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ATLA 42, 137145, 2014

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Comment
Every Silver Lining has a Cloud: The Scientific and Animal
Welfare Issues Surrounding a New Approach to the
Production of Transgenic Animals
Robert D. Combes1 and Michael Balls2
1Independent

Consultant, Norwich, UK; 2c/o FRAME, Russell and Burch House, Nottingham, UK

Summary The scientific basis and advantages of using recently developed CRISPR/Cas-9 technology for transgenesis have been assessed with respect to other production methods, laboratory animal welfare, and the scientific relevance of transgenic models of human diseases in
general. As the new technology is straightforward, causes targeted DNA double strand breaks
and can result in homozygous changes in a single step, it is more accurate and more efficient
than other production methods and speeds up transgenesis. CRISPR/Cas-9 also obviates the use
of embryonic stem cells, and is being used to generate transgenic non-human primates (NHPs).
While the use of this method reduces the level of animal wastage resulting from the production
of each new strain, any long-term contribution to reduction will be offset by the overall increase
in the numbers of transgenic animals likely to result from its widespread usage. Likewise, the
contribution to refinement of using a more-precise technique, thereby minimising the occurrence of unwanted genetic effects, will be countered by a probable substantial increase in the
production of transgenic strains of increasingly sentient species. For ethical and welfare reasons,
we believe that the generation of transgenic NHPs should be allowed only in extremely exceptional circumstances. In addition, we present information, which, on both welfare and scientific
grounds, leads us to question the current policy of generating ever-more new transgenic models
in light of the general failure of many of them, after over two decades of ubiquitous use, to
result in significant advances in the understanding and treatment of many key human diseases.
Because this unsatisfactory situation is likely to be due to inherent, as well as possibly avoidable,
limitations in the transgenic approach to studying disease, which are briefly reviewed, it is
concluded that a thorough reappraisal of the rationale for using genetically-altered animals in
fundamental research and by the pharmaceutical industry, and for its support by funding
bodies, should be undertaken. In the meantime, the use of CRISPR/Cas-9 to generate new transgenic cells in culture is to be guardedly encouraged.
Key words: animal wastage, CRISPR/Cas-9, non-coding regions, non-human primates, nonpredictive disease models, reduction, targeted effects, transgenesis.
E-mail address for correspondence: robert_combes3@yahoo.co.uk

Introduction
The new approach referred to in the title is called
CRISPR/Cas-9, and was originally developed in 2012
(1, 2). The silver lining is the fact that it optimises,
simplifies, and shortens the time taken for producing
new transgenic animal strains. But the cloud, for
those concerned about animal welfare at least, is the
fact that its application will not only increase the
range and diversity of transgenic rodent strains, but
will greatly expedite transgenesis in other species,
including non-human primates (NHPs; 3).

Since 1974, when the first genetically-modified


mouse was created, there have been many
attempts to improve the efficiency and speed of
transgenesis (4). To achieve stable integration and
expression of the transgene in host DNA, the
majority of transgenic mice are produced by the
microinjection of a DNA fragment equivalent to
several kilobases into the pronuclei of fertilised
eggs. The transgene is constructed by using recombinant DNA technology, during which it is inserted
into a vector (DNA fragment), and then amplified
to yield high copy numbers. The vector is engineered to carry an appropriate promoter, to permit

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the expression of the transgene after its integration into the host cells. However, a key problem
with this production method is that several copies
of the transgene can integrate at random positions,
with the possibility that many of the founder
offspring will not express the right gene, or will
express it at inappropriate levels.
An alternative to pronuclear microinjection has
been the use of embryonic stem (ES) cells, in which
the transgene integrates at a few sites by low level
homologous recombination (5). However, considerable animal wastage still occurs, and in addition,
ES cells are not readily available for certain
species, such as NHPs. This was evidently a major
impetus for searching for new methods, culminating in the development of the CRISPR/Cas-9
methodology (2).

