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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..


  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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