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SID 5

Research Project Final Report
Note
In line with the Freedom of Information
Act 2000, Defra aims to place the results
of its completed research projects in the
public domain wherever possible. The
SID 5 (Research Project Final Report) is
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research in a format that is easily
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SID 5 must be completed for all projects.
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SID 5 (Rev. 3/06)
Project identification
1.
Defra Project code
2.
Project title
SE1753
PREPARATION OF MORE SENSITIVE BIOASSAY
MODELS FOR THE IMPROVED DETECTION,
DIFFERENTIATION AND DIAGNOSIS OF THE BSE
AGENT
3.
Contractor
organisation(s)
VETERINARY LABORATORIES
AGENCY
NEW HAW, ADDLESTONE
SURREY KT15 3NB
54. Total Defra project costs
(agreed fixed price)
5. Project:
Page 1 of 20
£
2,029,615
start date ................
01/04/1999
end date .................
31/03/2009
6. It is Defra’s intention to publish this form.
Please confirm your agreement to do so. ................................................................................... YES
NO
(a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They
should be written in a clear and concise manner and represent a full account of the research project
which someone not closely associated with the project can follow.
Defra recognises that in a small minority of cases there may be information, such as intellectual property
or commercially confidential data, used in or generated by the research project, which should not be
disclosed. In these cases, such information should be detailed in a separate annex (not to be published)
so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report
without including references to any sensitive or confidential data, the information should be included and
section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No"
answer.
In all cases, reasons for withholding information must be fully in line with exemptions under the
Environmental Information Regulations or the Freedom of Information Act 2000.
(b) If you have answered NO, please explain why the Final report should not be released into public domain
Please note: This revised version of the SE1753 Research Project Final Report is being presented to
Defra for posting on the Defra website instead of the fuller, more detailed Final Report sent earlier (in
March 2009 and amended in June 2009), so that the full findings of the study (including methods, data,
results and analysis) are not presented on a publically available website prior to the submission and
publication of peer-reviewed scientific papers detailing the findings. This is due to the likelihood that
public disclosure of the results would jeopardise the publication of the findings in scientific journals.
Executive Summary
7.
The executive summary must not exceed 2 sides in total of A4 and should be understandable to the
intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together
with any other significant events and options for new work.
Transmissible spongiform encephalopathy (TSE) or prion diseases are a group of fatal neurodegenerative
diseases affecting man and animals, and include scrapie in sheep, bovine spongiform encephalopathy
(BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. The fundamental feature in the
pathogenesis of the TSE diseases, as proposed in the ‘protein only’ hypothesis is the posttranslational
conformational change of the host-encoded cellular prion protein (PrPC) of unknown function, to a diseaseassociated, partially protease resistant isoform (PrPSc), which accumulates in the brains and other tissues
of affected hosts.
Bioassay of TSE diseases in animal hosts is regarded as the gold standard method for the detection of
TSE infectivity and for the determination of TSE infectious titres by clinical end-point titration. However,
bioassays of TSE agents such as BSE and scrapie in natural hosts, i.e. cattle and sheep, are expensive
and time-consuming studies to conduct and are not suited to large-scale investigations. Transmission of
TSE agents to alternative animals models, such as conventional wild-type mice, may be affected by
species or transmission barriers due to a lack of homology between host PrP sequence and the infecting
TSE agents, leading to reduced transmission efficiencies and extended incubation times. As an example,
bioassay of BSE in wild-type RIII mice revealed an underestimation of BSE infectivity titres of ~1000-fold
in comparison to cattle.
Transgenic (Tg) mouse models expressing PrPs from other species, in the absence of endogenous
mouse PrP expression, can overcome the reduced efficiency of transmission of TSE agents from one
animal species to another. The generation of such models offers the prospect of faster, more economically
viable bioassays, which may be more sensitive to TSE disease detection than the natural host. Abrogation
of the species or transmission barrier was demonstrated in Tg mice overexpressing Syrian hamster PrP.
In these mice, the length of the prion incubation time was inversely proportional to the transgene
expression level in brain. Subsequently, Tg mouse models expressing PrP from other mammalian
species, including bovine, ovine and human, have been reported and have demonstrated different
susceptibilities and biochemical and neuropathological features in response to TSE infections.
Transgenetic approaches have contributed greatly to the improved understanding of TSE diseases and
provided major insights into the biology of these diseases, including the crucial role of PrP in disease
pathogenesis, the importance of PrP amino acid sequence in controlling disease susceptibility, the
relationship between PrPC expression and disease incubation times, the biochemical and genetic basis of
SID 5 (Rev. 3/06)
Page 2 of 20
species or transmission barrier effects, and the molecular basis of TSE strains.
This project, SE1753, represents a Defra-funded research project aimed at the generation and
assessment of transgenic PrP mouse models for the improved detection, differentiation and diagnosis of
ruminant TSE agents. The rationale for developing the project was to produce transgenic PrP mice that
could offer improved bioassay models of greater susceptibility and sensitivity to ruminant TSE agents
compared to conventional wild-type mouse lines. The aims were to assess the susceptibilities and
efficiencies of the generated transgenic PrP mouse models, overexpressing bovine, kudu and sheep PrPs,
for the improved detection of BSE, classical scrapie and atypical scrapie infectivity in transmission studies.
Potentially, such transgenic PrP models could provide improved bioassay models as alternatives to wildtype mouse lines and contribute to a reduction or replacement of such mice in TSE bioassay studies, as
well as contribute to a reduction in the economic costs and long disease incubation times associated with
conventional bioassay. The overall aims of the project, i.e. the generation of transgenic PrP mouse models
with demonstrated high susceptibilities to and reduced incubation times with TSE diseases compared to
conventional wild-type mouse lines were achieved.
Transgenic Prnp constructs were prepared as full-length bovine PrP, or as chimaeric Prnp genes
containing ovine and kudu PrP sequence flanked by mouse sequence, which were constructed in
overlapping PCR assays and cloned into a plasmid vector. Purified transgenes (coding for bovine, kudu
and sheep PrPs) were microinjected into fertilised oocytes of wild-type mice and the subsequent progeny
were PCR screened for the presence of transgenic DNA. Founder mice, identified as carrying the
transgenes, were bred with PrP null mice, which do not express mouse PrP, in order to transfer the
transgenes to a PrP ablated background. Subsequently, stable transgene-transmitting mouse lines
expressing bovine PrP, kudu PrP and ovine PrPAHQ and ovine PrPARR were produced, which were
characterised for transgene expression levels in brain (and other tissues) and for transgene copy
numbers. In the case of the ovine PrPAHQ mice, AHQ refers to amino acid residues alanine, histidine and
glutamine at codons 136, 154 and 171, respectively, and in ovine PrPARR mice, ARR refers to alanine,
arginine and arginine at the same positions. Where feasible, the generated Tg lines were bred to
transgene homozygosity for evaluation of disease susceptibility in TSE challenge studies.
Cryopreservation and test recovery of 11 of the generated Tg lines is in progress and will continue until
sufficient numbers of embryos from each line have been securely stored.
The breeding, screening and subsequent TSE challenges of Tg mouse lines were conducted in
accordance with the legal and ethical regulations of the Project Licence and in respect of review by the
local Ethical Review Process. Transgenic mice were monitored for signs of spontaneous disease or other
clinical signs due to intercurrent disease. Mice were observed daily, and clinically assessed on a weekly
basis, or more frequently if signs developed. Mice that developed adverse signs were euthanised on the
basis of veterinary advice and welfare considerations. One of the Tg(BoPrP) mouse lines, expressing a
high level of bovine PrP, was shown to develop a spontaneous neurological disease in young mice. The
most likely explanation was that the observed spontaneous degeneration was caused by the
overexpression of wild-type bovine PrPC protein.
