D9204.PDF

Rev. sci. tech. Off. int. Epiz.. 1 9 9 8 , 1 7 (1), 351-364
Lessons from qene knockouts
N.Osterrieder
(1)
&E. Wolf
( 2 )
(1) Institute of Molecular and Cellular Virology, Friedrich-Loeffler-lnstitute, Federal Research Centre for Virus
Diseases of Animals, D-17498 Insel Riems, Germany
(2| (Corresponding author) Institute for Molecular Animal Breeding/Gene Centre,
Ludwig-Maximilians-University, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany
Summary
The authors describe t h e t e c h n i q u e f o r t h e application of homologous
r e c o m b i n a t i o n in e m b r y o n i c s t e m cells, w h i c h is n o w w i d e l y used t o e n g i n e e r
m i c e w h i c h c a r r y s p e c i f i c k n o c k o u t s o f g e n e s . A s u m m a r y is g i v e n o f s o m e of t h e
k n o w l e d g e of the pathogenesis of and resistance t o infections w i t h parasites,
bacteria, or viruses w h i c h has accumulated during recent years, based o n the
i n v e s t i g a t i o n o f k n o c k o u t m i c e . S p e c i a l e m p h a s i s is p l a c e d o n k n o c k o u t a n i m a l s
w h i c h l a c k c o m p o n e n t s of t h e c y t o k i n e n e t w o r k , l a c k g e n e s w h i c h a r e c r i t i c a l f o r
t h e c o r r e c t p r e s e n t a t i o n o f antigens o r a r e d e f i c i e n t in different i m m u n e cell
s u b s e t s . In a d d i t i o n , a brief e x p l a n a t i o n is o f f e r e d of t h e possibilities f o r i n d u c i n g
t a r g e t e d deletions o r m u t a t i o n s in g e n e s of livestock s p e c i e s (e.g., b y n u c l e a r
t r a n s f e r o r b y m u t a g e n e s i s using" t h e alkylating a g e n t
Nethyl-/V-nitrosourea)
w h i c h could lead t o the breeding of animals w h i c h a r e resistant t o infectious
d i s e a s e s in t h e f u t u r e .
Keywords
Bacteria - Cytokines - Genetics - Knockout mice - Livestock - M u t a g e n e s i s - Parasites Viruses.
Gene targeting in embryonic
stem cells: a powerful tool for
analysing gene function
Embryonic stem (ES) cells are pluripotent cell lines which are
reproducibly derived from the inner cell mass of mouse
blastocysts ( 2 3 , 5 8 ) . Differentiation of ES cells is inhibited by
leukaemia inhibitory factor (LIF) or other cytokines of the
interleukin-6 family which allow long-term culture and
genetic manipulation of ES cells in vitro ( 1 0 0 , 1 0 1 , 1 0 2 ) .
Upon injection into blastocysts ( 1 2 ) or aggregation with
morula stage embryos ( 1 0 3 ) , ES cells may contribute to all
tissues of the resulting chimera, including its germ line
(Fig. 1). Therefore, the possibility exists - at least indirectly to reconstitute mice from cultured cells. The need to produce
chimeras can be circumvented by aggregating ES cells with
tetraploid embryos ( 6 2 ) . Using this technique, researchers
have obtained completely ES cell-derived offspring: however,
these were not fertile and thus not suitable to establish
transgenic lines.
ES cell technology is now widely established and offers a
powerful tool for functional analysis of genes. Based on the
principle of homologous recombination ( 1 9 ) , various
strategies have been developed to introduce targeted
mutations into ES cells (36) (Fig. 2 ) . Although the efficiency
of homologous recombination was low in early experiments,
the frequency of targeted integration was markedly increased
by using isogenetic DNA for the construction of targeting
vectors (87).
Initial studies were directed principally towards a complete
inactivation of gene function to evaluate the biological
relevance of the respective genes ( 1 3 , 14). The production
and use of these so-called 'gene knockouts' and the
corresponding knockout mice are now standard techniques in
many laboratories. In the past few years, new methods have
widened the spectrum of ES cell technologies by allowing the
introduction
of subtle site-directed mutations, large
chromosomal alterations and tissue-specific gene knockout
and repair. This allows analysis of gene function in a
lineage-specific way and also permits the analysis of genes
which lead to embryonic lethality if inactivated in early
embryonic development (69). The most widely used system
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Fig. 2
Strategies for introducing targeted mutation into the genome of
embryonic stem cells
The thick line represents the vector homology to the target locus; the broken
line represents the bacterial plasmid. The hatched rectangles are exons. The
positive selection marker is the neomycin phosphotransferase (neo) gene.
The thymidine kinase (TK)gene of herpes simplex virus is used as a negative
selection marker
a) Replacement vector: the positive selection marker interrupts the target
homology
b) Insertion vector: a positive selectable marker may be cloned into the
homologous sequences or the vector backbone. A double strand break is
generated in the target homology prior to transfection
scrapie agent ( 1 7 ) . With the second approach, the immune
response of the host to different pathogens can be investigated
by interfering with the immune system and by targeted
disruption or modification of genes which are involved in
immune mechanisms (Fig. 3 ) .
