the
Yet, a certain number of problems remain to be solved corresponding human genes would probably solve this
before the process is optimized. problem.
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A few experimental data suggest that reticulocytes some success (Etches et al., 1997; Kunita et al., 1998;
might be the source of recombinant non-secreted pro- Etches, 1999). The interest of this protocol still re-
teins. Efficient and specific promoters driving the ex- mains limited by the fact that the foreign gene is not
pression of foreign genes are available (Sharma et al., reproducibly transmitted to progeny.
1994). Enzymes or peptides might thus be stored in re- Retroviral vectors have been extensively studied
ticulocytes from transgenic animals and extracted after (Ronfort et al., 1997). When injected in the vicinity of
having been collected from slaughterhouse. primordial germ cells of developing chicken embryos,
the retroviral vectors are integrated into the genome,
Urine and the foreign gene are found in progeny with a low
but reasonable frequency. These tools recently led to
Urine is an abundant biological fluid already used to the production of foreign proteins in the egg white of
prepare proteins such as gonadotropins for pharma- transgenic chicken (Ivarie, 1999).
ceutical use. A recent work indicated that the human
growth hormone gene driven by the promoter of the Silk worm cocoon
mouse uroplakin II gene was expressed specifically
in urothelium. Up to 100–500 ng/ml of human growth The cocoon of insects contains large amount of a few
hormone was found in urine. This system may be use- proteins which form silk. From this point of view, this
ful if it happens that the foreign protein is matured in system shares a certain number of properties with milk
a more appropriate manner in urothelium than in the and egg white. Silk worm is an interesting biological
mammary cell or if the side-effects of the protein are model and it is also a natural producer of silk for
less deleterious for the animals (Kerr et al., 1998). industry. This urged several laboratories to generate
transgenic silk worms. This was achieved successfully
Seminal plasma after microinjection of linearized plasmids into eggs
(Nagaraju et al., 1996). The use of baculovirus vectors
Seminal plasma is a relatively abundant biological was also shown to be efficient (Yamao et al., 1999).
fluid in some species and it can be easily collec- The most promising approach seems presently to rely
ted. This is the case for pig. The promoter of the on the use of a piggy Bac transposon-derived vector
mouse P12 gene that promotes expression specifically (Tamura et al., 1999).
in male accessory sex gland has been used to syn- Silk worm can be easily reproduced into large
thesize the human growth hormone. The hormone was number of individuals. It is therefore, a good candi-
found in the seminal plasma from transgenic mice at a date to be a living fermentor. The efficiency of foreign
concentration as high as 0.5 mg/ml (Dyck et al., 1999). gene expression and protein maturation still remains
This system, as urine, may have specific advant- to be evaluated.
ages. In both cases, it is not known how complex
proteins are matured and secreted. Milk

Egg white Milk is currently the best available bioreactor

(Houdebine, 1994; Colman, 1996; Clark, 1998; Wall,
Egg white, as milk, is an abundant fluid containing 1999; Rudolph, 1999). Extensive studies have shown
huge amount of proteins and being secreted out of that it can be the source of a variety of recombin-
the body. It is thus expected to be an excellent sys- ant proteins, some of them being rather complex
tem to produce recombinant proteins. Yet, it has not molecules.
been implemented so far, essentially for technical Among the proteins which can be mentioned are:
reasons. human IGF1 (Zinovieva et al., 1998), human NGF-β
Generating transgenic birds remains a difficult task (Coulibaly et al., 1999), hGH (Devinoy et al., 1994),
with limited success. Microinjection of isolated gene human lysozyme (Lee et al., 1998), human lactoferrin
into on cell embryo followed by in vitro develop- (Platenburg et al., 1994), human erythropoietin (Mas-
ment is one possibility (Naito, 1997). The use of the soud et al., 1996), human thrombopoietin (Sohn et
transposon Mariner considerably enhances the yield al., 1999) and human parathyroid hormone (Rokkones
of transgenesis (Verrinder-Gibbins, 1998; Scherman et al., 1995). Interestingly, naturally complex pro-
et al., 1998). The use of ES cells to generate chi- teins have been secreted in milk in a fully functional
meric chicken is a very attractive approach which met form. This is the case for human fibrinogen composed
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of three subunits (Prunkard et al., 1996). This was gene and others are eliminated since they have only
also observed for human collagen obtained as a ma- the gene of interest, this method is presently the best
ture molecule after coinjecting the genes coding for approach to reduce at most the number of recipient
the two subunits and for the enzyme which modifies females. The green fluorescent protein (GFP) seems
the proteins posttranscriptionnally (John et al., 1999). presently the best marker. One major advantage is that
The human extracellular superoxide dismutase which this protein can be visualized with a non-invasive test.
is formed of two glycosylated homodimers containing Alternatively, fluorescent in situ hybridization (FISH)
one copper ion was also found in milk at the concen- performed in one isolated blastomer can reveal the
tration of several milligram per millilitre (Strömqvist presence of integrated foreign DNA (Echelard, 1997).
et al., 1997). Active recombinant immunoglobulin This protocol was applied successfully to generate
capable of neutralizing coronavirus was obtained in transgenic cows (Krimpenfort et al., 1991; Hyttinen
mouse milk (Castilla et al., 1998). An exotic pro- et al., 1994; Eyestone, 1998). It could be used as well
tein, spider silk, known for its exceptional mechanical for sheep and goat. Indeed, in vitro generation of one
properties has been recently produced in mouse milk cell embryos can also be achieved in these species.