TSE transmission studies were carried out in Tg(BoPrP), Tg(KuPrP), Tg(OvPrPAHQ) and Tg(OvPrPARR)
mouse lines, bred to transgene homozygous or hemizygous status on PrP null backgrounds, with a range
of TSE agents, including BSE, classical scrapie, atypical scrapie. Following challenge with TSE agents,
mice demonstrating fully developed clinical signs of disease were euthanised and brain and other tissues
were collected for confirmatory biochemical analysis, and for neuropathological examination of selected
cases. Full details of the results obtained in these studies will be presented in peer-reviewed scientific
journals.
The Tg(BoPrP) mice demonstrated wide susceptibility to BSE and scrapie infection as well as the capacity
to differentiate these TSE agents by biochemical and neuropathological means. The results also
suggested that the Tg(OvPrPAHQ) model may represent a highly appropriate bioassay model for detection
of atypical scrapie infectivity, not only due to its high susceptibility and rapid incubation times but also
because of its potential sensitivity. The model could be applied to the detection of atypical scrapie
infectivity in the tissues of experimentally infected and naturally infected sheep and goats. Tg(OvPrP AHQ)
mice were also susceptible to infection with classical scrapie and BSE.
In contrast, TSE challenges of Tg(KuPrP) mice demonstrated lower susceptibilities to scrapie and BSE
infection than Tg(BoPrP) mice. The majority of TSE-challenged Tg(OvPrPARR) mice were found to
demonstrate extended incubation periods or non-transmission with a range of TSE agents.
The overall aims of the project have been successfully achieved by the generation of transgenic PrP
mouse lines with demonstrated high susceptibilities and reduced incubation times in challenges with a
wide range of TSE agents. In particular, the Tg(BoPrP) and Tg(OvPrPAHQ) mouse models represent more
SID 5 (Rev. 3/06)
Page 3 of 20
susceptible and faster bioassay models (of potentially greater sensitivity) for BSE, scrapie and atypical
scrapie compared to wild-type mouse lines, and these mice offer the prospect of and could be applied to
the improved bioassay detection and differentiation of ruminant TSE diseases.
Specifically, the Tg(BoPrP) and Tg(OvPrPAHQ) mouse models produced could be applied to: investigating
naturally occurring and experimental TSE infections, the study and resolution of complex issues such as
mixed TSE infections, the genetics of susceptibility and resistance of sheep PrP genotypes to classical
scrapie and/or atypical scrapie infection in sheep, detection of low level and/or sub-clinical TSE infections,
detecting TSE infectivity in peripheral tissues of affected hosts, detecting BSE infection in sheep, and to
improved TSE strain differentiation including in atypical or unusual cases in ruminants and other species.
Potentially, the generated transgenic models could identify new risk materials for human and animal
health, which could lead to the implementation of new disease control measures for TSEs.
In addition, the generated transgenic PrP models will also contribute to the development of new projects
aimed at the improved differentiation and characterisation of TSE agents and in the evaluation of
transgenic PrP models with standardised TSE inocula. Furthermore, additional opportunities for the
collaborative use of the transgenic PrP mouse lines generated in this project will be sought.
Project Report to Defra
8.
As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with
details of the outputs of the research project for internal purposes; to meet the terms of the contract; and
to allow Defra to publish details of the outputs to meet Environmental Information Regulation or
Freedom of Information obligations. This short report to Defra does not preclude contractors from also
seeking to publish a full, formal scientific report/paper in an appropriate scientific or other
journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms.
The report to Defra should include:
 the scientific objectives as set out in the contract;
 the extent to which the objectives set out in the contract have been met;
 details of methods used and the results obtained, including statistical analysis (if appropriate);
 a discussion of the results and their reliability;
 the main implications of the findings;
 possible future work; and
 any action resulting from the research (e.g. IP, Knowledge Transfer).
1. INTRODUCTION.
Transmissible spongiform encephalopathy (TSE) or prion diseases are a group of fatal
neurodegenerative diseases affecting man and animals, and include scrapie in sheep, bovine
spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. The
fundamental feature in the pathogenesis of the TSE diseases, as proposed in the ‘protein only’
hypothesis (Griffith, 1967; Prusiner, 1991) is the posttranslational conformational change of the hostencoded cellular prion protein (PrPC) of unknown function, to a disease-associated, partially protease
SID 5 (Rev. 3/06)
Page 4 of 20
resistant isoform (PrPSc), which accumulates in the brains (DeArmond et al., 1987) and other tissues
(Bosque et al., 2002) of affected hosts.
Bioassay of TSE diseases in animal hosts is regarded as the gold standard method for the detection of
TSE infectivity and for the determination of TSE infectious titres by clinical end-point titration. However,
bioassays of TSE agents such as BSE and scrapie in natural hosts, i.e. cattle and sheep, are
expensive and time-consuming studies to conduct and are not suited to large-scale investigations.
Transmission of TSE agents to alternative animals models, such as conventional wild-type mice, may
be affected by species or transmission barriers due to a lack of homology between host PrP sequence
and the infecting TSE agents, leading to reduced transmission efficiencies and extended incubation
times. As an example, bioassay of BSE in wild-type RIII mice revealed an underestimation of BSE
infectivity titres of ~1000-fold in comparison to cattle (Wells et al., 1998).
The preparation of transgenic (Tg) mouse models expressing PrPs from other species, in the absence
of endogenous mouse PrP expression, can provide improved disease models for TSE diseases by
overcoming the reduced efficiency of transmission of TSE agents from one animal species to another.
The generation of such models offers the prospect of faster, more economically viable bioassays,
which may be more sensitive to TSE disease detection than the natural host. Abrogation of the species
or transmission barrier was first demonstrated in Tg mice overexpressing Syrian hamster PrP (Scott et
al., 1989). In these mice, the length of the prion incubation time was inversely proportional to the
transgene expression level in brain. Subsequently, Tg mouse models expressing PrP from other
mammalian species, including bovine (Scott et al., 1997; Buschmann et al., 2000; Castilla et al., 2003),
ovine (Vilotte et al., 2001; Crozet et al., 2001) and human (Telling et al., 1995; Korth et al., 2003) PrPs,
have been reported and have demonstrated different susceptibilities and biochemical and
neuropathological features in response to TSE infections.
These and other transgenetic approaches have contributed greatly to the improved understanding of
TSE diseases and provided major insights into the biology of these diseases, including the crucial role
of PrP in disease pathogenesis (Prusiner, 1992; Büeler et al., 1993), the importance of PrP amino acid
sequence in controlling disease susceptibility, the relationship between PrPC expression and disease
incubation times, the biochemical and genetic basis of species or transmission barrier effects (Scott et
al., 1989), and the molecular basis of TSE strains.
This project, SE1753, represents a Defra-funded research project aimed at the generation and
assessment of transgenic PrP mouse models for the improved detection, differentiation and diagnosis
of ruminant TSE agents. The rationale for developing the project was to produce transgenic PrP mice
that could offer improved bioassay models of greater susceptibility and sensitivity to ruminant TSE
agents compared to conventional wild-type mouse lines. The aims were to assess the susceptibilities
and efficiencies of the generated transgenic PrP mouse models, overexpressing bovine, kudu and
sheep PrPs, for the improved detection of BSE, classical scrapie and atypical scrapie infectivity in
transmission studies. Potentially, such transgenic PrP models could provide improved bioassay models
as alternatives to wild-type mouse lines and contribute to a reduction or replacement of such mice in
TSE bioassay studies, as well as contribute to a reduction in the economic costs and long disease
incubation times associated with conventional bioassay.
The overall objectives of the project, as described in this report and indicated below, were achieved:
01.