Knockout animals
Fig. 1
Schematic illustration of the production of germ-line chimeric mice by
morula aggregation or blastocyst injection
for introducing these fine-tuned mutations in mice is the
Cre/lox-P system from bacteriophage Pl ( 4 4 ) .
This repertoire of methods has been used extensively to
dissect the functions of genes involved in complex biological
systems, including those relevant for the immune system and
for resistance or susceptibility to infectious diseases.
Targets for gene disruption to
investigate the pathogenesis of
infectious diseases
To investigate the molecular pathogenesis of infectious
diseases and combat infectious diseases by breeding
measures, two different approaches can be followed. In the
first approach, the aim of generating transgenic or knockout
animals can be to prevent attachment, entry or replication of
the infectious agent into the respective hosts. The most
striking example of this approach is the generation of P r P
knockout mice which are resistant to infection with the
c
Discruption of receptors for
infectious agents or proteins
that are responsible for the
development of disease
Interference with the host
immune system, for example
by disrupting cytokine, major
histocompatibillity complex
or receptor genes
Fig. 3
Possible target genes for the generation of knockout animals to study
the pathogenesis of infectious diseases
To date, few reports and applications have dealt with the
prevention of entry of infectious agents into knockout
animals. This is largely due to the fact that the molecular
mechanisms involved in the early stages of infection of most
infectious pathogens remain unclear, and the lack of
information on these processes prevents the targeted
disruption of genes and gene products which would prevent
initial infection. However, as knowledge about the immune
system in man and animals has increased significantly during
the last years, the interaction of individual components of the
immune system, both on the level of the immune cells and the
soluble mediators which regulate the immune cascade
(cytokines and chemokines, as well as cytokine and
chemokine receptors) with pathogens was addressed using
knockout technology. Figure 4 gives a schematic overview of
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Ig
: immunoglobulin
APC : antigen-presenting cell
TCR : T-cell receptor
Fig. 4
Schematic illustration of the cytokines produced by different cells of the immune system
Black arrows Indicate the differentiation or activation of cells. Dashed arrows indicate positive feedback mechanisms, arrows marked with || indicate inhibitory
effects. Grey arrows indicate the cytokines produced by different cells
the current knowledge of the immune system and the
regulatory and modifying mechanisms underlying the defence
against infectious agents. As different infectious agents are
encountered by different immune measures of the host, the
first goal of targeted inactivation of genes is to determine
which factors in the complicated immune network have
essential, beneficial or detrimental effects on the capacity of
the host to control an infectious agent and the disease caused
by the pathogen. Virtually all molecules involved in the
immune network can be a target of gene disruptions.
However, depletion of single chemokines or chemokine
receptors, for example, may cause a general breakdown of the
immune system, might be compensated by other related
mechanisms, or may have specific effects on individual
infectious pathogens ( 4 7 ) . The following description of
selected reports represents examples of knockout animals
which were used to address specific questions of the
interaction of the host with infectious agents.
cytokine activities upon infection and of the interactions of
individual cytokines during acute or chronic infectious
disease has been obtained, at least in widely used mouse
models of infectious diseases (see below and Table I). In
addition, a large number of regulatory pathways underlying
the maturation of immune cells and their direction to the
active sites of the immune defence has been elucidated ( 2 5 ,
40).
One of the most important issues in using knockout models is
the understanding of the genetic basis for resistance or
susceptibility to infectious diseases. This has become an issue
of major interest in disease control and for the rational design
of novel vaccine approaches, which are extremely important
in a time of increasing numbers of immunocompromised
individuals. Hence, knockout models are used to more
precisely define:
- the mechanisms underlying acute and chronic disease
- the clearance of the pathogen from the organism
- immunity to infection or at least disease.
Lessons from knockouts: the
mouse as a model
As stated above, considerable progress has been made during
recent years in identifying mammalian genes and gene
products that influence the outcome of disease after infection
with different agents, including parasites, bacteria and viruses.
By using knockout technology, a better understanding of the
In acknowledging and using the great advantages of
transgenic and knockout models, however, it must be also
noted that pathogenesis and immunity models of animal or
human infectious diseases which rely on the use of knockout
animals must be substantiated by other experimental
procedures. This restriction of transgenic or knockout animals
is caused by the existence of subtle feedback mechanisms in
the organism which are influenced by targeted gene
Rev. sci. tech. Off. int. Epiz., 17 (1)
354
Table I
Examples of mice with targeted deletions of cytokine or cytokine receptor (R) genes, and infected with parasites, bacteria or viruses
Targeted deletion
Treated/infected with
Comments
IL-1 p
Lipopolysaccharide
Influenza virus
Escherichia coli
Lipopolysaccharide
Lower body temperature
Higher mortality
Partial resistance to challenge
Resistance to challenge
Breakdown of self-tolerance
IL-1 PR
IL-2
IL-3R
IL-4
IL-5R
IL-10
TNF-a
TNF-aR
IFN-y
ÍFN-yR
It
TNF .