(Karatzas et al., 1999). However, the techniques have not been defined in de-
These examples leave no doubt on the capacity of tails and optimized for these two species. On the other
the mammary gland to synthesize, mature and secrete hand, the embryos generated in vitro usually show a
foreign proteins. Apart from these successes, a certain lower rate of survival after transplantation in recipient
number of failures occurred for various reasons. Some females. All things being considered, superovulation
of them are purely technical such as the generation of has been retained for the generation of one cell sheep
transgenic mammals. Others, such as protein matura- and goat embryos rather than the in vitro protocol.
tion or secretion, are more fundamental. These points One of the major limitations of microinjection is
are considered in the following paragraphs. the low rate of foreign DNA integration. Works in
progress have shown that episomal vectors capable
of being maintained as circular genomes can be used
The generation of transgenic mammals to generate transgenic animals with a very high effi-
ciency (Attal et al., 1997; Vos, 1998). Alternatively,
Microinjection linear artificial chromosome might become available
in future (Willard, 1998). Significant improvements
The direct microinjection of linear DNA fragments of these vectors are required before they can become
into pronuclei defined in 1981 remains the method of relevant tools to facilitate preparation of recombinant
choice for the prolific species (mouse, rat, rabbit and proteins from milk. Episomal vectors capable of be-
pig). An improvement of the method was successful ing maintained during the life of the animal but not
for ruminants. In these species and mainly in cow, transmitted to progeny might probably be an attractive
the rate of foreign gene integration is very low. On compromise.
the other hand, embryos generated in vivo after super-
ovulation and recipient females are particularly costly. Cell transfection and embryo cloning
The preparation of one cell embryos after in vitro oo-
cyte maturation and fertilization considerably reduced The technique defined to generate cloned sheep by
the cost of the experiment. The possibility to develop transferring nuclei from somatic cells into enucleated
the microinjected embryos until the blastocyst stage oocytes was used successfully to obtain transgenic
allows the elimination of those which cannot survive. sheep harbouring the human factor IX gene (Schnieke
This reduces the number of recipient females. Ideally, et al., 1997). This approach proved to be efficient also
the identification of the putative transgenic embryos for goat (Baguisi et al., 1999). It can be extended to
would still reduce the number of recipient females. all species in which the cloning technique is efficient.
The PCR technique which cannot easily make a dis- This is obviously the case for cow.
tinction between the integrated and the free foreign This method presently appears as definitely more
DNA proved to be inappropriate (Page et al., 1995). attractive than microinjection. Indeed, about 2.5 times
The best way currently relies on the use of a marker less animals were required to generate the same num-
gene coinjected with the gene of interest. Although ber of transgenic sheep by cloning (Schnieke et al.,
some of the selected embryos harbour only the marker 1997). One of the advantages of the cloning approach
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is that the foreign gene is transferred in cultured cells. the fact that the biochemical status of multipotent
This allows the selection of cellular clones harbouring cells has never been defined clearly (Fléchon, 1997;
a limited number of integrated and intact genes. The Mc Laren, 2000). Publications suggest each year that
cells can be taken from females and thus generate fe- slight progress has been done. One of the most recent
males. This accelerates the production of milk from publications indicates that primordial germ cells from
the founder animals. Cells can be stored frozen and pig were able to generate animals with a high degree
reused to generate additional transgenics. of chimerism, suggesting that the multipotent cells
The overall yield of the method becomes depend- added to the embryos might have participated to the
ent essentially of the embryo cloning efficiency. The formation of germ cells of the chimera (Mueller et al.,
cloning of pig embryo which occured recently was ex- 1999).