02.
03.
04.
05.
Preparation of transgenic DNA constructs.
Microinjection of transgenic DNA constructs.
Screening and characterisation of transgenic mice.
Challenge of transgenic mouse lines with TSE agents and analysis of mice and results.
Completion and submission of annual and final project reports.
In addition, project SE1753 has allowed cooperation between our transgenic research team at VLA
(Weybridge) with two other research institutes, namely, the Friedrich-Loeffler Institute [(FLI); Federal
Research Centre for Viral Disease of Animals (FRCVDA), Greifswald-Insel Riems, Germany] and the
Institute National de la Recherche Agronomique (INRA, Jouy-en-Josas, France).
SID 5 (Rev. 3/06)
Page 5 of 20
2. MATERIALS AND METHODS.
Full details of the materials and methods used in these studies will be published in peer-reviewed
scientific papers, which are in preparation. Briefer details of the procedures used are presented in this
report.
2.1. Construction of Prnp transgenes.
In this project, a number of transgenic Prnp constructs were prepared:
 murine-bovine chimaeric Prnp genes with bovine 6 octapeptide repeats (bovine 6OR) and
bovine 5OR;
 a full-length bovine Prnp gene from a bacterial artificial chromosome (BAC) library clone;
 murine-kudu chimaeric Prnp genes with kudu 6OR and kudu 5OR;
 a murine-ovine PrPAHQ chimaeric Prnp gene containing alanine (A), histidine (H) and glutamine
(Q) at amino acid (AA) residues 136, 154 and 171
 a murine-ovine PrPARR chimaeric Prnp gene containing alanine (A), arginine (R) and arginine
(R) at AA residues 136, 154 and 171.
2.1.1. Preparation of chimaeric Prnp transgenes.
In the preparation of the murine-bovine, murine-kudu and murine-ovine Prnp transgenes, genomic DNA
was extracted from blood (bovine 6OR and 5OR; ovine PrPAHQ; ovine PrPARR), placenta (kudu 5OR) or
prepared synthetically (kudu 6OR). Extracted DNA was used as a template to amplify Prnp gene
sequences from bovine, ovine and kudu material and 3' untranslated region sequence from mouse, as
described previously (Buschmann et al., 2000). Chimaeric transgene sequences were generated by
overlapping PCR.
Due to a lack of appropriate kudu field material, chimaeric murine-kudu 6OR sequence was generated
synthetically (by a commercial company) and used in the preparation of the kudu 6OR transgene.
To prepare transgenes, chimaeric or synthetic DNA fragments were cloned into restriction enzymedigested half-genomic vector (phgPrP; Fischer et al., 1996). Recombinant plasmids containing the
correct sequences were expanded and purified. Transgene sequences were recovered by restriction
enzyme-digestion and purified for microinjection procedures.
2.1.2. Preparation of full-length bovine Prnp transgene.
The source material for the PrP transgene was a bovine bacterial artificial chromosome (BAC) clone
containing bovine DNA sequence, encompassing the bovine Prnp gene locus (Hills et al., 2001).
Following restriction enzyme digestion and purification procedures, a DNA fragment was recovered and
used as the full-length bovine Prnp transgene
2.2. Generation of transgenic PrP mice by pronuclear microinjection of Prnp gene constructs.
Microinjection of fertilised wild-type mouse oocytes was carried out for preparations of 6 different PrP
transgenes (murine-bovine 6OR; bovine full-length PrP; murine-kudu 6OR; murine-kudu 5OR; murineovine PrPAHQ; murine-ovine PrPARR) by arrangement with external institutes and commercial facilities.
2.3. Screening of transgenic PrP mouse lines mice.
Founder mice (Tg+/- Prnp+/+) identified by PCR at VLA were sent to VLA from the microinjection facilities
and bred with PrP null mice (Zurich I; Tg-/- Prnp0/0; Bueler et al., 1992) to establish hemizygous (Tg+/Prnp0/0; TgHE) and homozygous (Tg+/+ Prnp0/0; TgHO) transgene expression on a mouse PrP-ablated
background. PCR screening assays for the presence of the transgene, PrP null status (Bueler et al.,
1992) and for the absence of wild-type mouse Prnp (Büeler et al., 1992) were applied to the
subsequent progeny of transgenic mouse breeding colonies.
2.4. Breeding and maintenance of transgenic mouse lines.
As indicated, founder mice were bred with PrP null mice to produce F1 and F2 generations, with the
aim of breeding transgene hemizygous mice to transgene homozygosity. The breeding, screening and
subsequent TSE challenges of transgenic mouse lines were conducted in a containment level 2 facility
under a Home Office Project Licence in accordance with the legal and ethical regulations of the Project
Licence and in respect of review by the VLA Ethical Review Process. Transgenic mice were monitored
SID 5 (Rev. 3/06)
Page 6 of 20
for signs of spontaneous disease or other clinical signs due to intercurrent disease. Mice were
observed daily, and clinically assessed on a weekly basis, or more frequently if signs developed. Mice
that developed adverse signs were euthanised on the basis of veterinary advice and welfare
considerations.
A colony of PrP null mice (i.e. Zurich I Prnp0/0 mice; Bueler et al., 1992; 1993) was established at VLA
from breeding pairs obtained via INRA, with permission granted by the Institute of Molecular Biology,
University of Zurich. The colony was re-derived off-site and introduced into the VLA facility as breeding
stock with a disease-free status. C57BL/6 mice used in this study were obtained from already
established lines at VLA, whereas SJL mice were imported.
2.5. Characterisation of transgenic PrP mouse lines.
2.5.1. Estimation of transgene copy number by Southern hybridisation.
Southern hybridisation analysis was used to confirm transgenic status and estimation of transgene
copy number in genomic DNA extracted from tail samples from transgenic mice. Restriction enzymedigested DNA was electrophoresed, blotted onto PVDF membranes, probed with labelled Prnp gene
probes, and visualised by chemiluminescence. Comparison of transgenic Prnp fragments to normal
bovine or ovine brain Prnp DNA was carried out by densitometry analysis.
2.5.2. Western blotting characterisation of transgenic PrP expression in mice.
Transgene expression levels were examined by Western blotting analysis of brain and peripheral
tissues from mice in comparison to brain from normal bovine and ovine samples. Homogenised
samples were electrophoresed, transferred to PVDF membranes, probed with monoclonal antibodies
and visualised by chemiluminescence. Signals were captured on autoradiographic film or on an image
analyser. Comparative quantification of PrPC was performed by densitometry analysis.
2.6. Breeding of transgenic PrP mouse lines to transgene homozygosity.
To breed Tg mice to transgene homozygosity, pairs of hemizygous Tg sibling mice (Tg +/- Prnp0/0) were
selected and bred to produce, in accordance with classical genetics, a mixture of Tg homozygous (Tg +/+
Prnp0/0; referred to as TgHO), Tg hemizygous (Tg+/- Prnp0/0; referred to as TgHE) and non-transgenic
(Tg-/- Prnp0/0) offspring on a PrP null (Prnp0/0) background. Tg homozygous mice were identified by the
classical approach of backcrossing and by Southern hybridisation.
Backcrossing was used to identify TgHO bovine and kudu PrP mice. However, as backcrossing was
found to be a time-consuming method, identification of TgHO mice expressing the sheep PrP AHQ and
ARR alleles was carried out by Southern hybridisation analysis of gDNA from tail samples. Based on
comparisons to the signals produced by known TgHO and TgHT control samples, potential TgHO mice
were identified.