IFN
GM-CSF
Th2
:
:
:
:
:
References
24, 50
1
1
40
Effect on GM-CSF and IL-5; reduced eosinophilic counts
Nippostrongylus brasiliensis
Toxoplasma gondii
Leishmania donovani/L. mexicana
Plasmodium chabaudi
Trypanosoma brucei brucei
Higher mortality rates
Different disease progression
Reduced Th2-associated cytokines
Strain-dependent higher parasitemia
See IL-3R
Trypanosoma cruzi
T. gondii
Listeria monocytogenes
£ coli
L. monocytogenes
Lower parasite burden but earlier mortality
Lethal immune response
Impaired resistance and clearance
Mild effect
Impaired resistance and clearance
T. gondii
t. donovani
Legionella pneumophila
Influenza virus
Mouse hepatitis virus
Herpes simplex virus
Murine y-herpesvirus
Lymphocytic choriomeningitis virus
Plasmodium berghei
L. monocytogenes
Influenza virus
Earlier mortality
Uncontrolled visceral infection
Development of persistent disease
No effect
No effect on central nervous system spread
Enhanced suceptibility to lesions
Mild effect
No virus clearance in persistently infected mice
No cerebral malaria
Higher IL-4 production and susceptibility to disease
High virus burden
63
67
72
93, 94
3,4
42
33
65
1
65
73,74
86
38
34
51
106
71
89
70
83
43
interleukin
tumour necrosis factor
interferon
granulocyte macrophage colony-stimulating factor
Thelper 2
disruption or expression, and also by the nature of any
knockout and transgenic models which are very contrived per
se (9, 54). The second important restriction relates to the fact
that the information obtained is so far mainly restricted to the
mouse, which is used as a model for many infectious diseases.
The next paragraphs list some of the recent findings on the
outcome of disease after infection experiments with different
pathogens which have been elucidated using knockout mice.
The presented data will mainly concentrate on protozoan,
bacterial and especially virus diseases.
Resistance to infection with parasites
Parasitic infections have become a major issue world-wide.
This is partially - though not exclusively - caused by the
growing number
of individuals
who
suffer
from
immunosuppressions. Of particular importance in the
generation of an immunocompromised state in man are
infections with human immunodeficiency virus type 1
(HIV-1) and also allergic diseases, whereas in animals there is
a variety of causes of immunocompromised individuals.
Human infections with different protozoan pathogens such as
Eimeria
spp.,
Plasmodium
spp.,
Leishmania
major,
Trypanosoma spp., Toxoplasma
axe. also of great concern.
gondii or Pneumocystis
carimi
Research which applies the use of knockout animals to
investigate the outcome of disease with these pathogens has
concentrated on the beneficial or harmful effects of cytokine
action. An infection with T. gondii can cause an acute or latent
infection. Acute infections are caused by rapidly dividing
tachyzoites which harm virtually every cell of the body,
whereas latent infection is characterised by dormant
bradyzoite forms in tissue cysts ( 3 0 ) . With regard to the
molecular pathogenesis of T. gondii infection, there has been
speculation that infection is controlled primarily by
cell-mediated immune responses and interferon-y (IFN)-Y
activity ( 8 2 ) . The effect of IFN-y was thought to be
augmented by the effects of interleukin-12 (IL)-12 ( 3 2 ) . By
using IFN-y knockout mice, Scharton-Kersten et al. have
shown, however, that parasite control depends on IFN-y
alone and that in the absence of the cytokine, tachyzoite
replication is unlimited ( 7 3 , 7 4 , 7 5 ) . In contrast, IL-12 was
produced to levels found in wild-type mice, thus
demonstrating an indirect function of IL-12 through IFN-y in
control of T. gondii infection, since the infection could not be
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Rev. sci. tech. Off. int. Epiz, 17 (1)
controlled by IL-12 alone. Mice which no longer expressed
the IFN-y regulatory factor 1 (IRF-1) gene and which, as a
consequence, were unable to produce nitric oxide (NO), the
potent inactivator of intracellular (macrophage) parasites,
were more susceptible to infection, although NO was not
exclusively responsible for clearance of the pathogen (48). In
mice which were devoid of another cytokine, IL-10, T. gondii
infection. instantly caused high mortality which was
characterised by increased levels of IFN-y and IL-12. In
addition, other key cytokines, such as I L - 1 b and tumour
necrosis factor a (TNF-a) were augmented, which suggests
that the modifying cytokine IL-10 regulates other cytokines
and that in the absence of IL-10, IFN-y and IL-12 cause a
C D 4 (cluster of differentiation antigen) T-cell-dependent
immunopathological disease ( 3 3 ) . These findings were
substantiated by studies which analysed IL-4-deficient mice
and their susceptibility to T. gondii infection ( 6 7 ) . IL-4
knockout mice produced significantly lower levels of IL-10
but higher levels of IFN-y, and mortality rates were greatly
enhanced.