The Basis of the CRISPR/Cas-9 System


CRISPRs (clustered regularly interspaced short
palindromic repeats) are DNA regions that contain
multiple, short, direct repetitions of base
sequences (6). Each repetition contains a series of
base pairs, followed by the same or a similar series
in reverse (palindromes), and then by 30 or so base
pairs known as spacer DNA. Spacers are short
segments of DNA from a virus or plasmid, and
serve as a memory of past exposures. CRISPRs
are found in bacteria, where they act as an
immune system in conjunction with the products
of cas (CRISP-associated) genes (7). These products have endonuclease activity against the DNA
of newly-infecting phages, thereby conferring on
the host cell a form of acquired immunity.
The CRISPR system can be designed to bind
directly to genomic DNA, by using a custom-made
stretch of RNA (signal guide RNA or sgRNA),
which includes a 20 nucleotide-long sequence that
is complementary to the genomic target site to
which it therefore binds (8, 9). The complementary
sequence on the target DNA is called the protospacer. The three nucleotides located immediately
at the 3 side next to the proto-spacer sequence,
constitute the proto-spacer adjacent motif (PAM),
which is required to ensure high cleavage specificity in target sequences. This RNA system is held
together with the endonuclease, Cas-9, to give rise
to a genome-editing complex, which causes doublestrand breaks after binding to the target site.
Because sgRNAs can be generated for each region
of interest in the genome, the technique can be
used for specific genome disruption and replacement, in a flexible and simple system, resulting in
high specificity and low cell toxicity.
Cleavage of the DNA activates further DNA
repair in the form of non-homologous end-joining
(NHEJ), an error-prone mechanism, which
frequently inserts or deletes small fragments of

Comment

DNA at the target site (6). If these fragments are


targeted at coding regions, frame-shift mutations
can arise. Also, a transgene can be introduced into
a host genome by making use of another DNA
repair mechanism, called homology-directed repair
(HDR). This replaces broken DNA with homologous DNA, which is artificially introduced into the
cell and acts as a template for the new inserted
transgene.
In practice, a library of sgRNAs is often used,
each molecule of which recognises a specific target
of the host genome. Under optimal conditions, it
only takes a single base difference between the
target DNA sequence and that of the respective
sgRNA sequence for Cas-9 nuclease activity to be
inhibited. However, in reality, some basemismatching between the sgRNA and the DNA
target is tolerated. If this mismatching is extensive, it results in high levels of off-target effects, as
has been observed in mammalian cells (10). It
appears that the sources of sgRNAs and Cas-9
nuclease can greatly affect the accuracy of
targeting (11).
So much for the theory what about the practice?

The CRISPR/Cas-9 System in Practice


Following certain molecular modifications to the
gene-editing complex, the CRISPR/Cas-9 technique was first shown to work in eukaryotes, by
the co-injection of zebrafish embryos to produce
mutant offspring, and then, in a separate study in
mammals, by co-injecting mouse ES cells with
multiple sgRNAs and the CRISPR/Cas-9 expression vector targeted to five genes (12). It was found
that, in 10% of the tested clones, all eight alleles of
the five genes had the desired mutations, indicating the ability of the method to result in specific
mutations at multiple sites in a single step. Not
only was it possible to achieve a mutation efficiency of 6581% in tested ES cell clones, 77% of
them had mutations in both alleles of the genes. In
other words, the system had also induced homozygous effects in one step. Presumably, it is because
the genome-editing complex also specifically recognises the identical corresponding DNA target
sequence present at the same position on the
respective homologous chromosome in the
fertilised diploid egg cell, that homozygous
offspring can be developed in one procedural step.
Transgenesis without ES cells was achieved by
co-injecting mouse zygotes with Cas-9 mRNA and
different sgRNAs, as well as DNA vectors of
different sizes, in a one-step procedure (12). This
generated mice with mutations at a number of
specific targeted sites, such as conditional alleles
and endogenous reporter genes, with a low
frequency of off-site effects (13, 14). Therefore,