2.7. TSE transmission studies in transgenic PrP mice.
For transmission experiments, TSE inocula (BSE, classical and atypical scrapie, and ovine BSE) were
prepared as homogenates of bovine and ovine brain material, or from Tg mouse material for passaged
isolates. Groups of transgenic mice (generally 6-10 mice) aged 7-12 weeks old were inoculated under
anaesthesia by a combination of intracerebral (20 μl) and intraperitoneal (100 μl) routes or by the
intracerebral route only. In addition to routine daily health checks, following inoculation TSE challenged
mice were monitored at weekly intervals for the development of clinical signs in accordance with
established guidelines.
Mice demonstrating fully developed clinical signs of disease were euthanised and brain (and other
tissues) were collected for confirmatory analysis. In general, portions of brain were collected and stored
fresh frozen at –80°C for biochemical analysis and further transmission studies, and the remaining
portions were fixed in formalin for neuropathological examination of selected cases.
2.8. Western blotting analysis of transgenic PrP mice challenged with TSEs.
Western blotting detection of PrPres in brain extracts of clinically-affected or unaffected mice challenged
with TSE agents was used to confirm TSE disease. In transmission studies in TgHO mice and wild-type
controls, a Western blotting method adapted from a BioRad protocol was used to detect PrPres in brain
and other tissues.
SID 5 (Rev. 3/06)
Page 7 of 20
2.8.1. Western blotting detection of PrPres in TSE challenged mice.
In the Western blot method adapted from the TeSeE Western blot protocol (Bio-Rad), fresh frozen
brain samples from clinically-affected and unaffected inoculated mice were homogenised to produce
20% (w/v) preparations. In general, brain homogenates were extracted and treated with proteinase K
(PK) according to the manufacturer’s recommendations. For comparative PrPres control purposes,
homogenates of bovine BSE and ovine scrapie brain were treated similarly. Samples were boiled in
equal volumes of 2x SDS reducing buffer at 100°C for 5 min and electrophoresed and immunoblotted.
2.8.2. Deglycosylation of PrPres.
Where indicated, deglycosylation of homogenised, PK-treated and solubilised extracts of mouse brains
was carried out using peptide: N-glycosidase (PNGaseF), in accordance with the manufacturer’s
instructions. Final pellets were resuspended in SDS loading buffer and electrophoresed.
2.9. Neuropathology.
In selected cases, formalin fixed brain samples were examined by histopathological and
immunohistochemical (IHC) methods following paraffin embedding.
2.9.1. Histopathology.
For light microscopic evaluation of TSE-associated vacuolation, coronal sections were cut from 5 brain
regions and stained with haemotoxylin and eosin (H&E).
2.9.2. Immunohistochemistry.
Immunohistochemical (IHC) examination for PrPSc immunostaining was carried out on coronal sections,
similar to above, as described by Wells et al., 2003. Detection of PrPSc specific labelling was used as
the criterion to indicate a positive result.
2.10. Cryopreservation of transgenic PrP mouse lines.
The aim was to cryopreserve fertilised embryos from eleven of the SE1753-generated transgenic PrP
lines. Mice from each line were sent to a commercial facility for further breeding and superovulation.
Pregnant female mice at ~2.5 days gestation were sent to the Fertilised Embryo and Sperm Archive
facility for embryo harvest and cryopreservation. The process was repeated with the aim of cryostorage
of ~300 embryos per line. Following a period in storage, viability testing was conducted on a small
number of embryos. The resulting progeny mice were screened by PCR at VLA to assess transgenic
status. Cryopreservation of individual transgenic mouse lines was deemed to be satisfactory following
PCR confirmation of recovered mice and storage of a sufficient number of good quality embryos per
line.
3. RESULTS.
Full presentation of the results of these studies will be published in peer-reviewed scientific papers,
which are in preparation. A briefer outline of the results is presented in this report.
3.1. Preparation of transgene constructs and generation of transgenic PrP mouse lines.
In this project, Prnp transgenes based on full-length bovine PrP, bovine PrP with 6 octarepeats (bovine
6OR), bovine 5OR, kudu 6OR, kudu 5OR, ovine PrPAHQ and ovine PrPARR were prepared. Construction
of the chimaeric murine-bovine 6OR, murine-bovine 5OR, murine-kudu 6OR, murine-kudu 5OR,
murine-ovine PrPAHQ and murine-ovine PrPARR transgenes was conducted by use of the phgPrP vector
(Fischer et al., 1996), in which transgene expression was driven by a mouse PrP gene promoter. In
contrast, in the full-length bovine Prnp gene transgene, expression was under the control of the bovine
Prnp gene promoter.
Microinjection of four of the Prnp gene constructs, including repeated microinjections of re-prepared
and re-purified transgene preparations, led to the successful generation of transgenic PrP mouse lines.
These four transgenes (3 chimaeric: murine-kudu 6OR; murine-ovine PrPAHQ; murine-ovine PrPARR; and
the full-length bovine PrP transgene) produced stable transgene-transmitting and transgene-expressing
mouse lines.
SID 5 (Rev. 3/06)
Page 8 of 20
Microinjection of the prepared murine-ovine PrPARR transgene did not produce successful results.
However, an alternative phgPrP plasmid containing ovine PrPARR sequence (which was kindly provided
by INRA) was used to produce transgenic mice expressing ovine PrPARR.
Breeding of the founder lines produced from the four transgenes with PrP null mice (Zurich I; Tg -/Prnp0/0; Bueler et al., 1992) led to the successful establishment of 12 transgene hemizygous mouse
lines (Tg+/- Prnp0/0; TgHE) with expression of bovine PrP, kudu PrP, ovine PrPAHQ and ovine PrPARR on
a Prnp0/0 background.
3.2. Screening and characterisation of transgenic PrP lines.
3.2.1. PCR screening.
Mice produced from the microinjections and from subsequent breeding were screened by PCR for the
presence of the transgenes, PrP null status and wild-type PrP.
3.2.2. Transgene copy number.
Southern hybridisation was used to confirm the presence of transgenic Prnp in DNA extracted from
PCR transgene positive mice. This technique was also used to estimate transgene copy numbers in
founder lines by comparative densitometry in comparison to normal bovine DNA. Copy numbers were
estimated for the 12 transgenic mouse lines.
3.2.3. Expression of transgenic PrP.
Levels of transgene expression in brain and other tissues of bovine, kudu and sheep PrP AHQ and
PrPARR mouse lines were estimated by Western blotting analysis. Estimated expression levels of
transgenic PrP in the brains of hemizygous and homozygous mice, where bred, of 12 mouse lines were
determined. In addition to brain, a range of tissues were collected from different lines and examined by
Western blotting to estimate transgene expression levels.
3.3. Breeding of transgenic PrP lines to transgene homozygosity.
The aim was to produce lines of transgene homozygous mice (Tg +/+ Prnp0/0; TgHO) expressing bovine,
kudu and sheep transgenes for challenge with TSE agents. For each transgene, several mouse lines,
expressing different levels of the transgene, were maintained and bred, so that a selection of the most
suitable lines, based on transgene expression level and the stability of transgene transmission through
breeding could be made. All lines were monitored for the development of deleterious effects, including
the development of spontaneous disease, due to transgene status.
Eight transgenic lines expressing bovine, kudu, sheep PrPAHQ and sheep PrPARR were bred to
homozygosity. Several mouse lines could not be bred to homozygosity, including several higher
expressing lines, and other lines were found not to be stable as homozygous lines. These latter lines
were maintained as hemizygous breeding lines by crossing with PrP null mice.
Bovine and kudu PrP lines were bred to homozygosity following confirmation of the transgene
homozygous status of individual mice through backcrossing with PrP null mice. In the case of the ovine
PrPAHQ and ovine PrPARR mouse lines, Southern hybridisation was used to identify potentially TgHO
mice for breeding.