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Infections of animals or man with Trypanosoma cruzi (Chagas'
disease) and T. brucei lead to disturbance of the host immune
response and are characterised by an unspecific activation of
the immune system, which finally results in autoimmune
reactions and pathological consequences ( 1 5 , 3 5 ) . Resistance
to disease has long been known to depend on IL-10
production and its suppression of IFN-y production, and
infection is known to be aggravated by IFN-y (4, 6 0 ) . Using
mice which were deficient in IL-10, infection with T. cruzi
could be shown to lead to earlier and higher mortality,
although the parasite burden was lower when compared with
wild-type mice ( 4 2 ) . The same observation was made after
infection of mice lacking y ò T cells (a lymphocyte subclass
which is stimulated by IFN-y and downregulated by
application of IL-10) ( 5 2 ) , or by using mice which were
deficient in C D 4 or C D 8 cells (85). These results suggest a
complex interaction between immune cells in the control of
infection and their interdependence in parasite clearance and
disease outcome. IFN-y knockout mice displayed higher
resistance to T. brucei infection ( 4 ) , thus indicating that the
regulation of and mediation by this key cytokine ( T h l helper
cell activation) plays a dominant role in susceptibility and
resistance to infection with Trypanosoma spp.
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Other parasites of growing importance are L donovani and L.
major. These parasites cause leishmaniosis, a proliferative and
necrotic disease of the skin and inner organs which is
controlled primarily by the action of IFN-y and by a T helper
1 ( T h l ) type immune response (61, 6 6 , 8 0 ) . The knowledge
of Leishmania pathogenesis and the control of infection by
different types of immune cells and cytokines is largely based
on the use of knockout animal models and subsequent
infection experiments. In the absence of T h l cells and IFN-y,
macrophages which are responsible for parasite clearance are
not activated and protozoan replication is unrestricted ( 5 5 ,
96, 9 7 ) . Recent work, however, demonstrated that L. major
pathogenesis is not enhanced if major histocompatibility
complex (MHC) class II presentation is hampered in mice that
lack the invariant chain. Obviously, cytokines such as IL-12
and IL-4 were able to induce maturation of T cells in the
absence of functional MHC class II molecules, such that IFN-y
is secreted (16). These results were confirmed and extended
by the use of IFN-y knockout mice in which a
TNF-a-dependent action of IL-12 in the control of
leishmaniosis could be demonstrated (86).
Experiments comparable to those described above on the role
of yò T cells for T. cruzi have been performed with mice
lacking different immune cell subsets using the protozoan
parasites Eimeria spp. and Plasmodium spp. Mice which no
longer expressed either ct|3 or yò T cells exhibited increased
susceptibility to intestinal infection with Eimeria
spp.,
although this appears to be due to different mechanisms: the
lack of a | 3 T cells resulted in a reduced immunity to
reinfection, whereas lack of y ô T cells led to enhanced
intestinal damage ( 6 8 ) . As a result of B-cell depletion by
knockout technology, enhanced resistance to acute
Plasmodium spp. infection could be demonstrated. This
appears to be due to a rise in y ò T cells. In addition, a rise in
a | 3 T cells could be demonstrated in B-cell-deficient animals,
although this rise was not as marked as that in the y ô subset
(91). The authors conclude that this T-cell subset is largely
controlled by B-cell action, that this activation is independent
of IL-10 production, and that the expansion of y ô T cells
might be a target for further vaccine strategies which are able
to stimulate specifically this T-cell subset.
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Taken together, the reports summarised above have largely
improved the understanding of protozoan infection, and have
revealed a complex concert of cytokines in control of
resistance to and progression of infection. Thus, these findings
will no doubt be useful in future attempts to generate a new
generation of anti-parasitic drugs and efficacious vaccines.
Resistance to disease caused by bacteria
By generating transgenic and knockout animals, the
pathogenesis of a variety of bacterial diseases has been
elucidated. The role of B- and T-cell subsets and/or individual
cytokines involved in the resistance or susceptibility to
bacterial disease has been assessed, and special emphasis was
put on the molecular mechanisms underlying sepsis and
clearance of intracellular bacteria.
Sepsis caused by gram-negative bacteria is one of the most
important causes of mortality after surgical intervention, and
is caused by a shock syndrome which follows lipopolysaccharide (LPS) or endotoxin release ( 1 0 , 21). A critical step
in the development of LPS-caused shock is the release of the
key cytokines T N F - a and IL-1(3, which cause the attraction
and stimulation of immune cells locally in the
micro-environment. However, the same cytokines are able to
induce responses which are indicative of septic shock, not in
their paracrine but in their systemic functions ( 2 0 , 2 7 ) . The
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resistance to T N F - a - and IL-lp-induced shock was tested
after application of Escherichia coli or endotoxin in respective
knockout mice. Animals deficient in either cytokine were
shown to exhibit increased resistance to intravenous
application of endotoxin, thus indicating that acute and
systemic reaction and activation of immune T cells is
suppressed. In addition, in the absence of IL-1(3, LPS-induced
fever is no longer observed. Hence, this mechanism plays a
major role in disease progression ( 1 , 5 0 ) . Similarly, mice
deficient in CD14 (which serves as a receptor for LPS) are
much more resistant to the shock syndrome caused by
gram-negative bacteria, as shown in an infection model. In
addition, CD14-negative mice exhibited reduced levels of
bacteraemia, which suggests that the spread of gram-negative
bacteria at least is controlled by a CD14-dependent
mechanism ( 3 7 ) . The knowledge of the early interaction of
gram-negative bacteria with the host immune response could
be beneficial in the treatment or immune prophylaxis against
these pathogens, which are a frequent cause of mortalities in
the human and animal populations.