pected for several reasons and particularly to prepare
organ for xenografting to humans. It is not certain yet Gene transfer into sperms and oocytes
that this would compete favorably with the conven-
tional microinjection. The same is true for the other The introduction of foreign genes before fertilization
prolific species. into germ cells is a logical approach, which did not
The major advantage of the cloning technique is meet clear success until recently. The incubation of
that it can lead to the replacement of a targeted gene by sperm with DNA solution before fertilization gave
homologous recombination. Several lambs in which only a very small proportion of transgenic animals
a gene was replaced in cultured somatic cells were with rearranged genes in most cases. Another protocol
generated recently (Ayares, 1999). This theoretically originally defined for xenopus was shown to be effi-
allows the introduction of a foreign gene in the casein cient in mouse. The sperms are first treated by mild
locus, in whey protein genes or at any other selected detergents to destabilize their membranes. After an
site. incubation in a DNA solution, the sperms have to
Independently of the method of gene transfer, the be microinjected into oocytes to fertilize the oocyte
cloning technique may contribute quite significantly to (Perry et al., 1999; Robl, 1999). The efficiency of the
rapidly establish a herd of transgenic animals produ- method is therefore highly dependent of this of ICSI
cing pharmaceuticals from the animals producing the (intracytoplasmic sperm injection). This method met
highest level of the recombinant proteins. success in no other mammals than mouse so far.
Microinjection of linear DNA into oocytes never
The generation of chimeric animals from multipotent led to the reproducible generation of transgenic anim-
cells als. The use of retroviral vectors has recently led to
a success in cow (Chan et al., 1999) and it is being
Gene replacement has currently being performed in extended to non-human primates. Retroviral partic-
mouse for more than 10 years but not in other species. ules containing the envelope from vesicular somatic
Homologous recombination for gene replacement can virus were injected between zona pellucida and the
be obtained virtually in cell types but only multipo- membrane of the oocyte at a period when the nuc-
tent cell were really useful for this purpose. Indeed, lear membrane is absent. The efficient infection was
only multipotent cells lines from embryos (ES cells) followed by an easy access to the nucleus and a high
or from primordial germ cells (EG cells) have the rate of foreign gene integration. This method might
capability to participate to the generation of chimeric be more efficient than DNA microinjection. It is not
animals which are then mosaic for the genetic modi- certain that it will be, in time, more attractive than the
fication. Reliable multipotent cell lines have been method implementing the cloning technique.
obtained only from a few strains of mice. In other
species, the cells loose their multipotency during the
culture which is necessary to select the cells in which The expression of the transgenes
gene replacement occurred. These cells can then parti-
cipate only moderately to the development of chimeric Numerous experiments have shown that the level and
embryos and they do not transmit the genetic modi- specificity of expression of a gene construct used
fication to progeny. All the groups working with rat, as transgene cannot be easily predicted. DNA ad-
rabbit, chicken, pig or ruminants did similar obser- dition by microinjection generates lines of animals
vations. These repeated failures can be attributed to expressing the foreign gene at quite different levels.
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It is admitted that this phenomenon is due to a large the case for growth hormone (Devinoy et al., 1994),
extent to a position effect (Dobie et al., 1996). Trans- α1-antitrypsin (Wright et al., 1991) etc. On the con-
genes are poorly or not expressed when integrated trary, proteins such as human factor VIII which are
in centromeres or telomeres where DNA is inactive present at a low concentration in blood are not easily
and organized in heterochromatin. Experiments car- expressed in milk (Niemann et al., 1996; Paleyanda
ried out in vitro using cultured mammary cells and in et al., 1997). The same has been observed for proteins
vivo with transgenics revealed that the gene constructs not naturally secreted such as intracellular enzymes
optimized to work in transfected cells may be poorly or viral antigens. In these cases, the proteins might
efficient when transferred into mice (Petitclerc et al., not be efficiently recognized by chaperones in the en-
1995). doplasmic reticulum and Golgi apparatus or contain
These experiments show with no ambiguity that signals for a targeting to cell compartments other than
at least two independent points should be taken into the reticulum. For such proteins, little improvements
consideration to prepare potent expression vectors (i) are expected from modifications of the coding region.
the intrinsic capacity of the construct to transcribe ef- The minimum modification to be done is to add a sig-
ficiently the cDNA or the gene (ii) the potency of the nal peptide in the NH2 end of the protein to allow its
same vectors as transgenes. The first parameters can secretion.
be evaluated with transfected cells in vitro whereas Introns play an essential but complex role in gene
the second property of vectors can be estimated only expression. They are often not necessary for the ex-
in transgenic animals. This explains why only limited pression of a cDNA in transfected cells. Experiments
number of experiments have been performed to eval- carried out with several genes and promoters have
uate the efficiency of expression vectors in vivo. For shown that at least one intron, preferably added before
most research projects, transgenic mice synthesizing the cDNA, is required (Palmiter et al., 1991). The in-
limited amount of the foreign proteins are sufficient to trons have quite different efficiency (Petitclerc et al.,
give a satisfactory answer to experimenters. The same 1995). This may be due to two independent mech-
is not true when a high level of expression is needed anisms. The elimination of introns by exon splicing
and this is the case for the production of recombinant involves multiple signals on mRNA and proteins bind-
proteins. ing to specific sequences (Horowitz & Krainer, 1994).