3.4. Development of spontaneous neurological disease in a Tg(BoPrP) mouse line.
One of the generated transgenic bovine PrP mouse lines demonstrated development of spontaneous
neurological disease in young mice. Following biochemical and neuropathological examination of mice
affected by the disorder, it was concluded that the spontaneous disease was caused by the high level
of overexpression of bovine PrPC.
3.5. Challenge of Tg(BoPrP) and Tg(KuPrP) mouse lines with TSE agents.
In this project, transgene hemizygous (TgHE) and homozygous (TgHO) mice were challenged with
TSE agents (including bovine BSE, classical scrapie and atypical scrapie from sheep). In addition,
C57BL/6 and SJL mice were also inoculated with BSE and scrapie as inbred wild-type controls.
C57BL/6J mice were selected as one of the representative lines used at VLA, and one previously used
with several BSE and scrapie inocula. As an alternative wild-type line, SJL mice were selected for BSE
and scrapie challenge as controls on the basis that the line was reported to show reduced incubation
SID 5 (Rev. 3/06)
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times with BSE (Asante et al., 2002). Both C57BL/6 and SJL mouse lines have the same Prnp gene
coding sequence (Prnpa).
3.5.1. Challenge of Tg(BoPrP) and Tg(KuPrP) mouse lines with TSE agents.
Where homozygous transgenic PrP mice were produced, and in other lines maintained as hemizygous,
a wide range of inoculations were carried out with different TSE agents. The majority of challenges
were primary transmissions, but a small number of secondary transmissions were also conducted to
assess the extent of potential transmission barriers.
Differences in the incubation periods of TSE agents, such as BSE, were seen between the different
Tg(BoPrP) lines produced, which was most probably due to differences in the expression levels in
different mouse lines. Mice with higher expression levels demonstrated shorter mean incubation
periods. The fastest Tg(BoPrP) line demonstrated mean incubation periods that were >200 days faster
with BSE and >300 days faster with classical scrapie (pooled inoculum) compared to wild-type
C57/BL6 mice inoculated with the same TSE agents.
3.5.2. Analysis of Tg(BoPrP) and Tg(KuPrP) mouse lines challenged with TSE agents.
Brain samples from BSE or scrapie infected Tg(BoPrP) and Tg(KuPrP) mice were examined for
detection of PrPres by Western blotting, and in selected cases by deglycosylation of PrPres and by
histological and PrP IHC analysis. In some cases, other tissue samples (such as spleen and hindlimb
muscle) were recovered and examined.
3.5.3.1. Western blotting detection of PrPres in TSE challenged mice.
The PrPres profiles (fragment sizes) produced by BSE infection in Tg(BoPrP) mice were similar to the
bovine brain BSE control with a lower unglycosylated band at 19 kDa. In contrast, the PrPres pattern
produced by a classical scrapie pool inoculum in Tg(BoPrP) mice was similar to the sheep scrapie
control with a lower unglycosylated band at 21 kDa, with a similar finding in Tg(KuPrP) mice. At least
on first passage to bovinised mice, BSE and scrapie agents appeared to retain the same PrPres
glycoform profiles as in the naturally infected host.
Western blotting analysis of brain samples from one line of inoculated Tg(BoPrP) mice revealed that
mean incubation periods of BSE infection were >200 days faster than in C57BL/6 mice. Although BSE
transmissions to SJL mice were slower than to Tg mice, the mean incubation periods were comparable;
however, the attack rates in SJL mice were substantially lower.
Western blotting of tissue samples suggested the apparent detection of PrPres in spleen and hindlimb
muscle samples from BSE-infected Tg(BoPrP) mice. However, the results were not optimal and further
investigations will be required to confirm the detection of PrPres.
3.5.3.2. Deglycosylation of BSE and scrapie PrPres from Tg(BoPrP) mice.
Preliminary studies suggested that BSE and scrapie infection in Tg(BoPrP) mice could be distinguished
by deglycosylation patterns of PrPres, particularly in relation to the molecular mass positions of
unglycosylated bands, although this potential effect requires further validation.
Further investigations, including the use of different antibodies to characterise deglycosylated PrPres
fragments, could reveal whether the characteristics of inoculated TSE agents are retained or altered on
first and subsequent passage in transgenic mice.
3.5.3.3. Neuropathological analysis of BSE and scrapie infected Tg(BoPrP) mice.
Brain sections from Tg(BoPrP) mice and wild-type C57BL/6 mice infected with BSE were examined
histologically by H&E staining to reveal vacuolation. IHC analysis demonstrated differences in PrPSc
deposition in brain areas of Tg(BoPrP) mice compared to C57BL/6 mice. Differences in PrP Sc
distribution patterns were also seen between BSE and classical scrapie infection in Tg(BoPrP) mice. It
was concluded that in Tg(BoPrP) mice, differences in PrPSc types and their distribution patterns could
be used to identify different TSE sources / agents.
3.6. Challenge of Tg(OvPrPAHQ) and Tg(OvPrPARR) mouse lines with TSE agents.
Hemizygous and/or homozygous lines of Tg(OvPrPAHQ) and Tg(OvPrPARR) mice were challenged with a
range of TSE agents. Lines of each Tg(OvPrP) model were challenged with isolates, including atypical
SID 5 (Rev. 3/06)
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scrapie, classical scrapie and BSE. The majority of challenges were primary transmissions. Mice were
analysed by Western blotting detection of PrPres and in selected cases by neuropathological analysis.
3.6.1. Transmission of TSE agents to Tg(OvPrPAHQ) and Tg(OvPrPARR) mouse lines.
In the primary transmissions of atypical scrapie isolates to the Tg(OvPrPAHQ) mouse model, one of the
transgenic mouse lines demonstrated high susceptibility to the disease with all inoculated mice
developing symptoms of progressive neurological disease in relatively uniform mean incubation
periods.
Transmissions of the classical scrapie pool to Tg(OvPrPAHQ) mice also reflected differences in
expression levels between transgenic lines, with faster incubation periods in mice expressing at a
higher level. Incubation times of the classical scrapie pooled inoculum in the fastest Tg(OvPrPAHQ) line
were similar to those in the fastest Tg(BoPrP) line.
In contrast to the highly efficient transmission of the classical scrapie pool, two classical scrapie field
cases (VRQ/VRQ and ARQ/ARQ) demonstrated poor transmission to Tg(OvPrPAHQ) mice. This could
be explained by several factors, including the potentially low infectivity of the field cases compared to
the known high titre of infectivity of the classical scrapie pool, or the incompatibility of PrP genotypes of
the inocula and the recipient mice.
As found with the Tg(BoPrP) mouse lines, differences were seen in the mean incubation periods of
Tg(OvPrPAHQ) mice in response to TSE infection, depending on the expression level in the Tg mice.
With atypical scrapie infection, the highest expressing Tg(OvPrPAHQ) mouse line, which was transgene
homozygous, showed the shortest mean incubation periods. In contrast, a Tg(OvPrPAHQ) line that was
hemizygous for the transgene produced the longest mean incubation periods on TSE challenge.
Transmissions to Tg(OvPrPARR) mice are on-going, with some inoculated mice still potentially
incubating disease. Based on the results obtained so far, Tg(OvPrPARR) mice show an apparently low
susceptibility to TSE challenges, which could be due to a combination of factors, including a lower
expression level of transgenic PrP in these mouse lines compared to the other transgenic models, and
the potential resistance or low susceptibility of these mouse lines to the TSE inocula used. These
factors are likely to produce long incubation periods in susceptible mice or long survival times in
resistant mice. The full extent of the resistance or susceptibility of the Tg(OvPrPARR) mouse lines to
TSE agents has not yet been determined.
