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[SANET-MG] cows without Prions
January 5, 2007
Prof. Joe Cummins
Cows Without Prions
Prions are disease causing proteins causing wasting and
nerve damage in mammals. Toxic infectious prions result from
alterations in normal prion proteins produced at a prion gene (locus) in
mammals. Mammals are diploid so that there are two alleles of the prion
gene both active in producing prion protein. Diseases as varied as fatal
familial insomnia and kuru are believed to be associated with prions.
Other prion diseases include scrapie (a disease of sheep), chronic
wasting disease, (in deer and elk), variant Creutzfeldt-Jakob disease
(vCJD), and bovine spongiform encephalopathy (BSE or mad cow disease),
all caused by similar proteins in different species. The same gene is
responsible for spongiform encephalopathy
The protein that prions are made of is found throughout the body, even
in healthy people and animals. However, the prion protein found in
infectious material has a different structure and is resistant to
proteases, the enzymes in the body that break down proteins. The
diseased form directly interacts with the normal form to make it
rearrange its structure.
The two prion alleles were disrupted in transgenic mice
using homologous recombination to target gene disruption or disruption
and deletion . Disruption of the two alleles resulted in mice produced
using cloning with no gross abnormalities that neither produced prions
nor harbored prion infection However, deletions that extended into
flanking genes caused the knock out mice to suffer ataxia and purkinje
cell loss in the adults (1). One study found mice devoid of prion
protein were found to have cognitive deficits which could be rescued by
reconstitution of prion genes in neurons (2). Mice devoid of prion
genes are free of prions and resist prion infection , the mice are
nearly normal.
Sheep cell lines from which prion protein alleles were
removed by homologous recombination were produced . However, when
nuclei from the cloned the four lambs that were born soon succumbed,
there at birth and one after 12 days (3).
Using bovine fetal fibroblast cell lines , the two prion
protein alleles were disrupted sequentially using homologous
recombination. In the fist step a vector bearing puromycin antibiotic
selection marker flanked by and short prion gene sequence and lox
sites (to allow the cre recombinase, earlier inserted into the calf
cell using an expression plasmid to remove the marker and prion genes
sequences from the disrupted prion gene sequence leaving a deletion to
prevent restoration of the prion gene by recombination ). The second
allele of the prion gene was similarly disrupted and deleted. The
nuclei from the fibroblasts lacking prion genes were used to clone
calves (4).The extensive report on cattle produced using the clones from
the cattle cloned from the nuclei of fetal fibroblast disrupted and
deleted as described in reference 4 showed that the cloned cattle were
apparently normal.
Brain cells of the cloned cattle failed to propagate prions and both
normal and infectious prions were absent from the animals. The cloned
cattle appeared normal in all regards other than their lack of normal
prions (5). The lox sites introduced in preparation of the cloned cells
may create a persistent source of chromosome instability that bears
fuller scrutiny., Certainly the cre expression plasmid should have been
eliminated from the cloned animals but there does not seem to be a
report on the presence or absence of the recombinase in the cattle, The
persistence of cre recombinase is known to produce chromosome damage in
mammalian cells (6) both at lox sites and cryptic lox sites
The use of homologous recombination and gene disruption and
deletion used to produce prion free cattle is different from the
illegitimate recombination used in plant crop genetic engineering.
There is, in principle , much less left over and scrambled DNA
sequences in the cloned cattle than is found in genetically modified
crops. However it remains to be seen whether there is as yet
unidentified problems with removing normal prions from the cattle and
how long the resistance to prions will persist in the cattle. Of course
a bull with prion genes adept at fence jumping could set back the prion
free herd in no time at all..
References
1. Weissmann C and Flechsig E. PrP knock-out and PrP transgenic mice
in prion research. Br Med Bull. 2003;66:43-60
2.Criado JR, Sanchez-Alavez M, Conti B, Giacchino JL, Wills DN,
Henriksen SJ, Race R, Manson JC, Chesebro B and Oldstone MB. Mice devoid
of prion protein have cognitive deficits that are rescued by
reconstitution of PrP in neurons. Neurobiol Dis. 2005 Jun-Jul;19(1-2):255-65
3.Denning C, Burl S, Ainslie A, Bracken J, Dinnyes A, Fletcher J, King
T, Ritchie M, Ritchie WA, Rollo M, de Sousa P, Travers A, Wilmut I and
Clark AJ Deletion of the alpha(1,3)galactosyl transferase (GGTA1) gene
and the prion protein (PrP) gene in sheep. Nat Biotechnol. 2001
Jun;19(6):559-62
4. Kuroiwa Y, Kasinathan P, Matsushita H, Sathiyaselan J, Sullivan EJ,
Kakitani M, Tomizuka K, Ishida I and Robl JM. Sequential targeting of
the genes encoding immunoglobulin-mu and prion protein in cattle. Nat
Genet. 2004 Jul;36(7):775-80
5. Richt JA, Kasinathan P, Hamir AN, Castilla J, Sathiyaseelan T, Vargas
F, Sathiyaseelan J, Wu H, Matsushita H, Koster J, Kato S, Ishida I, Soto
C, Robl JM and Kuroiwa Y. Production of cattle lacking prion protein.
Nat Biotechnol. 2006 Dec 31; [Epub ahead of print] doi:10.1038/nbt1271
6. Loonstra A, Vooijs M, Beverloo HB, Allak BA, van Drunen E, Kanaar R,
Berns A and Jonkers,J. Growth inhibition and DNA damage induced by
Cre recombinase in mammalian cells. Proc Natl Acad Sci U S A. 2001 Jul
31;98(16):9209-14.
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