Another well-known problem with transgenesis is The choice of an intron must take this parameter into
their faculty to be expressed at a low level in various account. Introns and specially the first intron of genes
tissues in which the utilized promoter is not expected contain binding sites for transcription factors. This
to work. It is admitted that this ectopic expression is contributes to maintain chromatin in an open and act-
due to a position effect. Indeed, chromatin contains ive form around the cap. These introns must be used
many transcription enhancers, which can stimulate preferably. Alternatively, sequences for the binding of
expression of transgene integrated in their vicinity. transcription factors may be added within the intron
These observations suggest that quite different (Petitclerc et al., 1995).
problems must be solved to generate satisfactory ex- One of the functions of introns is to favor the trans-
pression vectors in a reliable manner. location of mRNA from the nucleus to the cytoplasm.
Sequences playing this role have been found in sev-
The optimization of the transcribed region of the eral genes devoid of introns (Huang & Carmichael,
transgenes 1997). The real efficiency of these sequences added
to transgenes has not been described so far.
Each group of living organisms have a preferential use A certain number of cDNAs contain cryptic spli-
of codons (Fox, 1987). Using synthetic genes may cing sites which recombine with the splicing donor site
allow the best adaptation of the codons to the mam- of the introns located in the upstream. This was ob-
mary cell machinery. The initiation AUG codon is served with the human factor IX cDNA (Clark, 1998).
better utilized when it is surrounded by the consensus Mutation in the cDNA may eliminate the splicing site.
Kozak sequence GCCA/GCCAUG G. If absent, this It is not known if the above mentioned sequences
sequence may be added by in vitro mutation. might be an alternative to mutation of the splicing
In a certain number of cases, it has been observed site.
that proteins naturally synthesized and secreted at a The 50 UTRs (untranslated region) of the mRNA
high rate are abundantly produced in milk. This is allow a better translation efficiency when they are
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poorly structured. Sequences favoring translation such expression of the transgenes. Unexpectedly, these sig-
as IRES (internal ribosome entry site) may be added nals loose a large part of their efficiency when foreign
before the initiation codon (Houdebine & Attal, 1999). cDNAs are introduced within the genes.
The 30 UTRs sometimes contain sequences contribut- The real capacity of the different milk protein gene
ing to stabilization of mRNA. Available informations promoters cannot be easily evaluated. A comparison
on this point are too rare to be presently useful. can be done. The human GH gene has been used as
Transcription terminators have different potency. a reporter by several groups (Günzburg et al., 1991;
Those from growth hormone or β-globin genes are Reddy et al., 1991; Tojo et al., 1993; Devinoy et al.,
reputed to give satisfactory results. 1994; Ninomiya et al., 1994; Wen et al., 1995; Hira-
One possible strategy may consist in introducing bayashi et al., 1996; Cerdan et al., 1998; Oh et al.,
the foreign cDNA within a milk protein gene. Mini- 1999). From these studies, it appears that the rab-
genes containing the first and the last introns have also bit WAP promoter (6.3 kb) is more efficient than the
been used. This proved to be a good approach in some mouse WAP promoter (2.6 kb). The variations of the
cases but not in others. It cannot therefore, be retained expression level in the individual mice indicate that
as the best solution. none of these promoters can, alone, give expression of
In a certain number of cases, several cistrons must transgenes in a reproducible manner.
be expressed simultaneously. Bicistronic constructs A co-injection of a gene construct with the whole
may in principle give satisfactory results. This implies β-lactoglobulin known to be highly expressed was
that IRESs are added between both cistrons. In prac- shown to rescue the expression of the foreign co-
tice, it appears that IRESs have quite variable potency integrated gene. This effect was dependent on the
and that they must be added at about 100 nucleotides transcription of the β-lactoglobulin gene but not of its
after the termination codon of the first cistron to be translation (Yull et al., 1997). Although helpful, the
fully efficient. Moreover, bicistronic mRNA may not addition of β-lactoglobulin gene appears insufficient to
be translated at a high rate (Houdebine & Attal, 1999). bring expression of associated genes to a satisfactory
It seems therefore, preferable to co-inject the con- level in all cases.
structs to be expressed simultaneously or to link them
within the same plasmid if high level of expression are The distal regulatory elements
wanted. Experiments carried out with different genes have
indicated that long genomic DNA fragments (100–
The factors controlling transcription of transgenes 300 kb) are highly efficient to express the gene they
contain in transgenic mice. This proved to be the case
The promoters for human (Fujiwara et al., 1999a) and goat (Stinnakre
To express foreign genes into the mammary gland, et al., 1999) α-lactalbumin contained in YAC and BAC
milk protein gene promoters must be used. The known vectors, respectively. In both cases, the genes were
genes have described by Mercier and Vilotte (1997). expressed in all transgenic mices in a copy dependent
These promoters showed different potency. Those of manner.
