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the termmal stage of disease m the 129/01a PrP”- mice (280 d) and compared to the
termmal 129/01a PrP+˜+ profile (150 d) The brain areas assessed were ( I) medulla, (2)
cerebellum, (3) superior coll˜culus, (4) hypothalamus, (5) thalamus, (6) hippocampus,
(7) septum, (8) thalamtc cortex, (9) forebrain cortex. Dashed line, endpoint PrP+˜+ Sohd
line; end pomt PrP+˜-

4. Normal Function of PrP
One of the major reasons for the numerous gene targeting expertments
undertaken m the last few years has been to investigate gene function. The
normal functron of PrP has not yet been established. The PrP gene is expressed
at high levels m neuronal cells of the adult brain (4,28) and recently has been
detected m astrocytes and ohgodendrocytes (29). Lower levels of PrP mRNA
can be detected m other tissues, such as heart, lung, and spleen (5,30). PrP
mRNA has also been detected during mouse embryogenesrs. By 6.5 d gene
expression can be detected in the extraembryomc tissue and by 11.5 d m
the developing central and peripheral nervous system (31). These studies have
suggested a role for PrP m promotmg neuronal cell differentiation and m
mamtammg neuronal function m the differentiated neurons. However, the
expression of the PrP gene 1snot limited to neuronal cell populattons since tt
has been detected m astrocytes and ollgodendrocytes, during kidney develop-
ment, tn the developmg tooth bud, and m placenta ammon and yolk sac, sug-
gesting the role of PrP may be more widespread, perhaps as part of a cell
245
PrP-Deficient Mice
signaling system required for differentiation of specific cells or as a cell adhe-
sion molecule.
It was hoped that the generation of PrP null mice would address the question
of the normal function of PrP. The initial analysis of the PrP null mice (20,21),
however, showed that without PrP, mice appeared to develop and reproduce
riormally. It has now been shown there is little or no phenotyplc effect m many
null mutant mice produced by gene targeting. This lack of phenotype 1sthought
to be owing to the organism compensatmg for the loss of a gene by alteration m
expression of other genes or use of alternative developmental pathways.
More recently, however, PrPO™O mice were shown to display weakened long-
term potentiation (LTP) in the hippocampal CA 1 subfield (32). Abnormal1 ties
in the LTP were also detected m the inbred PrP null mice that consistently
showed an absence of LTP m the CA1 region of the hlppocampus and in Its
place a short-term potentlatton, which decays to control levels within 1 h (33).
Since the two lines of mice have been produced by different targetmg strate-
gies, these results indicate that the impairment of synaptic plasticity 1sa result
of the loss of PrP and not an artifact of gene targeting and subsequent mouse
production. Reintroduction of the high copy numbers of the human PrP gene
into PrP null mice has been shown to restore the LTP response to that seen
in the wild-type controls (34). The effect on LTP therefore can be attributed to
an absence of PrP, but it remains difficult to ascribe the differences between
knockout and wild-type mice to a specific function of PrP or to compensatory
effects of the organism m the absence of PrP.
5. Introduction of Point Mutations
into the Endogenous Murine PrP Gene by Gene Targeting
Variant forms of the PrP protein have been shown to be associatedwith human
TSE (13-25) and amino acid differences have been shown to be associatedwith
different incubation periods of scraple in mice (8), hamsters (ZO),and sheep (12).
Recent advances m the gene targeting technology now have made it pos-
sible for the role of the specific alterations in the PrP gene to be analyzed by
introducing point mutations into the endogenous PrP gene (35). In this way
the relationship between Sine and PrP can be clearly defined by introducing
the 108 and 189 sequencesassociated with the Sine p7 allele into the PrP gene
from a Sine s7 mouse.
This approach also can be used to produce transgenic models for the human
TSEs. Standard transgenic approaches have been used to produce mice expres-
smg high levels of a chimeric hamster murine PrP gene with the codon 101
proline to leucine mutation (18). The equivalent mutation (codon 102) in
humans has been shown to be associated with Gerstmann-Straussler Syndrome
(13,24). Transgenic mice overexpressing the mutant PrP protein have been
246 Manson