Comment

since the CRISPR/Cas-9 methodology permits the


creation of homozygous animals in a single step, it
is unnecessary to undertake further breeding, as is
normally required when using traditional transgenesis methodology, which produces heterozygotes
which have to be crossed to generate homozygotes,
to provide for expression of the mainly recessive
mutations introduced.
The new method can be used to produce knockout, knock-in or conditional knock-out mice, by
direct pronuclear injection in embryos. It does not
require the use of selectable markers, thus
allowing the precise insertion of point mutations.
Crucially, and somewhat surprisingly, given the
fact that double-strand DNA breaks are induced,
the co-injection of CRISPR/Cas-9 and sgRNA into
zygotes resulted in very low fetal toxicity. Equally
surprising, is the fact that a method based on a
process that is a natural defence mechanism in
prokaryotes, seems to operate equally well in
eukaryotes, through the ectopic expression of Cas9, without the presence of a promoter element for
the gene. The ability of the CRISPR/Cas-9 system
to produce transgenic mice has been confirmed,
and the technique works also in rats and in
Drosophila (15, 16).
The scientific advantages of the new technique
are truly formidable not only do its accuracy and
its ability to directly induce homozygous mutations
substantially reduce the number of surplus
animals produced, but in addition, fewer animals
with lethal genotypes will be lost and fewer
animals with unwanted new genotypes will have to
be killed. Also, as multiple genes can be altered in
the same step, the generation of complex transgenic animals, with several genetic alterations, is
also facilitated. We also understand that, technically, the new methodology is not excessively
demanding, and in one case, at least, it has
enabled the time for producing a transgenic mouse
line to be shortened from a maximum of 18 months
to just 11 weeks (Horst Spielmann, personal
communication).

Animal Wastage during Transgenesis


The considerable levels of animal wastage due to
transgenesis have long been a controversial animal
welfare issue (1719). The extent of wastage is
difficult to quantify accurately, as details are
rarely published, but an impression of numbers
required for breeding was obtained in a separate
study, from analysing abstracts summarising
applications for licences to undertake animal
studies on vision and related neurobiology
research over the period 20052013, published by
the UK Home Office (20). Abstracts relating to
studies involving the development and use of new
transgenic mice typically stated that the majority

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of animals, out of estimated totals of about 1,000,


were to be used just for breeding purposes. These
animals are not required for any other purpose in
the project, and are euthanised.
Such wastage is counter-intuitive with regard to
reduction, and is highly regrettable for financial
cost reasons. However, because the unwanted
animals are usually killed before their use has had
any direct benefit, they are unlikely to have been
subjected to any procedures, which is a minor, but
positive, welfare benefit. The need for breeding
animals in transgenesis, together with the use of
transgenic animals, have both been implicated as
major factors in reversing the year-on-year decline
in published statistics for the numbers of animals
used for experimentation a reversal seen in
statistics compiled by several countries over the
last few years (21). However, it should be remembered that most countries do not record the
numbers of animals used for breeding (22).
It was noted that breeding of genetically-altered
animals remained the largest proportion of procedures in the UK statistics for 2011, published by
the Home Office (23). However, the Home Office
report for 2012 (24) states that: ...4.11 million
scientific procedures were started in Great Britain,
an increase of eight per cent (+317,200 procedures)
compared with 2011. ...the rise was mainly attributable to a 22 per cent (+363,100) increase in the
breeding of genetically-modified (GM) animals and
harmful mutants (HM). ...for the first time, the
number of procedures involving GM animals (1.91
million) was greater than the number performed on
normal animals (1.68 million). Therefore, wastage
due to breeding is still a major concern, at least in
the UK.
Transgenesis is also inefficient for several other
procedural reasons. It has been estimated that
only 25% of the organisms arising from pronuclear
injection are transgenic (25), and only a few of
these will turn out to be satisfactory for further
study, after screening with PCR and Southern
blotting. Another study found that only 7% of
implanted embryos gave rise to a transgenic
animal, though success levels would be expected to
vary case-by-case (18).
In addition, many embryos have to be implanted
for each successful pregnancy, and many pregnancies are required for the birth of a single transgenic
animal. Furthermore, it is necessary to check for
germline transmission of the transgene, and to
subject the resulting heterozygotes to more
breeding, to generate a homozygous genetic background for the expression of the recessive genetic
alteration. In addition, large numbers of surplus
animals can accumulate as a result of the need to
keep colonies to maintain lines of transgenic
strains, for possible use in future experiments.
However, this source of wastage can be minimised
by cryopreserving embryos and gametes (26). Loss