K and αS2-casein are particularly weak. All the other Interestingly, the DNA fragment containing the
promoters are being used with variable success. The human α-lactalbumin was highly efficient to express
MMTV LTR is also a candidate. Its potency and its hGH gene (Fujiwara et al., 1999b).
specificity may be insufficient for this purpose. Some of the regulatory elements located far up-
Among the different milk protein genes tested and stream or downstream of the genes have initially been
containing only a few kilobytes of DNA as promoters, named insulators. Indeed, it is generally believed that
only two of them ovine β-lactoglobulin and rat whey a transgene is unduly activated or extinguished by the
acidic protein (WAP), were expressed in a satisfactory action of host genomic regulators located in its vicin-
manner as transgenes. Their expression occurred in all ity. A certain number of experiments indicate that the
mice as a function of the copy number with no ec- situation is far more complex.
topic transcription (Withelaw et al., 1992; Dale et al., The insulating effect was first attributed to MAR
1992). In both cases the promoters were of variable (matrix attached region) sequences present in the DNA
and unpredictable efficiency when associated with for- fragments surrounding the genes. These fragments
eign genes. This suggests that signals of unknown were shown to enhance associated transgene expres-
nature and located within the genes contribute to the sion. MARs are generally AT rich regions, which
312

bind to the nuclear matrix where many transcription observed with transgenes. Chromatin openers found
factors are concentrated. It is admitted that genes are in LCR and enhancers seem essentially to reduce the
bound to the nuclear matrix when they are actively intensity of variegation (Walters et al., 1996). Altern-
transcribed. Additional studies have demonstrated that atively, the idea that insulators have the capacity not
the insulating effect was not solely due to MARs but only of preventing the activation by neighour enhan-
to other unidentified sequences located in their vicin- cers but also of blocking the formation of inactive
ity (Sippel et al., 1997; Bonifer, 1999). MARs seem chromatin is attractive (Bell & Felsenfeld, 1999).
still to contribute the expression of associated genes The question remains to know why transgenes are
and transgenes by modifying DNA structure through so frequently silent. Some general rules are progress-
DNA topoisomerase action (Adachi et al., 1989) and ively emerging. A transgene seems to have less chance
inhibition of methylation (Forrester et al., 1999). to be expressed efficiently if it contains non animal se-
Gene insulators are DNA sequence, which pre- quences, if it is devoid of intron and if it is integrated
vent action of the regulatory elements of a gene on a in multicopy (Dorer, 1997; Garrick et al., 1998). An
neighbour gene. Such sequences have been identified interesting hypothesis is that the cellular mechanisms
in Drosophila genome (Dorsett, 1999). Experiment- which inactivate retroviral and transposon sequences
ally, insulators are defined as elements preventing the by DNA methylation and deacetylation of histones are
activation of a promoter by a neighbour enhancer. also those which extinguish transgenes (Fire, 1999).
An insulator was found in the 50 HS4 region of the The effect of the chromatin openers and distal enhan-
LCR (locus control region) from the chicken β-globin cers might be to inhibit the mechanism of transgene
locus (Recillias-Targa et al., 1999). Insulators are not silencing (Santoso et al., 2000). To be efficiently ex-
considered as capable per se of stimulating gene or pressed, a transgene should therefore contain introns,
transgene expression. to be devoid of plasmid or synthetic sequences, not
The effect of insulators on transgene expression is to be integrated in multicopies or to be associated with
generally considered as being essentially a protective an appropriate LCR. This situation is encountered with
effect against the action of genomic extinguisher. A the long genomic DNA fragments.
certain number experiments do not support this view. Another mechanism has not been described in de-
Transgenic rabbits harbouring the human DAF and tail. Ectopic expression of transgenes is thought to be
CD59 under the control of the human EF1α gene do due to the action of neighbour genomic enhancers. The
not express the foreign genes at a high rate in the six addition of appropriate insulators to the transgenes
lines examined (Taboit-Dameron et al., 1999). It is un- should reduce or even suppress this effect. Alternat-
likely that in all cases the transgenes were integrated in ively, the ectopic expression may result from a cryptic
the vicinity of genomic extinguishers. The addition of basal transcription at the site of integration. Such basal
the 50 HS4 from chicken β-globin locus to the vectors transcription has been observed in different genomic
was sufficient to allow a high expression of the foreign regions (Travers, 1999) namely in the human β-globin
genes in all the lines of transgenic rabbits (Taboit- locus (Ashe et al., 1997).