Most other reports on the genetic resistance to bacterial
infections have dealt with organisms which replicate
intracellularly or which interfere with host cells other than by
endo- or exotoxin production. Among the bacterial infections
which were analysed using knockout animals are those with
Listeria spp., Brucella spp., Mycobacterium spp. or Francisella
tularensis. The deletion of the (32-microglobulin gene leading
to an absence of a functional CD8-dependent cytotoxicity,
and also the deletion of functional C D 4 cells, were shown to
cause delayed clearance of both Listeria monocytogenes and
Mycobacterium
bovis bacillus Bilié-Calmette-Guérin (BCG)
from infected animals. However, infection with either
bacterium was not fatal in these animals, thus suggesting a
certain redundancy in the host response to the pathogens (46,
47). The disruption of the IFN-y receptor gene led to
decreased levels of TNF-a, and Listeria infection was more
severe when compared with that in control mice. Obviously
IL-4, which is not measurable after L. monocytogenes infection
in wild-type mice but which is produced by mice which lack
the IFN-y receptor, is responsible for the more severe
symptoms. In contrast, IL-12 did not appear to be involved in
Lisieria-caused disease (83). Using mice which are deficient in
the macrophage type I and II class A scavenger receptors
(proteins which are involved in binding of bacteria), disease
after infection with L. monocytogenes
was shown to be
augmented, which demonstrates that receptor-mediated
incorporation and presentation of the antigens by
macrophages is essential in the host defence against
intracellular pathogens (81).
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In similar animal models (i.e., using mice with MHC class I or
II knockouts), researchers were able to demonstrate that
Brucella abortus clearance and resistance to disease is caused
mainly by the cytotoxicity of C D 8 T cells (79). By identifying
the host effectors and the targets in the bacterium, the authors
intend to produce rationally designed novel vaccines against
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this zoonotic pathogen. Using animals which are deficient in
different T-cell subsets, the resistance and immunity to
F. tularensis
infection was analysed. The antibacterial
response was shown to be based mainly on a | 3 T cells,
whereas depletion of C D 4 cells or y ô T cells did not alter
disease control appreciably. The authors concluded that in
general, resistance to intracellular bacteria is largely
dependent on the a(3 T-cell subset, but that C D 4 or C D 8
cells can control the disease alone and independently (105).
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IFN-y knockout mice have been used to analyse the outcome
of infection with Legionella pneumophila. These experiments
demonstrated that IFN-y action is essential for clearance and
control of pulmonary infection with Legionella.
The
antimicrobial activity of IFN-y after infection with this
pathogen appears to be dependent on the synthesis of the
inducible nitric oxide (NO) synthetase, which is upregulated
by IFN-y and which is obviously responsible for intracellular
killing of the bacterium which prevents systemic spread of
Legionella to virtually all tissues (38).
Resistance to virus infection and disease
Recently, intensive research concerning the resistance to
infection or disease caused by infectious pathogens has been
performed with viruses. This is probably caused by the nature
of the pathogens which - due to their relatively small genomes
- are well characterised on a molecular basis. As a
consequence, unlike the situation with more complex
organisms (e.g., parasites or bacteria), a pathogen-caused
effect can often be reduced to one or few viral gene products,
and thus the 'choice' of transgenic or knockout animal models
is facilitated. A large number of reports on genetic resistance
to viral disease have been based on the analysis of knockout
animals: a small and non-representative collection of this
research will be presented below.
Most reports concerning the effect of targeted knockouts and
their influence on the outcome of disease deal with the role of
different immune cell subsets, of antigen presentation by
either MHC class I or II, and of individual cytokines as
described above for protozoan and bacterial infections. The
use of C D 4 - / - and C D 8 - / - mice and infection with influenza
viruses demonstrated that neither cell type appears to be
required for the clearance of the virus at the effector stage
(22). In addition, the same authors could demonstrate by the
use of J-chain knockout mice (which are no longer capable of
forming secretory multimeric immunoglobulin A [IgA]) that
immunity to influenza is not dependent on the presence of
secretory IgA. Mice lacking mature B cells after the deletion of
the Ig p chain also did not alter appreciably the efficiency of
immune responses to influenza viruses, or the generation and
maintenance of helper T cells ( 9 0 ) . In contrast, mice which
are deficient in the key cytokine IL-1 exhibited higher
mortality rates after infection, which were apparently caused
by the inability of these animals to develop fever (50).
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In the well-defined lymphocytic choriomeningitis virus
(LCMV) system, which represents a member of the
Arenaviridae, various reports on the use of knockout animals
and the effects on disease resistance have been published. In
MHC class I-deficient mice, no C D 8 T-cell response is
observed, which in turn leads to an increased expansion of
C D 4 T cells that exhibit cytotoxic activity. Therefore in
wild-type animals, the C D 8 T cells appear to control C D 4
T cells and thus the resistance to disease (92). In addition, by
the depletion of C D 4 T cells, an exhaustion of the immune
system to LCMV could be demonstrated, in that long-term
virus loads were uncontrolled in MHC class Il-knockout mice
- in contrast to the clearance of acute virus loads - which was
at least partially caused by the absence of cytotoxic T
lymphocyte (CTL) memory cells ( 5 9 , 8 8 ) . In the context of
the co-operative effect of different immune cells in LCMV
control, the interaction of CD40 and CD40 ligand (CD40L)
was addressed by the use of CD40 or CD40L knockout mice.
