Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies.

Prusiner, Stanley B.; Groth, Darlene; Serban, Ana; Koehler, Ruth; Foster, Dallas; Torchia, Marilyn; Burton, Dennis R.; Yang, Shu-Lian; DeArmond, Stephen J.

doi:10.1073/pnas.90.22.10608

Cited by 420 publications

(274 citation statements)

References 48 publications

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“…Moreover, neither accumulation of PrP Sc nor prion propagation could be detected in their brains. Other investigators also showed similar results using other lines of PrP-/-mice (11)(12)(13). These results indicate that PrP C is essentially required for prion propagation, and that prion propagation is linked to accumulation of PrP Sc , strongly supporting the protein-only hypothesis.…”

Section: Protein-only Hypothesis and Mice Devoid Of Prpsupporting

confidence: 70%

“…We and others showed that PrP Sc failed to be produced in mice devoid of PrP C (PrP-/-) (11)(12)(13)(14), indicating that PrP C is essential for the generation of PrP Sc . Moreover, it was reported that PrP Sc -like PrP could be generated from PrP C in certain conditions in vitro (15).…”

Section: Abnormal Isoform Of Prpmentioning

confidence: 99%

“…Since PrP-/-mice were resistant to these diseases (11)(12)(13)(14), it is considered that the conformational conversion of PrP C to PrP Sc plays an essential role in the pathogenesis of the diseases. However, the exact nature of this role has not been fully understood.…”

Section: Findings In Prp-null Mice and Patho-genesis Of Prion Diseasesmentioning

confidence: 99%

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Molecular biology of prion protein and its first homologous protein

Sakaguchi

2007

J. Med. Invest.

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Section: Protein-only Hypothesis and Mice Devoid Of Prpsupporting

confidence: 70%

Section: Abnormal Isoform Of Prpmentioning

confidence: 99%

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Molecular biology of prion protein and its first homologous protein

Sakaguchi

2007

J. Med. Invest.

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“…Disruption of PrP C expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities [2][3][4][5] . However, the impact of ablating PrP C function in natural host species of prion diseases is unknown.…”

Section: Introductionmentioning

confidence: 99%

Production of cattle lacking prion protein

et al. 2006

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Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrP C , such as PrP BSE in bovine spongiform encephalopathy (BSE) in cattle and PrP CJD in Creutzfeldt-Jakob disease (CJD) in humans 1 . Disruption of PrP C expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities [2][3][4][5] . However, the impact of ablating PrP C function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrP C -deficient cattle produced by a sequential gene-targeting system 6 . At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification 7 . PrP C -deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins.To generate PrP C -deficient (PRNP −/− ) cattle, we transfected a male Holstein primary fetal fibroblast line 6594 with first and second knockout (KO) vectors (pBPrP(H)KOneo and pBPrP (H)KOpuro vectors) 6 to sequentially disrupt the two alleles of PRNP. PRNP −/− fetal cell lines were established at 40-60 d of gestation and three of the PRNP −/− fetal cell lines (5211, 5232 and 4296) were recloned to produce calves (Table 1 and Fig. 1a). To verify that the calves possess the PRNP −/− genotype, we collected ear biopsies and established fibroblast cell lines for genotyping. Genotyping was done by genomic PCR specific to each gene targeting event 6 (primer pairs: neoF7 × neoR7 and puroF14 × puroR14, Fig. 1b COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Biotechnology website for details).Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access Fig. 1c) and confirmed the disruption of PRNP-specific mRNA expression in PRNP −/− calves. For protein expression analysis, we performed PrP-specific western blot analyses on fibroblasts (Fig. 1d), peripheral blood lymphocytes (Fig. 1e) and brain stem (Fig. 1f) from wild-type and PRNP −/− calves using the mouse anti-bovine PrP monoclonal antibody F89. We detected PrP-specific bands in the wild-type calves, whereas no reaction was observed in PRNP −/− calves and negative control mouse fibroblasts. These data clearly demonstrate that the PRNP gene is functionally inactivated in the PRNP −/− calves.PRNP −/− cattle were monitored for growth and general health status from birth to 20 months of age. Mean birth weight was 46 kg and average daily gain was 0.91 kg/d to 10 months. Both values were in the normal range for Holstein bulls. Serum chemistry was evaluated at 6 months of age and compared with published reference ranges. All the values for PRNP −/− calves (n = 12) were well within the reference range (Supplementary Table 1) and obvious abnormalities w...

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“…The conversion from normal PrP C to PrP Sc is recognized as the key event to develop prion disease based on the ''protein-only'' hypothesis [5]. It has been justified that PrP C -deficient (PRNP -/-) mice are resistant to TSE and do not propagate infectious PrP Sc without apparent developmental or behavioral defects [6][7][8][9]. Therefore, suppression of endogenous PrP C expression could inhibit endogenous or exogenous PrP Sc formation and accumulation and thereby protect individuals from prion diseases.…”

Section: Introductionmentioning

confidence: 99%

Knockdown of the prion gene expression by RNA interference in bovine fibroblast cells

Wang

Zhang

et al. 2009

Mol Biol Rep

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PRNP is the gene encoding prion protein whose misfolded and b-sheet-rich isoform is the infectious agent of transmissible spongiform encephalopathy (TSE). TSE, also called prion diseases, cause fatal neurodegenerative and transmissible disorders in human and animals. Among these diseases, bovine spongiform encephalopathy (BSE) has tremendous impact on economy and human health in the world. In the present study, we hypothesize suppression of the PRNP gene expression could raise resistance to BSE in cattle by using vectorbased small interfering RNA (siRNA) expression systems. Therefore, the objective was to screen effective DNAencoding short hairpin RNAs (shRNAs) which could knockdown the PRNP gene expression in bovine fibroblast cells. Human U6 promoter was employed to drive shRNA transcription from the DNA vector, and seven shRNAs, that designed to target coding region and 3 0 untranslated region of the PRNP gene, were selected. Four out of seven shRNAs tested were found to be effective in inhibiting the PRNP gene expression, and the most significant suppression level was as much as 62.9% evidenced by real-time RT-PCR. Furthermore, the protein abundance was obviously reduced compared to the control. Overall, the present study demonstrated that vectorbased siRNA expression systems is an efficient approach to knockdown the PRNP gene expression in bovine fibroblast cells and thereby provide donor cells for somatic cell nuclear cloning to produce cattle that is resistant to prion related diseases.

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Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies.

Abstract: Mice, homozygous for prion protein (PrP) gene ablation (Prn-p°/°), develop normally and remain well >500 days after inoculation with murine scrapie prions. In contrast, wild-type mice developed scrapie <165 days after inoculation and most Prn-p°/+ mice, heterozygous for disrup-

Cited by 420 publications

References 48 publications

Molecular biology of prion protein and its first homologous protein

Molecular biology of prion protein and its first homologous protein

Production of cattle lacking prion protein

Knockdown of the prion gene expression by RNA interference in bovine fibroblast cells

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