Dameron et al., 1999). A similar effect was observed Independently, cryptic transcription of both DNA
with the transerythritin gene promoter in liver (Wang strands might generate double strand DNA which in-
et al., 1996) and the bovine αS1-casein gene promoter duces RNA interference which leads to a specific
in the mammary gland (Echelard, 1998). This suggests degradation of the mRNA coded by the transgene
that the 50 HS4 region contains not only an insulator and to its apparent silencing (Fire, 1999; Bosher &
but also elements capable of maintaining chromatin in Labouesse, 2000).
an active form. It is more and more believed that these
elements are chromatin openers which locally induce The possible vectors for a special control of
an hyperacetylation of histones and a demethylation transgene expression
of DNA (Pikaart et al., 1998; Bonifer, 1999). It be- The gene constructs may be introduced within a milk
comes, therefore, more and more difficult to make a protein gene by conventional homologous recombin-
clear distinction between chromatin openers and clas- ation. The use of restriction sites, which cleave both
sical enhancers. They both seem to act by increasing DNA strands, may improve the efficiency of homo-
the frequency of chromatin opening rather than by logous recombination (Cohen-Tannoudji & Babinet,
interacting directly with the complex of transcription 1998). A simplified protocol may rely on the use of the
initiation. Variegation in expression has often been Cre-loxP system. This system is generally implemen-
 313

ted to eliminate a given integrated sequence flanked by carboxylation, amidation, etc. These mechanisms are
two loxP sequences. The recombination of the loxP dependent on cellular enzymes, which are present at
sequences is triggered by the presence of the Cre re- variable concentration in the different cell types.
combinase (Wagner et al., 1997). A loxP sequence Glycosylation is undoubtedly one of the most im-
preintegrated in a milk protein gene may be the tar- portant post-translational event for therapeutic pro-
geted site of integration for a gene construct flanked teins (Fussenegger et al., 1999). Glycosylation may be
by two loxP motives (Rucker & Piedrahita, 1997; Kolb required for the biological activity of the proteins. This
et al., 1999). is the case for gonadotropins and to some extent for
The advantage of these protocols is that relatively antibodies (Wright & Morrison, 1997). Glycosylation
simple and standard gene constructs are expected to be is essential for the stability of many proteins in blood
sufficient since they are integrated in genomic locus circulation.
containing the required regulatory elements. The in- The mammary cell naturally secretes proteins
tegration of a foreign gene in a milk protein gene which are N-or O-glycosylated. Recombinant proteins
implies the inactivation of the latter. It has been shown found in milk are glycosylated but not always in an ap-
that the inactivation of β-casein gene does not al- propriate manner. Human antithrombin III was shown
ter milk secretion. On the contrary, milk devoid of to be not stoichiometrically sialylated. Yet, this pro-
αs1-casein is no more secreted with high efficiency tein extracted from the milk of transgenic goats is
(Chanat et al., 1999). expected to be on the market in 2000. Indeed, the
Several systems allowing the control of transgene lack of sialylation reduces the stability of the molecule
expression by exogenous inducers not acting on en- in vivo but it does not hamper its biological activity
dogenous genes are available. The most popular re- (Meade, 1999). Other proteins such as α1-antitrypsin
lies on the use of tetracycline and analogues. This (Clark, 1998) or EC superoxide dismutase (Strömqvist
system allowed the conditional expression of goat α- et al., 1997) were glycosylated in a manner almost
lactalbumin gene in transgenic mice (Soulier et al., similar to this of the native proteins. Unexpectedly,
1999). In the best conditions, the transgenic may in the bile salt-stimulated lipase was defective of O-
this way be expressed only in the mammary gland glycosylation (Strömqvist et al., 1996). This fact has
during lactation. The promoter which governs the not been explained. Indeed, this protein is naturally
expression of the gene of interest is potent and it secreted in milk in the O-glycosylated form. The reas-
drives the synthesis of the corresponding protein at ons why some recombinant proteins are not correctly
a high level. In practice, a background expression glycosylated are particularly complex. The glycosylat-
due to an imperfect control of the gene coding for ing enzymes may be limiting, specially when very
the tetracycline-sensitive transcription factor and to high amounts of the foreign proteins are synthesized.
ectopic expression out of the mammary gland may The addition of the genes coding for the glycosylating
reduce the advantage of this system. A selection of enzymes to the animals by transgenesis is conceivable.
the mouse lines expressing the activator in an ap- This approach met some success with some cultured
propriate manner must be previously done. Improved cell lines. A moderate expression of the genes coding
versions of this tool have been recently proposed (Blau for glycosidases is probably needed to avoid disturb-
& Rossi, 1999; Forster et al., 1999). It implies the ance of the cellular machinery. The poor glycosylation
simultaneous use of an activator and a repressor both of some proteins may result from their inappropriate
sensitives to tetracycline or doxycycline. This sys- folding in the endoplasmic reticulum and the Golgi
tem may eliminate the background but not the ectopic apparatus not providing the enzymes with a free access
expression. to the glycosylation sites.