The results demonstrated that the CD40-CD40L interaction is
important in the interaction between B and T cells, but is far
less important in T-cell activation. This is due to a failure of
CD40- and CD40L-deficient animals to perform a switch in Ig
isotypes and to generate antibody to T-cell-dependent
antigens ( 6 4 ) . Furthermore, the presence of C D 8 cells is
absolutely critical in both the acute and chronic clearance of
LCMV infection, as has been demonstrated by the use of
|32-microglobulin knockout mice (5, 5 9 ) . This higher
susceptibility of animals to LCMV is dependent on the
production of IFN-y and subsequent virus clearance by the
induced C D 8 cytotoxic T cells ( 8 9 ) . These results are
supported by the findings of Walsh et al., who showed the
inability of perforin-deficient mice to clear LCMV because
they are unable to eliminate infected cells. This inability to
clear LCMV-infected cells is even potentiated in the absence of
FAS-mediated apoptosis ( 5 6 , 9 5 ) . Thus, two complementary
cytotoxic mechanisms are used in the elimination of LCMV
from infected cells and for the resistance to particularly
long-term infection.
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An important infection of neonates is rotavirus diarrhoea,
which causes damage to the gastrointestinal tract. However,
immunity to rotaviruses is poorly understood. By using
(32-microglobulin knockout mice, researchers demonstrated
that acute virus infection can be cleared in the absence of CTL
responses and that CTL are not necessary for the development
of a sustainable immunity. In contrast, as shown by the use of
B-cell-deficient mice, immune B cells play a major role
(probably by the generation of IgA) in the clearance of acute
rotavirus infection and longevity of immunity (28). Recently,
these findings were corroborated by the same authors and
defined more precisely: using perforin-, FAS-, and
IFN-y-deficient mice, no increased susceptibility to rotavirus
infection was observed, although C D 8 cells appear to be
involved in virus clearance from the body by a mechanism
that is independent from their cytotoxic activity (29).
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Coxsackie viruses, which are members of the genus
Enterovirus of the family Picornaviridae, can cause fatal heart
disease, especially in immunocompromised individuals, as a
result of acute or chronic myocarditis. The question of
whether direct virus effects or immunopathological processes
cause the diseases associated with the virus is unresolved.
Knockout mice which were deficient in C D 4 cells or
|32-microglobulin were shown to be less susceptible to acute
disease. In addition, it could be clearly demonstrated that,
although high virus titres in mouse hearts post infection were
observed after C D 8 cell depletion, these mice recovered
earlier from infection and death rates were markedly reduced
(39): the same results were seen in mice with a targeted
deletion of the T-cell tyrosine kinase ( 5 3 ) . Thus, Coxsackie
virus damage to the heart appears to be largely dependent on
immunopathological
mechanisms, although
following
knockout of the IFN-y receptor, a high susceptibility to acute
infection, which is caused by the absence of any inducible NO
synthetase - a prerequisite for clearance of intracellular
pathogens, especially in antigen-presenting cells - could be
observed (53).
+
+
Some information on HIV-1 pathogenesis and immunity has
been obtained using knockout mice and murine leukaemia
viruses as a model virus-host system for human infection. In
the absence of B cells, initial virus replication was diminished.
This reduction in virus replication is probably due to the
absence of the primary target cells, which consequently leads
to a reduced virus burden and T-cell and macrophage
infection ( 4 9 , 7 6 ) . Further work identified C D 8 cells as the
main effector cells in the resistance to mouse acquired
immune deficiency syndrome (AIDS), which is at least
partially dependent on perforin function. However, in both
^-microglobulin and perforin knockout mice, no
progression to lymphoproliferation was observed, thus
suggesting that other mechanisms besides C D 8 T-celldependent cytotoxicity are involved in resistance of the
animals to the retrovirus (84). Recently, the myeloencephalopathy which is caused by murine leukaemia virus, and which
is characterised by a paralysis and neurodegenerative disease
indistinguishable from that caused by prions, has been shown
to be independent of the absence or presence of the P r P
protein. These results suggest that, although clinically and
histologically similar diseases are observed after infection with
either prions or murine leukaemia virus, the two agents act in
different ways. Some scientists have speculated that murine
leukaemia virus might use the same pathway as prions but
interfere at a point downstream of the P r P (45).
+
+
c
c
A relatively large number of reports on the use of knockout
animals deals with the pathogenesis of and immunity to
herpes simplex virus type 1 (HSV-1). Despite the intensive
and thorough characterisation of this virus, the pathogenesis
of herpetic lesions, latency and the short duration of
immunity are less well defined. Using knockout animals and a
murine model of HSV-1 infection, some revelations on the
cytokines and the subsets of immune cells which are involved
Rev. sci. tech. Off. int. Epiz., 17 (1)
358
in the control of infection have been obtained. Results
demonstrated that in the absence of C D 4 cells - in contrast to
the absence of C D 8 cells - a marked increase in susceptibility
to herpetic lesions was evident (57). These results confirmed
and extended previous observations that cytotoxic C D 4 cells
are primarily responsible for the control of HSV-1, especially
in neuronal tissues. However, the absence of TFN-y, which
was drought to play a major role in the pathogenesis of ocular
lesions, did not alter the disease appreciably. The findings
showed that IFN-y is involved in virus clearance but that
other cytokines and immune responses are also functionally
active in HSV-l-caused disease ( 1 1 , 1 0 6 ) . Nonetheless,
IFN-y-deficient mice were able to develop sustainable
anti-HSV-l immune responses (106), which agrees with the
complex cascades that orchestrate anti-HSV-1 immunity.