shown to spontaneously develop neurodegeneratlon, sponglform changes m
the brain, and astrogliosls (18). Whether this disease can be attributed speclfi-
tally to the PrP mutation or to overexpression of the PrP gene IS difficult to
establish. More defirutlve transgenic models have now been produced by
introducing specific alterations into the endogenous PrP gene using gene tar-
geting techniques. Using a double replacement strategy, mice have been pro-
duced in which amino acid 101 of the endogenous PrP gene has been mutated
from Pro to Leu. Both heterozygous and homozygous lines of mice carrying
this mutation have been produced (36). No spontaneous neurodegeneratlve dls-
ease has been observed m these animals m up to 250 d of age.
6. Future Prospects
Gene targeting has provided us with an extremely powefil approachto analyze
thesediseases.The mtroductlon of specific mutations will allow the role of these
mutations m PrP to be defined. Genetargeting technology 1snow being developed
through bacteriophage Pi cre/lox site-specific recombmase systems (37-39)
to allow mduclble gene expression, both tissue specifically and temporally.
Inducible expression will allow the development of more specific models
that ablate PrP gene expresslon at different time-points in development These
models will be able to differentiate between normal function of the PrP protein
m the adult mouse and compensatory effects resulting from the complete abla-
tion of PrP during development.
Tissue-spectfic expression will allow the role of PrP gene expression m spe-
cific cell types and specific tissuesto be analyzed. These models will be able to
address questions relating to agent replication, transfer of infectivity from the
periphery to central nervous system, and the role of PrP expression m specific
neuronal cell populations m the development of the disease pathology.
Transgenic models can also be developed using this approach, which ˜111
allow expression of mutant and wild-type PrP genes at different time-points
during the disease process. These models will allow m vivo analysis of interac-
tions between different PrP molecules and the precise mechanisms leading to
the development of the pathology of the disease to be defined
References
1 Stahl, N., Borchelt, D. R., Hslao, K., and Prusiner, S. B. (1987) Scraple prlon
protein contamsa phosphomositolglycohpid Cell 51,229-240
2. Bruce, M , McBride, P A., and Farquhar, C F (1989) Precisetargeting of the
pathology of the slaloglycoprotem, PrP, and vacuolar degeneration in mouse
scraple.Neuroscl. Lett 102, l-6
3 DeArmond, S J , Mobley, W. C , DeMott, D L., Barry, R A., Beckstead,J H.,
and Prusiner, S. B (1987) Changesin the localisationof the brain prion proteins
during scraplemfectlon. Neurology 37, 1271-128 1
PrP- Deficien t Mice 247
4 Manson, J , McBrtde, P , and Hope, J. (1992) Expressron of the PrP gene in the
bram of Smc congemc mice and its relationship to the development of scrapre
Neurodegeneratron 2,45-52.
5 Oesch, B., Westaway, D., Walchr, M , McKinley, M P., Kent, S. B H., Aebersold,
R , et al (1985) A cellular gene encodes scrapte PrP 27-30 protem Ceil 40,
735-746
6 Dickinson, A G , Meikle, M V , and Fraser, H (1968) Identrficatton of a gene
which controls incubation period of some strains of scrapre m mice J Comp
Pathol. 78,293-299
7 Carlson, G A., Kingsbury, D T., Goodman, P A , Coleman, S , Marshall, S T ,
DeArmond, S , et al (1986) Linkage of prton protem and scrapte rncubatton ttme
genes. Cell 46, 503-5 11
8 Westaway, D., Goodman, P. A., Mrrenda, C., McKinley, M. P , Carlson, G A ,
and Prusmer, S B. (1987) Distinct prron proteins m short and long incubation
period mice Ceil 51,65 l-662.
9 Hunter, N., Hope, J., McConnell, I., and Dickson, A G. (1987) Linkage of the
scrapre associated fibrtl protem(PrP) gene and Sine using congenrc mace and
restrrctton length fragment polymorphisms analysis J Gen Vwol 68,27 11,27 12.
10 Hunter, N., Dann, J. C , Bennett, A D., Somervtlle, R. A , McConnell, I, and
Hope, J (1992) Are Sznc and the PrP gene congruent? Evidence from PrP gene
analysts m Sine congemc mice. J Gen. Vwol 73,275 l-2755.
11 Lowenstein, D H , Butler, D A , Westaway, D , McKinley, M P., DeArmond, S.
J , and Prusmer, S. B (1990) Three hamster species with different mcubatton
times and neuropathologtcal features encode different prion proteins. Mol Cell
Biof 10, 1153-I 163
12 Goldmann, W., Hunter, N., Benson, G., Foster, J., and Hope, J. (1991) Scrapie-
associated fibril proteins are encoded by lines of sheep selected for drfferent alle-
les of the Sip gene. J Gen. Vrrol 72, 241 l-2417.
I3 Doh-ura, K., Tateishi, J , and Sasakt, H. (1989) Pro-Leu change at position 102 of
prton protein is the most common but not the sole mutation related to Gerstmann-
Straussler syndrome. Blochem Blophys. Res Commun 163,974-979.
14 Collmge, J , Harding, A. E , Owen, F., Poulter, M., Lofthouse, R , Boughey, A.
M., et al. (1989) Diagnosis of Gerstmann-Straussler syndrome m familial
dementia with prron protein gene analysis Lancet 2, 15-l 7.
15. Owen, F., Poulter, M., Shah, T., Colhnge, J., Lofthouse, R., Baker, H., et al. (1990)
An in-frame insertion m the prion protein gene m familial Creutzfeldt-Jakob dis-
ease. Mol. Brain Res. 7,273-276.
16. Scott, M., Foster, D., Mirenda, C., Serban, D , Coufal, F., Walchli, M., et al. (1989)
Transgenm mice expressmg hamster prron protem produce species-spectfic
scrapie infectivity and amyloid plaques. Cell 59, 847-857
17 Westaway, D., Mirenda, C. A., Foster, D., ZebarJadian, Y., Scott, M., Torchra,
M., et al. (199 1) Paradoxical shortening of scrapre mcubatron trmes by expression
of prion protein transgenes derived from long incubation period mice. Neuron 7,
59-68
Manson
248