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of animals can also occur naturally during prenatal and post-natal development. Unfortunately,
there is a lack of published data on mortality rates
and ages at which deaths occur during the production of genetically-modified animals, and the use of
public databases detailing such information has
been called for (26).
A further cause of wastage arises as a result of
the need to remove variant animals exhibiting
inherent phenotypic instability, in order to maintain a high level of homogeneity in the colony (27).
In addition, commercial breeders might experience
variability in demand, to such an extent that they
have to maintain large stocks of animals, many of
which are unlikely to be sold (2830).
Previous attempts to address the low efficiency
of transgenesis include the injection of the transgene and appropriate regulatory sequences in the
form of an artificial chromosome vector (31), with
variable success. Other methods, particularly to
increase specificity for eliciting targeted DNA
changes, include gene-trap vectors, Cre-Lox recombination, TALENs (transcription activator-like
effector nucleases), and zinc finger nucleases. Like
CRISPR/Cas-9, the last-named technique induces
targeted double-strand breaks in DNA, and it has
been used to generate transgenic rats, as well as
transgenic mice (32). It is not the intention here to
describe these other methods in detail: suffice it to
say that each has its drawbacks, while the lastnamed three, in particular, have had variable
success in reducing the occurrence of random
insertion and other genetic effects.

Transgenic NHPs
It could be argued that, from a purely scientific
perspective, the area of transgenesis perceived to be
in most need of increased efficiency, is the production of transgenic NHPs, especially due to the high
financial costs of maintaining each animal, the long
time required for breeding, and problems in
acquiring the necessary ES cells. In the past, the use
of viral vectors has been the only way to generate
such animals. However, the CRISPR/Cas-9 method
has led to renewed interest in developing genetically-altered NHPs strains, as studies with them
have become financially and logistically more
manageable. One report has already been published
(33), demonstrating that such strains can be
produced by using the CRISPR/Cas-9 technique
instead of viral vectors. The genome-editing
complex, containing Cas-9 coding sequences and a
library of sgRNAs specific for three target genes,
were co-injected into one-cell-stage embryos of
cynomolgus monkeys. This yielded simultaneous
mutations in two of the target genes, as identified by
DNA sequencing. The latter technique was used to
verify the germline transmission of both of the trans-

Comment

genes in twins born after the transfer of geneticallymodified embryos into surrogate females.
The above data show that multiple mutations can
be induced in a single step in NHPs by using the
CRISPR/Cas-9 system. Moreover, no genetic alterations were detected in any off-target genomic sites,
confirming the high specificity of the process in
NHPs. However, these results should be regarded as
very preliminary. The apparent success of the
methods used, particularly the lack of unintended
genetic alterations, has yet to be confirmed.
Moreover, Niu et al. (33) commented that preliminary sequence data of both cultured embryos and
founder animals showed multiple genotypes, which,
they suggested, might be because nuclease cleavage
had occurred many times, at different stages of
embryogenesis. This issue would need to be resolved
by further genotyping. However, as Niu et al. themselves note, this will have to wait for the infant
founders to become older, to ensure the availability
of sufficient biopsy material. If this were to turn out
to be necessary for each new transgenic strain
produced in the same manner, it would increase the
time required and partially negate the advantages of
the CRISPR/Cas-9 technology. Also, any further
studies would be hampered by the five-month
gestation period.
Interestingly, a paper published very recently
(34), reports the first successful application of the
TALENs gene editing technique to produce transgenic NHPs the species in question being a
female cynomolgus monkey born with mutations
targeted to her methyl-CpG binding protein 2 gene
(MECP2), following the failure of several pregnancies. Although the founder animal is now several
months old, the same caveats apply to transgenesis effected by both this and the CRISPR/Cas-9
methods. In reality, there are few published data
for either method on the numbers of animals or the
time required. The CRISPR/Cas-9 study used 11
monkeys as egg donors, and 29 acted as surrogate
recipients, each one being injected with three eggs
(giving a total of 87 micro-injected embryos). This
compares with a corresponding figure of 200 eggs
which were needed to create ANDi, the first transgenic monkey (35). However, assuming that there
was confirmation of the preliminary analyses, of
the founders arising in both studies, it is possible
that many of the welfare problems and concerns
inherent in the two techniques, arising as a consequence of performing procedures on the animals,
as raised by Bottrill (35) with regard to the production of ANDi, would have been, at best, overcome,
or, at worst, alleviated.