+
+
+
elimination have been elucidated. Tumourigenesis has been
shown to be essentially dependent on cell cycle progression
and on the failure of apoptotic mechanisms induced by
several independent pathways. In addition, the suspected
involvement of the double-stranded RNA-dependent protein
kinase induced by type I interferons in tumour suppression
could not be confirmed (77, 7 8 , 1 0 4 ) .
Targeted mutagenesis of genes in species other
than the mouse
Although a number of ES cell-like cell lines have been
reported in species other than the mouse, successful germ-line
transmission of these cells has not been proven for any of
these species (2, 3 1 ) . However, recent progress in cloning
Although the use of knockout animals in carcinogenesis
studies is widely accepted and has improved the
understanding of different effector and immune mechanisms,
few reports on virus-induced cancer and the use of knockout
models are available. Mouse mammary tumour virus
(MMTV)-ras transgenic animals which lack the tumour
suppressor p53 were affected with tumours earlier than
control mice. Using this model, it could be demonstrated that
p53 can act in an apoptosis-independent manner in the
elimination of tumour cells ( 4 1 ) . The absence of C D 4 or
C D 8 cells led to a higher incidence of polyomavirus-induced
tumours, which was even higher in C D 4 C D 8 double
knockout animals (7, 8 ) , thus indicating that both classes of
immune T cells play important and interdependent roles in
the control of virus infection and tumourigenesis. By using
different knockout animals and models of tumour induction
by viruses or viral gene products, several mechanisms
underlying the progression of tumour cells or their
+
+
+
+
animals by nuclear transfer offers new strategies for targeted
mutagenesis of genes in livestock species. Potential practical
uses of nuclear transfer in livestock species include both the
multiplication of embryos produced from selected matings
and the ability to produce offspring from cells which can be
maintained in culture prior to embryo reconstruction, thereby
providing a route for precise genetic modification.
The latter approach appears to be feasible in the future, as live
offspring have been obtained from sheep following nuclear
transfer from cell lines of embryonic (18, 98), foetal and even
adult origin ( 9 9 ) (Fig. 5 ) . Whether this technology can be
transferred to other species remains to be shown: however,
successful cloning in calves using cultured cells from the inner
cell mass of blastocysts as nuclear donors seems promising
(26). These attempts may provide the possibility to generate
livestock with resistance to different diseases.
Fig. 5
Diagram showing the cloning of embryos by nuclear transfer using embryonic cells or cell lines established from embryonic, foetal or adult tissues
Rev. sci. tech. Off. int. Epiz., 17 (1)
359
Fig. 6
Depiction of the systematic screening for new genes and their functions after ethylnitrosourea mutagenesis of mice
Complementary approaches for
the discovery of genes relevant
for infectious diseases
- ENU induces
mutations.
The mutants
gain-of-function and complete loss-of-function mutants can
also be expected.
The
The most important tool to obtain insight into the biological
function of genes is the use of mutants. The use of ES cells and
homologous recombination allow the systematic production
of mouse mutants for any gene which has been cloned.
Complementary to such a 'gene-driven' approach, 'phenotypedriven' approaches will be necessary to identify new genes or
gene products through a search for new mutants with specific
defects, for example in immune function and resistance to
infectious diseases.
mainly point
produced will therefore be mainly hypomorphic, although
systematic production and analysis of ENU mutants
involves a number of different steps (Fig. 6 ) . F males of an
0
inbred strain are treated with ENU and, after a transient
period of sterility, are bred with females of the same genetic
background to produce F
1
pedigrees. F
x
and F
3
offspring and subsequently F
3
mice are scored phenotypically for
dominant and recessive mutations, respectively. Once a
mutant has been confirmed phenotypically and genetically,
the chromosomal location of the mutation can be mapped
using mice from an outcross/backcross panel produced with
another inbred strain by genome-wide microsatellite-typing of
Mutagenesis
of
mice
using
the
alcylating
agent
ethylnitrosourea (ENU) is a powerful approach for the
systematic production of mouse mutants for a number of
reasons, as follows (6);
- Protocols are available which allow a very efficient
mutagenesis rate in mice. The frequency of mutant recovery is
about 1/1,000 for a specific locus which can be scored
phenotypically, although strain as well as dosage and
treatment regimens do influence the mutagenesis rate.
'mice showing versus mice not showing' the particular
phenotype. Mutations induced by ENU will not be tagged
molecularly. Although this is initially a disadvantage with
respect to the cloning of the responsible genes, the availability
of point mutations will be very important for a more detailed
functional analysis. Furthermore, the advances that
are
currently made in the field of genomics, particularly the
production of high resolution genetic, physical and transcript
maps will reduce the difficulties inherent in the cloning of the
genes mutated after ENU treatment.