3.6.2. Analysis of Tg(OvPrPAHQ) and Tg(OvPrPARR) mouse lines challenged with TSE agents.
Brain samples were obtained from Tg(OvPrPAHQ) and Tg(OvPrPARR) mice that were challenged with
TSE agents and were euthanised due to demonstration of clinical (terminal) signs of TSE disease or
due to intercurrent disease or for other welfare reasons. For confirmation of TSE disease, Western
blotting analysis was carried out to detect PrPres, and in selected cases neuropathological analyses
were conducted.
3.6.2.1. Western blotting detection of PrPres in TSE challenged Tg(OvPrPAHQ) mice.
The transmission of atypical scrapie isolates, derived from sheep with different PrP genotypes, to
Tg(OvPrPAHQ) mice was highly efficient, and particularly to the mouse line with the highest expression
level. Examination of brain samples from affected mice by Western blotting analysis revealed detection
of PrPres. Typically, PrPres profiles consisted of 4 bands similar to those described previously (Benestad
et al., 2003). In particular, the atypical scrapie isolates transmitted to Tg(OvPrPAHQ) mice demonstrated
a prominent low molecular mass band at ~11 kDa, which was absent from classical scrapie and bovine
BSE PrPres profiles.
The PrPres profiles produced were distinct from bovine BSE and ovine scrapie but were similar to the
profiles produced by atypical scrapie transmission in the transgenic tg338 mouse model (expressing
sheep PrPVRQ), as described by Le Dur et al. (2005) and as determined and reported in project
SE1850. The sensitivity of the atypical scrapie PrPres produced in Tg(OvPrPAHQ) mice to proteolytic
digestion with proteinase K was not examined in this study. However, following passage of atypical
scrapie isolates in Tg(OvPrPAHQ) mice, the resulting PK-resistant PrPres fragments were detectable at
the levels of PK treatment recommended in the Western blot BioRad method.
SID 5 (Rev. 3/06)
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3.6.2.2. Neuropathological analysis of Tg(OvPrPAHQ) mice infected with atypical scrapie isolates
from sheep.
Coronal sections of brain from Tg(OvPrPAHQ) mice infected with atypical scrapie isolates were
examined by IHC, which revealed positive detection of PrPSc in certain brain regions.
3.7. Cryopreservation of transgenic PrP mouse lines.
Cryopreservation of eleven selected SE1753-generated transgenic PrP lines is on-going at the project
end-date (31-03-09). As part of the process of archiving the selected lines in a cryopreservation
repository, the superovulation of transgenic mice to produce embryos for cryostorage is being
conducted via a commercial facility. Until cryopreservation of the selected transgenic mouse lines has
been completed, including test recovery of cryopreserved embryos and confirmed genotyping of
resulting mice, it will be necessary to maintain transgenic mouse lines at VLA. Highlighting the
difficulties encountered in the cryopreservation process, the yield of good quality embryos for
cryopreservation could not be predicted, and production of low quality embryos, which has occurred in
several transgenic lines, has led to delays in the cryopreservation process.
4. DISCUSSION.
Full discussion of the results produced in these studies will be published in peer-reviewed scientific
papers, which are in preparation. A briefer discussion of the results is presented in this report.
Transgenic mouse lines expressing foreign PrP genes, including bovine (Scott et al., 1997; Buschmann
et al., 2000; Castilla et al., 2003) and ovine PrPs (Vilotte et al., 2001; Crozet et al., 2001) have been
reported previously and have demonstrated susceptibilities to infection with BSE and/or scrapie.
Expression levels of transgenic PrP as well as PrP sequence homology between recipient PrP and the
incoming agent have been shown to have a potent effect on susceptibility to prion diseases as well as
on disease incubation times (Scott et al., 1989). However, high levels of transgene expression can
result in deleterious disease, including spontaneous neurological disease as reported by Westaway et
al. (1994) and as found in one of the transgenic bovine PrP mouse lines produced in this project.
The overall aim of this project was to produce stable transgenic PrP mouse models with improved
susceptibility and exhibiting reduced disease incubation times in detecting TSE disease agents in
comparison to wild-type mouse lines. This was achieved with the generation and assessment of the
Tg(BoPrP) and Tg(OvPrPAHQ) mouse models.
4.1. Generation of transgenic PrP mouse lines.
Preparation and microinjection of Prnp transgenes resulted in the successful generation of a number of
stable transgenic mouse lines expressing bovine, kudu and sheep PrPs, which were characterised by
molecular biological techniques. Several of these lines were bred successfully to transgene
homozygosity, whereas in the case of several other Tg(PrP) lines, despite attempts, homozygous
transgenic mice could not be produced. In addition to the planned generation of bovine and ovine PrP
models, preparation of a kudu PrP transgenic model was undertaken, as this species (kudu) has
demonstrated a shortened clinical phase of infection with the BSE agent.
Whereas chimaeric Prnp constructs, based on modifications to the half-genomic (phgPrP vector;
Fischer et al., 1996), were prepared for kudu and ovine PrPs, a different approach was adopted in the
preparation of the full-length bovine PrP transgene, in which a larger fragment derived from a bovine
genome BAC library clone was used. The advantage of using longer transgenic constructs is that such
constructs are likely to be more stable for germline transmission in transgenic mice than shorter
constructs. Transgenic mice expressing a 120 kb sheep PrPVRQ BAC construct, as reported by Vilotte
et al. (2001), demonstrated a markedly reduced incubation time with one scrapie isolate, but a longer
incubation with another scrapie isolate. In the model, expression of sheep PrPVRQ was related to
transgene copy number and was position independent.
In this project, transgenic PrP constructs were microinjected into wild-type mouse fertilised oocytes, as
these have improved survival rates compared to microinjected PrP null oocytes. In addition, it has been
noted that direct microinjection of PrP transgenes onto a PrP null genetic background can result in
transgenic mice with a low breeding efficiency. Thus, the action taken in this study could provide
advantages for the long-term breeding of the SE1753-generated transgenic mouse lines.
SID 5 (Rev. 3/06)
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Following successful generation of stable transgenic PrP mouse lines expressing bovine, kudu and
ovine PrPs, transgenic mice were bred successively with PrP null mice to transfer transgene
expression on to a mouse PrP-ablated background. Characterisation of transgene expression enabled
estimations of expression levels in brain and peripheral tissues. Stable lines were bred to transgene
homozygosity to facilitate TSE challenge studies.
Despite the overexpression of transgenic PrP in brain and other tissues in these models, spontaneous
neurological disease was only seen to occur in one of the Tg(BoPrP) lines overexpressing the
transgene at a high level. The occurrence of spontaneous disease has been described previously in
other overexpressing transgenic models (Westaway et al., 1994; Hsiao et al., 1990 & 1994; Chiesa et
al., 2001), in which conversion of the transgenic PrP to PrPSc was either detected or not detected and
where differences in pathological phenotypes were also reported. These spontaneous disorders or
proteinopathies occurring in transgenic mice as a result of overexpression of wild-type PrP, were
described by Westaway et al. (1994) as representing a new category of prion diseases.
As indicated above several models expressing bovine PrP have been reported, however, the
Tg(BoPrP) mice generated in this study are likely to represent a model with different characteristics or
attributes due to the expression of full-length PrP driven by a bovine Prnp gene promoter. The other
transgenic PrP mouse lines generated in this study, expressing kudu PrP, sheep PrPAHQ and sheep
PrPARR, appear to be unique models and have not been reported by others in the scientific literature.
4.2. TSE transmissions to transgenic PrP mice.
TSE transmission studies were carried out in Tg(BoPrP), Tg(KuPrP), Tg(OvPrPAHQ) and Tg(OvPrPARR)
mouse lines bred to hemizygous or homozygous status. The majority of TSE infection studies were
successfully completed, including the Western blotting analysis of challenged mice for confirmation of
TSE disease by detection of PrPres. However, at the end-date of the project (31-03-09), some
transmissions are still in progress, which may be due to the lower susceptibility or resistance of the
inoculated mice, to infection with TSE agents.