18. Hstao, K K , Scott, M., Foster, D , Groth, D F., DeArmond, S J , and Prusmer, S
B. (1990) Spontaneous neurodegeneration m transgemc mice wtth mutant prlon
protein Sczence 250, 1587-l 590
19 Westaway, D , DeArmond, S J., Cayetano-Canlas, J., Groth, D , Foster, D., Yang,
S.-L , et al (1994) Degeneration of skeletal muscle, peripheral nerves and central
nervous system m transgemc mice overexpressmg wild type prton proteins Cell
76, 117-129
20 Bueler, H., Frscher, M , Lang, Y , Bluthmann, H , Lrpp, H.-L , DeArmond, S J ,
et al. (1992) The neuronal cell surface protein PrP IS not essenttal for normal
development and behavtour in mice. Nature 356,577-582
21 Manson, J C , Clarke, A R , Hooper, M , Attchtson, L., McConnell, I , and Hope,
J , et al (1994) 129/ala mace carrying a mutation m PrP that abohshes mRNA
productton are developmentally normal Mol. Neurobzol 8, l-5
22 Manson, J C , Clarke, A R , McBride, P A , McConnell, I , and Hope, J (1994)
PrP gene dosage determines the timing but not the final mtenstty or dlstrtbutton of
lesions m scrapte pathology Neurodegeneratron 3,3 1 l-340
23 Bueler, H , Aguzzt, A , Satler, A , Gremer, R -A , Autenretd, P., Aguet, M , et al
(1993) Mace devoid of PrP are resistant to scrapte Cell 73, 1339-l 347
24 Prusmer, S B , Groth, D , Serban, A , Koehler, R , Foster, D , Torchta, M , et al
(1993) Ablation of the prton protein (PrP) gene m mice prevents scraple and
factmates the productton of anti-PrP antibodies Proc Nat1 Acad SCI USA 90,
10,608-10,612
25 Carlson, G A , Ebelmg, C., Yang, S -L., Telling, G., Torchta, M., Groth, D., et al
(1994) Prion protem specified allotyprc interactions between cellular and scrapte
prton protems m congemc and transgemc mice Proc Nat1 Acad Scl USA 91,
5690-5694
26 Bueler, H., Raeber, A., Sailer, A , Ftscher, M., Aguzzt, A., and Weissmann, C.
(1994) High prton and PrPSClevels but delayed onset of disease in scrapte-mocu-
lated mice heterozygous for a disrupted PrP gene Mel Med 1, 19-30
27 Sailer, A , Bueler, H , Fischer, M , Aguzzt, A., and Wetssmann, C (1994) No
propagation of prtons m mice devoid of PrP. Cell 77,967,968
28 Kretzchmar, H A , Prusmer, S. B., Stowrmg, L. E., and DeArmond, S J. (1986)
Scrapte prion proteins are synthesised m neurones Am. J. Pathol. 122, 1-5.
29 Moser, M , Colello, R J., Pott, U , and Oesch, B. (1995) Developmental expres-
sion of the prton protein gene m ghal cells. Neuron 14,509-5 17
30 Caughey, B., Race, R., and Chesebro, B. (1988) Detection of prlon protein mRNA
m normal and scrapte-infected tissues and cell lines. J Gen. Vzrol69, 7 1 l-7 16
31. Manson, J., West, J D., Thomson, V., McBrtde, P., Kaufman, M H., and Hope, J
(1992) The prron protem gene a role m mouse embtyogenests? Development 115,
117-122
32 Collmge, J , Whtttmgton, M , Sidle, K , Smith, C , Palmer, M , Clarke, A , et al
(1994) Prion protein IS necessary for normal synaptic function. Nature 370,295--297
33. Manson, J., Hope, J., Clarke, A , Johnston, A., Black, C., and MacLeod, N (1995)
PrP gene dosage and long term ootentratton Neurodeszeneratlon 113-l 15
4.
/V-Deficient Mice 249