Animal Welfare Considerations


Several other key welfare issues relating to NHP
transgenesis also need to be addressed, not the

Comment

least of which is the ethics of artificially altering


the genomes of such evolutionarily-advanced
animals. This issue has been debated extensively
elsewhere (3639), and, for the present discussion,
we shall assume that such moral arguments are
not going to halt the production of transgenic
NHPs. However, we must stress that many primatologists and behaviour experts believe strongly
that primates experience the world in ways that
are very similar to those of humans, that they have
sensory capacities similar to humans, and can
suffer in similar ways to humans (40, 41). In addition, NHPs have an acute awareness of their
surroundings, and are more likely than lesssentient species to suffer by losing interaction and
bonding with other individuals in their group,
when they have to be separated for experimental
purposes. While these characteristics are undoubtedly closest to those of humans in the case of the
Great Apes, there is no evidence to refute the
assumption that the NHPs more-commonly used
in the laboratory, such as macaques and
marmosets, are substantially different, despite
their lack of highly-sophisticated mental abilities
equivalent to those exhibited by Great Apes (38).
From this, it follows that there is a very strong
case for applying the same, or very similar, moral
arguments to the artificial and deliberate alteration of the genotypes of NHPs and humans.
Added to the above are the negative welfare
costs associated with: a) re-use, a practice that is
commonly employed with NHPs; b) the effects of
any deleterious genetic alterations arising from
transgenesis; c) the procedures and experimentation inherent in the studies in which they are being
used; and d) handling and manipulation, including
long periods of isolation and/or restraint (42).
The above considerations were discussed in
detail at an ECVAM workshop held 17 years ago
(43), and it was concluded that: Certain uses of
transgenic animals (for example, the use of higher
non-human primates) should be considered, in
principle, to be unacceptable. We were both participants at that workshop, and see no reason to
moderate the considered opinion of the participants in the workshop. On the contrary, we believe
that all NHPs should be included in the statement.
This conclusion was reached after carefully
weighing up the scientific and welfare advantages
and disadvantages of the new methods for transgenesis, in the context of the costs inherent in
using NHPs in laboratories.
Our concerns are shared by others. For example,
Dr Ian Ragan, a board member of the National
Centre for the Three Rs, stated recently (3) that:
Ethical concerns around the use of monkeys mean
that any method that makes it relatively easy to
alter them genetically must be properly justified
...this wont stop some researchers trying to add GM
monkeys to their toolbox though on the basis that

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they will inevitably provide better models of human


disease than GM rodents. In some instances this
might be true, but the case for creating GM
monkeys needs to be based on much more than a
bland and sweeping generalisation.

Where are the Medical Benefits of


Transgenesis?
Transgenesis has been widely practised for over 25
years, and many thousands of laboratory animals
have been used. Their use was based on the
assumption that there would be significant
advances in the understanding of the mechanisms
and treatment of disease. Unfortunately, this has
not happened, and publicity material produced by
the pharmaceutical industry, in support of transgenesis, is rich on promises and thin on achievements (45, 46). Sadly, the truth is that knowledge
about the mechanisms of many conditions, especially neurodegenerative diseases, remains very
limited, and effective therapies and cures have, in
many cases, yet to be developed. In fact, instead of
a promised profusion of new drugs, there has been
a substantial deceleration in the numbers passing
from the laboratory to the clinic.
There are many possible reasons for the above
situation, such as the extreme complexity of the
disorders concerned, and the fact that the start of
the slowdown in the development of new drug therapies coincided with a shift in emphasis toward
target approaches to candidate identification, and
selection (4648). It is also very possible that the
assumption that transgenic rodents serve as good
models of human disease, because they carry relevant genetic alterations and/or human genes, does
not hold in many cases. This would not be
surprising after all, the introduction of one or
two small genetic alterations in an effort to
humanise a mouse, will not alter the fact that the
resulting animal is still overwhelmingly a mouse.
The major use of transgenic animals in pharmaceutical development is in target validation.
However, even in the case of Alzheimers disease,
which is now hailed as being a success story for the
use of GM animals, since the discovery of the first
chemical able to arrest loss of brain cells, the
success of using transgenic organisms to increase
our knowledge of the mechanistic basis of the
human condition, in order to define new drug
targets and effective therapies, has been limited
thus far (49, 50). In 1995, a transgene was introduced into mice to direct expression of the
Alzheimer A peptide to neurons (51). The data
showed that the peptide was neurotoxic in vivo,
suggesting that apoptosis may be responsible for
the accompanying neuronal loss emulating the
principal underlying cellular feature of the
disease. However, while further preclinical studies