The human protein C produced in the milk of mice
was poorly active. A study of the molecule revealed
The post translational events that it was not quantitatively cleaved. Subunits gener-
ation and assembly could thus not occur. The action
The maturation of the proteins of the furin transgene allowed the native proteins C
to be processed and active (Drews et al., 1995). As
After their biosynthesis, many proteins are subjec- opposed, the human protein C from transgenic pig was
ted to biochemical modifications including specific fully active. The human factor IX was carboxylated by
cleavage, folding, subunit assembly, glycosylation, the mammary cell (Clark, 1998).
314

The stability of transgene expression were observed with human erythropoietin (Massoud et
al., 1996) with hGH (Devinoy et al., 1994) with bGH
In mouse, the high expression of a transgene reduced (Thépot et al., 1995) with zona pellucida glycoprotein
somewhat the synthesis of endogenous milk proteins mZP3 (Litscher et al., 1999) and probably with some
(Mc Clenaghan et al., 1995). This fact is not really others. In several cases (Devinoy et al., 1994; Thépot
surprising. Indeed, during lactation in the rabbit, up et al., 1995; Bishoff et al., 1992), the concentration
to 20% of the milk protein mRNAs remain free and of the recombinant proteins was much higher in blood
not associated to form polysomes (Houdebine unpub- during lactation. In one mouse line at least, the hGH
lished data). This suggests that the translation ma- was found in blood only during lactation. The idea
chinery is saturated. Exogenous mRNAs are then ex- that the lactogenic hormones enhanced the ectopic ex-
pected to compete with the milk protein mRNAs. The pression of the transgenes is unlikely. Rather, a small
same phenomenon has not been observed in ruminants proportion of the recombinant proteins escapes from
(Colman, 1996). the mammary gland and migrates to blood. This may
The expression of a transgene was essentially at result from a leakiness of the mammary epithelium or
a constant level in the different individuals of a line to a non-strictly vectorial secretion at the apical side
over number of generations (Colman, 1996). The of the cells. It is interesting to note that whey pro-
co-suppression of transgene expression observed re- teins, namely WAP, are normally found in blood of
peatedly in plants is therefore, at most marginal in lactating animals (Grabowski et al., 1991). Caseins
animals. An exception was reported: the bovine α- organized in large micelles are not present in blood
lactalbumin transgene in mouse was expressed at a during lactation. Only whey and recombinant proteins
highly variable degree in the individuals of a given line in solution in the lactoserum can cross to some ex-
(Bleck & Bremel, 1994). Another important point is tent the mammary epithelial barrier. This suggests that
the stability of the transgene itself in the long term. a protein having a deleterious side-effect in the an-
Experiments carried out in mouse indicated that trans- imal may not be produced without any problem in
geneses are generally quite stably integrated in host milk even if the transgene is strictly controlled by the
genome (Aigner et al., 1999). tetracycline-dependent system. More specific effects
were observed at the mammary gland level. WAP im-
The in vitro and in vivo systems to predict the paired mouse and pig mammary gland development
efficiency of a gene construct (Shamay et al., 1992). Human EC superoxide dis-
mutase gene expressed in rabbit mammary gland re-
A few mammary cell lines are available and extens-
duced quite significantly milk production (Houdebine,
ively used in different laboratories. The most pop-
1998).
ular is the HC11 mouse line. The cells can at best
predict the intrinsic potency of a construct for tran-
scription but not the level of expression in transgenic The purification of recombinant proteins
animals. The cell lines are not expected to be able
to reflect all the events, which mature the proteins
post-transcriptionally. The purification of the recombinant proteins from milk
The direct introduction of a gene construct into the raises generally no particular problem. Milk contains
mammary gland of a lactating animal has been per- little amount of proteases. Casein can be removed eas-
formed with a retroviral vector (Archer et al., 1994) ily by non-drastic procedures. Chromatography may,
or with a gene gun (Kerr et al., 1996). It can at on a case by case basis, lead to a high purity of the
best provide relevant informations on the post trans- proteins (Wright & Colman, 1997; Van Cott et al.,
lational modifications of the recombinant proteins but 1996). However, it should be kept in mind that milk
not predict the transcription efficiency of the construct. is a relatively complex biological fluid and that the
complete elimination of some of its components may
The side-effects of the recombinant proteins on the not be easy in some cases.
animals Particular difficulty may be encountered when the
recombinant protein is similar to an abundant milk
The recombinant proteins produced in milk are gener- protein. This is the case of human serum albumin
ally to be used in humans. Their chance to be active which cannot be separated easily from the same pro-
in the animals is high. Side-effects on the animals tein from the animals.
 315

Specific breeding conditions and selection of ani- genes can be secreted from recombinant CHO cells at
mals among herd non-contaminated by prions are a concentration as high as 20 µg/ml of medium per
considered as sufficient to prepare safe products. 1 × 106 cells for a period of 24 h (Berdoz et al., 1999).