34. Whtttington, M., Sidle, K., Gowland, I., Meads, J., Hrll, A., Palmer, M , et al
(1995) Rescue of neurophysrologrcal phenotype seen in PrP null mice by transgene
encoding human prton protein. Nature Genet 9, 197-201.
35. Stacey, A , Schmeke, A, McWhtr, J , Cooper, J , Colman, A , and Melton, D W
(1994) Use of double replacement gene targeting to replace the murme alpha lactal-
bumin gene with its human counterpart m embryonic stem cells m mace Mel Cell
Bzol 14, 1009-1016
36. Moore, R., Redhead, N , Selfridge, J., Hope, J., Manson, J , and Melton, D (1995)
Double replacement gene targeting for the productron of a series of mouse strams
with different prron protein gene alterations. Bzotechnology 13,999-1004
37. Orban, P. C , Chui, D , and Marth, 3 D (1992) Trssue- and site-specific DNA
recombmatron in transgemc mice Proc Nat1 Acad Scz USA 89,6861-6865
38. Lasko, M , Sauer, B., Mosmger, B , Lee, E., Manning, R., Yu, S -H , et al (1992)
Targeted oncogene actrvatron by sate-specific recombmatron m transgemc mice.
Proc Nat1 Acad Scz USA 89,6232-6236
39 Gu, H , Marht, J , Orban, P., Mossman, H., and RaJewsky, K (1994) Deletron of
a DNA polymerase B gene segment in T cells usmg cell type specific gene target-
mg. Science 265, 103-l 06.
15
Transgenic Approaches
to Prion “Species-Barrier” Effects

David Westaway


1. Introduction
Like conventronal vtruses, priori isolates exhibit distinctive, and often
restricted host-ranges. However, the molecular events that shape the host-
ranges of these two classesof pathogen are dissimilar, reflecting their funda-
mentally drfferent life cycles. As discussed by Rtdley and Baker (Chapter l),
many lines of experimentation indicate that PrPSC, aberrant form of a host-
an
encoded neuronal sialoglycoprotein PrPC,is the major constituent of the scrapte
priori. Resistance to protease digestion m vitro 1s a convenient hallmark of
PrPSC. PrPC or a closely related protease-sensitive macromolecule 1sconverted
to PrPSC a posttranslational event: This event may correspond to a conforma-
in
tional change templated by PrPSC molecules (1,2). Whereas the host-range and
cellular tropism of mammalian viruses frequently reflects their binding to cell-
surface proteins to gam entry mto cells via endocytosis, PrPsc-receptors have
yet to be identified: Although PrPc plays a critical role m determmmg prton
host-range (see thefollowzngl and 1sdisplayed on the cell surface via a gly-
colipid anchor (3), three observations suggest that it cannot be considered a
“receptor” m the conventional sense,e.g., in the sense that CD4 is the receptor
for HIV. First, the cell surface molecules co-opted by mammahan viruses cycle
within the endocyttc pathway and are destined for degradation: During prton
repltcatton PrPC 1s converted to PrPsc and changes a variety of biophysrcal
properties (4). It 1snoteworthy that PrPSC a half-life of >48 h m pulse-chase
has
experiments and may thus be protease-resistant in vtvo, as well as in vitro (5).
Second, PrPC that IS not displayed on the cell-surface to a sigmficant degree
(owing to ablation of Asn-linked glycosylatton sites) can also become pro-

From Methods m Molecular Medme Pnon Dfseases
Edlted by H Baker and R M Ridley Humana Press Inc , Totowa, NJ

251
252 Westaway

tease-resistant (6) Third, aggregates of purified PrPSC taken mto cultured
are
cells, presumably by phagocytosis (7).
It is also unlikely that the immune system plays any substantial role m deter-
mining prion host-range It has long been noted that scrapie infection fails to
induce inflammatory (8) or humoral immune responses (9) Although one gene
affecting Creutzfeldt-Jakob disease (CJD) incubation times was mapped to the
maJor histocompatibihty complex of mouse (ZO), other workers using different
congenic strains of inbred mice maculated with scrapie prions failed to repro-
duce this finding (II).

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