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for this drug target in animal models were promising, these results have yet to be translated to the
clinic (52).
A similar story applies to Huntingtons disease
(53), seizures and epilepsy (54), as well as
Parkinsons disease (55), the transgenic animal
models for which have been criticised elsewhere
(56). Indeed, any progress that has been made, has
often been based more on the use of conventional
animal models, and studies involving patients (3).
Also, in a scientific appraisal of transgenic mouse
models of human disease, published eight years
ago, it was concluded that many such systems have
limited scientific relevance, and even fewer have
positively contributed to the development of novel
human medicines. Sadly, little seems to have
changed in the interim period, and the use of
transgenic animals clearly has a long way to go
before its claimed potential will, if ever, be even
remotely realised.
The reasons why a candidate drug, that shows
positive indications in a transgenic animal model
of the respective disease, fails to exhibit similar
therapeutic activity against the same disease in
volunteers, include: an incorrect genetic background; lack of a human-specific cytoplasmic factor
necessary for drug activity; use of a model based on
an inappropriate target/mechanism; lack of the
required levels of biotransformation of a pro-drug
to an active form in the mouse, or drug inactivation
in humans; and lack of transport in humans to the
target site (a particular problem with neuro-active
compounds and the effects of species-specificity on
passage across the bloodbrain barrier). Some of
these issues are inherent problems in the use of
transgenic rodents that might be difficult to overcome, while others are due to problems of the low
fidelity of mouse models in general.
We have previously speculated that genetic
alteration of mice, by ENU mutagenesis, or via
transgenesis, could change regulatory (non-coding)
regions of DNA (42, 56). We also noted that,
without full genomic sequencing, the occurrence of
such alterations in regulatory genes would remain
cryptic, in the absence of the target coding genes
being controlled, because the former have no
phenotypic effects on their own. This means that it
is more difficult to detect the involvement of regulatory genes, compared with coding sequences, in
the aetiology of disease, thereby increasing the
chance that transgenic strains are produced with
inappropriate targets and genetic backgrounds, in
cases where changes in regulatory genes are actually responsible for a disease phenotype. In these
circumstances, it would be expected that use of
such a mouse model of the disease would not result
in successful target validation. On the other hand,
successful validation should occur with a transgenic strain in which the correct regulatory gene
and genetic background had been used.