The results obtained in the TSE transmission studies indicate the successful outcome achieved with
Tg(BoPrP) and Tg(OvPrPAHQ) mouse lines, which in comparison to wild-type mouse lines demonstrated
improved susceptibility and reduced incubation times in challenges with a wide range of TSE agents.
Tg(BoPrP) mice were highly susceptibility to BSE and classical scrapie infection and demonstrated
greater susceptibility and reduced incubation periods compared to C57BL/6 and SJL wild-type mice.
PrPres profiles revealed that on primary passage of both BSE and scrapie the banding patterns found in
the originating hosts were reproduced in the Tg(BoPrP) mice.
IHC analysis also revealed that BSE and scrapie in the Tg(BoPrP) mice could be distinguished by
difference in PrPSc distribution patterns in certain brain regions. Further biochemical analysis revealed
differences in the deglycosylation profiles of PrPres from Tg(BoPrP) mice infected with BSE or scrapie.
To further validate these preliminary findings, additional deglycosylation and epitope mapping studies
of PK cleavage products from transgenic mice infected with TSE agents should be conducted, as
carried out in the characterisation of Nor98/atypical scrapie by Klingeborn et al. (2006) and Gretzschel
et al. (2006).
The advantage of breeding the transgenic mouse lines to homozygosity was shown in Tg(BoPrP) mice,
where a marked reduction in BSE incubation period was seen in homozygous mice compared to a
previous challenge of hemizygous mice. In addition, in TSE-susceptible lines, mice with higher
transgene expression levels demonstrated faster incubation periods than those showing lower
expression.
The Tg(BoPrP) model produced here, expresses full-length PrP under the control of the bovine Prnp
promoter. Other bovinised mouse models have also been reported (Scott et al., 1997; Buschmann et
al., 2000; Castilla et al., 2003), in which the models produced by Buschmann et al. (2000) and Castilla
et al. (2003) were based on the use of the phgPrP vector, whereas the mice reported by Scott et al.
(1997) were generated by use of the CosSHa.tet vector. It is likely that differences between these
models (such as full-length or chimaeric PrP and the effects of different promoters) could result in
SID 5 (Rev. 3/06)
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different effects (TSE susceptibilities, incubation periods, biochemical and neuropathological
phenotypes) in TSE transmissions.
For example, in contrast to the high susceptibility of the transgenic full-length bovine PrP model
generated here to BSE and scrapie infection, with a similar broad susceptibility described for the Scott
et al. (1997) model, it was reported that boTg110 mice (Castilla et al. (2003) demonstrated a restricted
or selective susceptibility to BSE and were less susceptible to scrapie. It was also noted that a
neurological syndrome involving hindlimb motor impairment was seen to occur in older boTg110 mice
(Castilla et al., 2003), which was similar to that seen in older PrP knockout mice (Edinburgh, PrP -/-)
generated by Manson et al. (1994). In the Tg(BoPrP) model reported here, the efficient transmission of
sheep scrapie is consistent with the role of glutamine at codon 171 in controlling scrapie susceptibility.
In contrast to Tg(BoPrP) mice, Tg(KuPrP) mice produced in this study demonstrated a lower efficiency
of transmission of classical scrapie and BSE.
In 2003, Benestad et al. reported the first detected cases of an atypical form of scrapie in sheep in
Norway, which occurred predominantly in sheep carrying PrP genotypes associated with relative
resistance to classical scrapie. Since that time, atypical scrapie has been described in sheep and goats
in many European countries and elsewhere (reviewed by Benestad et al., 2008). The origin of the
disease and whether it is an infectious or spontaneous disease under natural conditions is unknown.
However, experimental transmissions to mice (Le Dur et al., 2005) and sheep (Simmons et al., 2007)
have demonstrated the infectious nature of atypical scrapie agents.
An important finding in this project was that the generated Tg(OvPrPAHQ) mouse model was highly
susceptible to atypical scrapie infection. In addition, the model demonstrated susceptibility to classical
scrapie and BSE. Overall, the findings suggest that the Tg(OvPrPAHQ) model may represent a more
appropriate bioassay model for detection of atypical scrapie infectivity, not only due to its high
susceptibility and rapid incubation times but also because of its potential sensitivity. This model could
be applied to the detection of atypical scrapie infectivity in the tissues of experimentally infected and
naturally infected sheep and goats.
In comparison to the TSE challenges of other transgenic mouse lines, the transmissions of a range of
atypical scrapie, classical scrapie and BSE isolates to Tg(OvPrPARR) mice have been poor or
unsuccessful. However, some mice are still incubating disease or showing resistance to disease at
~600-700 days post-inoculation.
4.3. General comments.
The overall aims of the project have been successfully achieved by the generation of certain transgenic
PrP mouse lines with demonstrated high susceptibilities and reduced incubation times to a range of
TSE agents. The results of TSE transmission studies have shown that particular Tg(BoPrP) and
Tg(OvPrPAHQ) mouse lines represent more susceptible and faster bioassay models (of potentially
greater sensitivity) for BSE, scrapie and atypical scrapie compared to wild-type mouse lines, and that
these mouse models could be applied to the improved bioassay detection and differentiation of TSE
diseases.
As indicated, full details of the results obtained with the Tg(BoPrP) mouse model and its usefulness in
the improved detection and differentiation of ruminant TSE agents will be presented in a peer-reviewed
scientific paper.
Similarly, full details of the results obtained for the Tg(OvPrPAHQ) mouse model, which suggest that the
model offers the prospect of improved detection and differentiation of ruminant TSE strains within the
same PrP genotype model, will be presented in a peer-reviewed scientific paper.
In this study the Tg(BoPrP) and Tg(OvPrPAHQ) mouse models have demonstrated improved TSE
bioassay characteristics compared wild-type mouse lines. Use of these models could lead to faster,
more efficient TSE bioassays, and reduce the economic costs, long disease incubation times and lower
susceptibilities associated with conventional bioassay.
SID 5 (Rev. 3/06)
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5. MAIN IMPLICATIONS OF THE FINDINGS.
Project SE1753 has delivered the main aims of the research: the generation of stable, overexpressing
transgenic PrP mouse models with demonstrated high susceptibilities and reduced incubation times to
ruminant TSE diseases compared to wild-type mouse lines. In particular, the Tg(BoPrP) mouse lines
generated show high susceptibility, reduced incubation periods and differentiation of BSE and classical
scrapie agents in the same model, and the Tg(OvPrPAHQ) mouse lines generated show high
susceptibility and early detection of atypical scrapie isolates, as well as susceptibility to classical
scrapie and BSE agents. The potential benefits associated with generating and applying these
transgenic mouse models are numerous and indicated below.
The transgenic models generated in this study will reduce the need to undertake large-scale
experiments in the corresponding natural host species, such as cattle and sheep. By demonstrating
reduced disease incubation times and greater susceptibilities to TSE agents, the transgenic models
produced represent improved models for TSE bioassays and could replace (or reduce) the use of
conventional wild-type mouse lines, which would be in line with ethical policies and lead to reduced
economic costs.
The transgenic models produced here offer the advantage of wide susceptibility and detection of TSE
agents as well as differentiation within the same transgenic models. In contrast to conventional wildtype mouse lines, the transgenic mice generated in this project are likely to represent more suitable
models for investigating naturally occurring and experimental TSE infections, and could be useful for
the study and resolution of complex issues, such as mixed TSE infections, within the same transgenic
model.