Other competitor systems may emerge in future.
Recent works have shown that peptides not directly
Conclusion and perspective related to human erythropoietin (EPO) can mimic the
action to the hormone (Wrighton et al., 1996). Non
The techniques leading to the production of recombin- peptide analogues may also mimic EPO action (Qures-
ant proteins in milk have now reached a certain degree chi et al., 1999). This approach is very attractive since
of maturity. The fact that human antithrombin III from the synthetic molecules may be potentially admin-
goat milk is expected on the market in 2000 illustrates istered by the oral route. On the contrary, most of the
this point. proteins have to be injected repeatedly. Progresses are
These techniques may and must be improved. The being made to protect proteins from degradation in the
cloning of embryos with transfected cells as nuclear digestive tract and to favor their absorption by the gut.
donors has greatly facilitated gene transfer into ru- The production of recombinant proteins by bioreactors
minants. Yet, the availability of reliable episomal vec- at a low cost might be a decisive advantage in that
tors would still simplify the generation of transgenic case.
animals in all species. The direct transfer of a gene to a living organ-
Expression vectors have still great progress to ism may lead to a significant secretion and action
make. Gene constructs still often lead to unexpec- of the corresponding protein. Pig growth was in this
ted results in transgenics (Ilan et al., 1996a,b; Barash way accelerated by the transfer of the GHRH gene
et al., 1999). The use of episomal vectors independent (Draghia-Akli et al., 1999). This procedure has many
of host chromatin would probably alleviate some of advantages over protein injection. These sophisticated
the expression problems. systems have little chance to be implemented for many
The present trends consist in using long genomic proteins. The production of recombinant proteins thus
DNA fragments as vectors. This is the case for α- will most likely remain an important industrial activity
lactalbumin gene (Fujiwara et al., 1999; Stinnakre, for several decades. Several hundreds of proteins for
1999). Works in progress suggest that regions from the many different purposes are expected to be prepared
casein cluster could have LCR properties and allow a by bioreactors at a competitive price (Wall, 1999,
high and reliable expression of foreign genes (Rijnkels Harris, 1999). A few recombinant proteins synthes-
et al., 1999). The same reasoning may be valuable for ized by bioreactors are currently subjected to clinical
β-lactoglobulin and WAP genes. tests.
In future, the essential elements controlling the ex- Antibodies seem to be the kind of proteins which
pression of milk protein genes might be combined in will be the most frequently used. Human monoclonal
an optimum manner to generate compact and highly antibodies may, for example, be a good alternative
efficient promoters. Such an approach has recently to antibiotics for some infection diseases. Bacteria
led to the definition of a regulatory region quite spe- and yeast can produce simplified antibodies (ScFv and
cific of muscle cells and more potent than the natural Fab) but less easily Fab0 2 or complete molecules. Milk
promoters (Li et al. 1999; Somia et al., 1999). (and possibly other bioreactors) proved to reach this
The mammary gland is presently the only really goal in several cases.
available animal bioreactor. It has a certain num- Certain categories of proteins have not been pro-
ber of competitors which have been depicted above. duced yet or marginally in milk. This is the case for
Transgenic plants are also potentially other potent sys- peptides having antimicrobial activities. These mo-
tems to produce recombinant proteins (Fisher et al., lecules may be an attractive alternative to antibiotics
1999). However, the purification of foreign proteins (Yarus et al., 1996; Latham, 1999).
from plants is not always easy and some of the post- Recombinant membrane proteins can be obtained
translation modifications of animal proteins are not from milk fat globules. This was the case for CFTR
correctly achieved in plant cells. (Di Tullio et al., 1992). The amount of the protein
Cells in culture may become more competitive sys- was very low. Yet, milk may potentially be the way
tems in the future. It is impressive that functional to obtain membrane receptors in sufficient amount to
humanized IgA synthesized by using four independent define their structure after crystallization. This ap-
316

proach may be essential to define synthetic molecules Note added in proof

acting on the receptors. It is clear that milk may be
the source of proteins not only for direct therapeuti- Human α-1 antitrypsin was produced in sheep
cal use but also for the study of proteins proper. milk after the introduction of the cDNA under β-
Small animals, namely rabbit, may be the bioreac- lactoglobulin gave promoter by homologous recom-
tors of choice for this purpose. Mice may in some bination in fetal fibroblasts subsequently used to gen-
cases provide experimenters with enough protein. In- erate transgenic annuals by cloning (McCreath et al.
deed, the incubation of the mammary gland on ice (2000) Nature 405 1066–1069).
may release more than one milliliter of milk in a
few hours (Stinnakre et al., 1992). The advantages
and drawbacks of the different species have been
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