Comment

The likelihood of the above situation occurring in


practice is increased by the fact that a growing
number of human diseases, such as multiple sclerosis, Alzheimers disease, alpha-thalassaemia,
Human Spinocerebellar Ataxia Type 8 (SCA8) and
myotonic dystrophies, are being shown to be due to
changes to regulatory non-coding sequences
(5759). A further reason is that the majority of
the genome is comprised of non-coding DNA
while over 80% of the human genome is biochemically active, less than 3% encodes proteins, and
part of the non-coding DNA is actually transcribed
to produce mRNAs of varying lengths, that are not
translated (60). Of particular interest are long noncoding RNA (lncRNA) molecules exceeding 200
nucleotides in length, which have been associated
with so-called dark matter of the genome, whose
exact function has yet to be fully elucidated (58,
59).
Patients with Human Spinocerebellar Ataxia
Type 8 (SCA8) have a trinucleotide expansion in
an lncRNA molecule, named ataxin 8 opposite
strand (ATXN8OS), which is antisense to the
KLHL1 gene. The involvement of this mutation in
SCA8 disease progression has been confirmed in a
transgenic mouse model, and mice with this repeat
expansion showed a similar progressive neurological phenotype to humans with SCA8 (61).
Non-coding, regulatory DNA sequences often
flank coding genes with complex expression
patterns, acting as highly conserved breaks
(barrier elements) to separate one co-regulated
sequence from the next one downstream, both
sequences of which would otherwise be contiguous
(58). Changes to these barrier elements would be
expected to result in abnormal expression patterns, if deletions lead to mis-regulation through
the extension of regulatory influence from control
of a neighbouring regulatory domain to immediately downstream coding genes.
That this is indeed possible, and that such
effects can cause human disease, was shown
recently by Spielmann et al. (62), in a study of the
autosomal dominant developmental defect,
Liebenberg syndrome. This results in an upperlimb malformation, with abnormal elbows and a
partial homeotic transformation of the upper
extremities to lower extremities. Two deletions
and a translocation upstream of the PITX1 gene
were identified as the genetic alterations responsible for this.
Pitx1 is a homeobox gene transcription factor,
normally only switched on in the hind-limb. The
authors showed that generation of mice with a
transgene for an enhancer element, with regulatory control of fore-limb and hind-limb activity,
used as a driver for Pitx1 in transgenic mice,
resulted in phenotypic characteristics analogous to
Liebenberg syndrome. It was concluded that the
enhancer was sufficient to produce the malforma-

Comment

tion, if it could regulate the expression of PITX1, as


would be possible by removal of the barrier
element separating the PITX1 regulatory domain
from neighbouring regulators. Without this
barrier, the enhancer was free to switch on PITX1
for expression of both fore-limb and hind-limb
developmental genes.
The above examples serve to illustrate some of
the many pitfalls and complexities in developing
and using transgenic animals as disease models,
and they stress the need to consider more than just
the coding genome as a target for transgenesis. In
addition, where the involvement of regulatory
genes has been taken into account, it is always
possible that successful therapeutic interventions
in the respective mice may not work in the clinic,
due to species differences in regulatory control
mechanisms, although the general consensus is
that regulatory gene mechanisms show a high
degree of conservation.

Conclusions
While the new CRISPR/Cas-9 technology reduces
the level of animal wastage resulting from the
production of each new strain, any long-term
contribution to reduction will be offset by the
overall increase in the numbers of transgenic
animals likely to result from its widespread usage.
Likewise, the contribution to refinement of using a
more-precise technique, thereby minimising the
occurrence of unwanted genetic effects, will be
countered by a probable substantial increase in the
production of transgenic strains of increasingly
sentient species.
Therefore, we still believe that the ECVAM
workshop statement should be the default position
for any assessment of proposed work on developing
and using transgenic NHPs in general. In other
words, such assessments, like those conducted by
ERPs (ethical review processes) and IACUCs
(institutional animal care and use committees), as
well as other regulatory and legislative bodies,
should lead to project approval only in extremely
exceptional circumstances. This conclusion has
also been taken in light of what we perceive to be
the inadequate protection of NHPs from general
usage in laboratories, afforded, for example, by
Directive 2010/63/EU (63).
In the meantime, we urge that further consideration be given to monitoring and exploring in
greater detail, the welfare consequences for all
species of every available method of transgenesis,
and to the wide application of the most promising
of these, such as CRISPR/Cas-9, for increasing the
diversity of cell lines for use in in vitro studies.
In addition, for both animal welfare and scientific reasons, there needs to be an in-depth critical
assessment of the rationale for using transgenic

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animals in general to study and treat human


diseases, based on past achievements and likely
future successes. Such a review should include a
systematic analysis of the possible reasons for the
lack of therapeutic efficacy in the clinic of drug
candidates that have previously given encouraging
indications in transgenic organisms.
The time is surely ripe for the pharmaceutical
industry and the major funding bodies to re-assess
their near-obsession with genetically modifying
animals, by holding an inquest into the reasons for
the slow progress in developing effective drugs
against major diseases that still cause suffering
directly to many people each year, and indirectly,
to many thousands of laboratory animals.

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