- ENU mutagenises premeiotic spermatogonia of F males
which can be bred to produce a large number of F offspring
or F pedigrees, respectively.
0
L
3
•
Rev. sci. tech. Off. int. Epiz., 17 (1)
360
Les leçons de l'invalidation de gènes
N. Osterrieder & E. W o l f
Résumé
Les a u t e u r s d é c r i v e n t la t e c h n i q u e d e r e c o m b i n a i s o n h o m o l o g u e a p p l i q u é e a u x
c e l l u l e s s o u c h e s e m b r y o n n a i r e s , t r è s utilisée a u j o u r d ' h u i p o u r o b t e n i r d e s souris
« k n o c k o u t » d o n t c e r t a i n s g è n e s s p é c i f i q u e s o n t é t é é l i m i n é s . Ils p r é s e n t e n t une
s y n t h è s e d e s c o n n a i s s a n c e s sur la p a t h o g é n i e d e s i n f e c t i o n s p a r a s i t a i r e s ,
b a c t é r i e n n e s ou v i r a l e s , qui se sont multipliées c e s d e r n i è r e s a n n é e s , à partir de
r e c h e r c h e s e f f e c t u é e s a v e c d e s souris « k n o c k o u t » , e t sur la r é s i s t a n c e à c e s
i n f e c t i o n s . Ils m e t t e n t n o t a m m e n t l ' a c c e n t s u r l e s a n i m a u x « k n o c k o u t »
d é p o u r v u s d e c e r t a i n e s c o m p o s a n t e s du r é s e a u d e s c y t o k i n e s e t d e c e r t a i n s
g è n e s i m p o r t a n t s pour u n e b o n n e p r é s e n t a t i o n d e s a n t i g è n e s o u d o n t les
d i f f é r e n t e s s o u s - p o p u l a t i o n s de c e l l u l e s du s y s t è m e i m m u n i t a i r e p r é s e n t e n t d e s
d é f i c i e n c e s . Les a u t e u r s p r é s e n t e n t é g a l e m e n t un bref e x p o s é s u r les possibilités
d'induire d e s d é l é t i o n s ou d e s m u t a t i o n s c i b l é e s d a n s l e s g è n e s d ' e s p è c e s
a n i m a l e s ( p a r e x e m p l e , p a r t r a n s f e r t n u c l é a i r e ou p a r m u t a g e n è s e à l'aide de
l'alkylant N - e t h y l - N - n i t r o s o u r é e ) qui p o u r r a i e n t p e r m e t t r e d e s é l e c t i o n n e r , à
l'avenir, d e s a n i m a u x g é n é t i q u e m e n t r é s i s t a n t s a u x m a l a d i e s i n f e c t i e u s e s .
Mots-clés
Bactéries -
Bovins -
Cytokines -
Génétique -
Mutagenèse -
Parasites -
Souris
« knockout » - Virus.
Enseñanzas de las supresiones específicas de genes
(gene knockouts)
N. Osterrieder & E. W o l f
«
Resumen
Los a u t o r e s d e s c r i b e n la t é c n i c a p a r a a p l i c a r la r e c o m b i n a c i ó n h o m ó l o g a en
células primordiales embrionarias, actualmente
genética
para
crear
e s p e c í f i c a s (knockouts)
ratones
portadores
de
m u y utilizada e n i n g e n i e r í a
determinadas
supresiones
d e g e n e s . El artículo c o n t i e n e un r e s u m e n d e los
c o n o c i m i e n t o s s o b r e la p a t o g é n e s i s d e las i n f e c c i o n e s p a r a s i t a r i a s , b a c t e r i a n a s
o v í r i c a s y la r e s i s t e n c i a a t a l e s i n f e c c i o n e s q u e h a n ido d e p a r a n d o e n los últimos
a ñ o s las i n v e s t i g a c i o n e s c o n r a t o n e s m u t a n t e s ( r a t o n e s knockout).
Se hace
e s p e c i a l h i n c a p i é en los m u t a n t e s d e s p r o v i s t o s d e a l g ú n c o m p o n e n t e d e la r e d de
c i t o q u i n a s o d e g e n e s c r u c i a l e s p a r a la c o r r e c t a p r e s e n t a c i ó n d e a n t í g e n o s , y en
los q u e s o n d e f i c i e n t e s en a l g u n a s s u b c l a s e s d e c é l u l a s d e l s i s t e m a i n m u n i t a r i o .
Se c o m e n t a n a d e m á s s u c i n t a m e n t e las p o s i b i l i d a d e s d e inducir d e l e c i o n e s o
m u t a c i o n e s p r e d e f i n i d a s e n d e t e r m i n a d o s g e n e s d e e s p e c i e s g a n a d e r a s (por
transferencia
nuclear
N-etil-N-nitrosourea,
o
por m u t a g é n e s i s
mediante
el
agente
alquilante
por e j e m p l o ) , lo q u e haría posible en un f u t u r o la cría
selectiva de animales resistentes a e n f e r m e d a d e s infecciosas.
Palabras clave
Bacterias - Citoquinas - Ganado - Genética - M u t a g é n e s i s - Parásitos «knockout»-Virus.
Ratones
361
Rev. sci. tech. Off. int. Epiz., 17(1)
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