In addition, the transgenic models could be applied to the detection of low level and/or sub-clinical TSE
infections, to detecting TSE infectivity in peripheral tissues of affected hosts, to detecting BSE infection
in sheep, and to improved TSE strain differentiation including in atypical or anomalous cases in
ruminants and other species.
Application of the Tg(BoPrP) and Tg(OvPrPAHQ) mouse models to the detection of TSE infectivity in the
tissues of clinically-affected and asymptomatic animals from naturally occurring or experimental TSE
infections, and particularly to tissues that were negative in previous less sensitive bioassays based on
conventional mouse lines, could identify new risk materials for human and animal health, which could
lead to the implementation of new disease control measures for TSEs.
6. POSSIBLE FUTURE WORK.
There are a number of potential applications of the transgenic PrP mouse models generated in project
SE1753. The Tg(BoPrP) and Tg(OvPrPAHQ) models, in particular, could be applied to the potentially
improved detection and differentiation of TSE field cases, such as classical and atypical BSE and
scrapie cases or unusual cases from hosts of varying PrP genotypes. This could enable the improved
molecular pathogenetic characterisation of such cases and contribute to an evaluation of the potential
risks to human and animal health.
The Tg(BoPrP) model could be applied to the differentiation of ovine BSE and classical scrapie disease
in sheep, and in addition could be suited to the identification and characterisation of variant CreutzfeldtJakob disease (vCJD) cases of human prion disease.
The Tg(OvPrPAHQ) model could be applied to identifying the distribution of prion infectivity in tissues
from experimentally infected and naturally occurring cases of atypical scrapie in sheep and goats in
comparison to the distribution of classical scrapie, and could also be applied to investigating the carrier
status of apparently healthy sheep.
The broad susceptibility of the Tg(BoPrP) and Tg(OvPrPAHQ) models to a range of TSE agents would
suggest that these mice could be suited to the study of mixed TSE infections in experimentally and
naturally occurring disease. Importantly, materials or products intended for consumption in the human
SID 5 (Rev. 3/06)
Page 15 of 20
and animal food chains could be examined in these transgenic models to identify potential risks of TSE
infectivity.
The application of the transgenic models produced in SE1753 could facilitate collaborative and
standardised approaches to the detection and differentiation of TSE agents and diseases across
different laboratories and allow comparative studies with other transgenic PrP models.
7. ACTIONS RESULTING FROM THE RESEARCH.
Following the successful generation of transgenic PrP mouse models with demonstrated susceptibilities
to ruminant TSE agents, embryos from eleven of the generated transgenic PrP lines are being
deposited in a cryopreservation respository. The results of the challenges of the transgenic PrP mouse
lines with TSE agents, as well as general characterisation of the lines, will be published in peerreviewed scientific journals (manuscripts in preparation).
At the end-date of the project (31-03-09), two aspects of the project remained uncompleted. These
were the continuing incubation of transgenic mice challenged with TSE agents, which had not yet
developed disease, and the continuing process of cryopreserving 11 of the generated transgenic PrP
lines. These issues were raised in the SID5A document accompanying this Final Project Report.
In addition, to the use of selected transgenic lines from project SE1753 in two internally (seedcorn)
funded VLA projects, and utilisation of selected lines in two further Defra-funded research projects: (i)
in a project (SE2014) aimed at improved differentiation and characterisation of TSE agents and (ii) a
project proposal aimed at the evaluation of transgenic PrP models with standardised TSE inocula,
additional opportunities for collaborative use of the transgenic PrP mouse lines generated in SE1753
will be sought.
8. REFERENCES.
Asante, E.A., Lineham, J.M., Desbruslais, M., Joiner, S., Gowland, I., Wood, A.L., Welch, J., Hill, A.F., Lloyd,
S.E., Wadsworth, J.D. & Collinge, J. (2002). BSE prions propagate as either variant CJD-like or sporadic CJD-like
prion strains in transgenic mice expressing human prion protein. EMBO J. 21, 6358-6386.
Benestad, S.L., Sarradin, P., Thu, B., Schönheit, J., Tranulis, M.A. & Bratberg, B. (2003). Cases of scrapie with
unusual features in Norway and designation of a new type, Nor98. Vet Rec 153, 202-208.
Benestad, S.L., Arsac, J.-N., Goldmann, W. & Nöremark, M. (2008). Atypical/Nor98 scrapie: properties of the
agent, genetics and epidemiology. Vet Res 39, 19.
Bosque, P.J., Ryou, C., Telling, G., Peretz, D., Legname, G., DeArmond, S.J. & Prusiner, S.B. (2002). Prions in
skeletal muscle, Proc Natl Acad Sci USA 99: 3812-3817.
Bueler, H., Fischer, M., Lang, Y., Bluethman, H., Lipp, H.P., DeArmond, S.J., Prusiner, S.B., Aguet, M. &
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References to published material
9.
This section should be used to record links (hypertext links where possible) or references to other
published material generated by, or relating to this project.
SE1753 POSTER PRESENTATIONS
1. Griffiths PC, Plater JM & Plowright L (2000).Transgenic mouse models for bovine spongiform
encephalopathy and scrapie. Poster presentation (Poster and abstract D8). TSE Joint Funders’
Conference, Keele University, 4-6 April 2000.
2. Griffiths PC, Plater JM & Perez Nadales EP (2002). Preparation of more sensitive bioassay
models for the improved detection, differentiation and diagnosis of the BSE agent. Poster
presentation (Poster and abstract 31). TSE Joint Funders’ Conference, Durham University,
March 2002.
3. Griffiths PC, Plater JM, Field ME & Rice PB (2004). Preparation of more sensitive bioassay
models for the improved detection, diagnosis and differentiation of the BSE agent. Poster
presentation (Poster and abstract 115). TSE Joint Funder’s Conference, University of York, 1-3
September 2004.
4. Griffiths PC, Plater JM, Hazelby OJ, Hills D, Williams J, Spiropoulos J, Johnson L and Windl O
(2005). Spontaneous neurological disease in transgenic bovine PrP mice. Poster presentation
(Poster and abstract D-20). Prion 2005 Conference, Dusseldorf, Germany, 19-21 October 2005.
5. Griffiths PC, Plater JM, Hazelby OJ, Jayasena D, Green RB, Spiropoulos J, Johnson L, Wells
GAH and Windl O (2006). Preparation of transgenic PrP mouse models for BSE and scrapie.
Poster presentation. Joint Funders’ Conference, University of Warwick, September 2006.
6. Griffiths PC, Plater JM, Chave A, Jayasena D, Tout AC, Cawthraw S, Scholey S, Dexter I,
Lockey R, Green RB, Spiropoulos J & Windl O (2007). Transmission of ruminant TSEs to
transgenic mice expressing bovine, kudu and sheep PrPs. Poster presentation. Prion 2008
Conference, Edinburgh, Scotland, UK, September 2007.
7. Griffiths PC, Plater JM, Chave A, Jayasena D, Tout AC, Scholey S, Dexter I, Lockey R,
Spiropoulos J & Windl O (2008). Generation and use of transgenic PrP mice as improved
bioassay models for ruminant TSEs. TSE Joint Funders’ Conference, University of Warwick.
8. Griffiths PC, Plater JM, Chave A, Jayasena D, Tout AC, Scholey S, Dexter I, Lockey R,
Spiropoulos J & Windl O (2008). Generation and use of transgenic PrP mice as improved
bioassay models for ruminant TSEs. Prion 2008 Conference, Madrid, Spain.
SE1753 MANUSCRIPTS IN PREPARATION
Due to the time period involved in producing the transgenic PrP models and the further time
period required to conduct TSE challenge studies, previous publication of results from this study
has not been feasible, however, data is now available to allow publication of the results of the
studies.
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