PrP C. PrP SC PrP. PrP. mrna

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1 PrP C PrP SC PrP C PrP C PrP SC PrP C PrP SC PrP C PrP SC PrP PrP SC PrP SC PrP PrP SC PCR PCR mrna PrP PrP C RT-PCR PrP mrna PrP zhaodm@ cau.edu.cn 1

2 SPF Ecoli DH 5α TRIZOL RNA Gibco dntpstakara Taq DNA TaKaRa M-MLV Promega DEPC Promega RNASEOUT RibonucleasePromega SYBR Green qpcr KitFINNZYMES ECOR TaKaRa pgem -T Easy Vector SystemsPromega E.Z.N.A Gel Extraction Kit Promega PCR PTC-150 MJ Research PCR OPTICON 2 MJ Research h TRIZOL RNA RNA RNA PrP mrna Genbank K02234 Primer Primer5.0 P1GACCGCTACTACCGTGAA P2ACCATCAGG AAGATGAGAAA bp RNAµL mmol/ldntps2µloliga dt 18 2µLDEPC µl PCR min min RT µl PCR h M-MLV RT µlrnasinµlm-mlv µluµl PCR µlbuffer µldntpsµlp 1 P 2 µmol/l µlex Taq µlcdna µl µl s s s min E.Z.N.A Gel Extraction Kit PCR pgem -T Easy Vector Systems pgem -T-easy µlpgem -T Easy Vector50ng5µLPCR µlt 4 DNA µl µl µl DH 5α Amp - LB h Amp + X-Gal IPTG LB h PCR Amp + LB 200r/min µl PCR 2

3 RT-PCR RNase A 260 RT-PCR OPTICON 2 PCR cdna µl Opticon analysis software(version 2.01) Opticon analysis software(version 2.01) PrP mrna SPSS10.0 T- P0.05 RNA A 260 /A 280 RT-PCR bp ECOR pgem -T Easy ECOR bp bp DNA PCR bp mrna RT-PCR DNA DNA Pubmed blast 2000bp 1000bp 500bp 7750bp 250bp MMarker DL ECOR PCR Figure 1 Agarose gel electrophoresis of recombinant T-easy-vectors following digestion with Ecol. M: marker DL2000; 1 and 2: EcoR digested recombinant T-easy-vectors; 3 and 4: products of PCR using recombinant T-easy-vectors as template copies/µl copies/µl 100bp 3

4 r 2 = B 2C RNA ng/mg ng/mg RT cdna n=10 2% ng RNA PrP mrna ng RNA PrP mrna ng RNA PrP mrna PrP mrna mg PrP mrna ng RNA PrP mrna Central nervous system Peripheral organs Tissues 1 RNA PrP mrna Table 1: Quantification of total RNA and PrP mrna in golden hamster tissues No. of samples Total RNA(ng/mg tissue) CV% PrP mrna copies/ng RNA CV% Neocortex Cerebellum Thalamus Obex Spleen Lymph node Heart Lung Liver Kidney 4

5 A copies/reaction copies/reaction copies/reaction copies/reaction B C PrP mrna RT-PCR PrP mrna y=-0.29x r 2 =0.999, 5

6 P0.05 Neocortex Cerebellum Thalamus Obex Spleen Lymph node Heart Lung Liver Kidney Neocortex Cerebellum Thalamus Obex Spleen Lymph node Heart Lung Liver Kidney ng RNA PrP mrna RT-PCR mrna SYBR Green I PCR DNA SYBR Green I PCR PrP mrna ng RNA PrP C PrP mrna Cashman Aucouturier Robakis Northern Blot Ales Tichopad PrP PrP mrna Harris Northern Blot Ales Tichopad PCR Northern Blot PrP mrna PrP PrP Liemann S, Glockshuber R. Transmissible spongiform encephalopathies [J]. Biochem Biophys Res Commun, 1998, 250: [2] Prusiner S B. Novel proteinaceous infectious particles cause scrapie [J]. Science, 1982,216:

7 Stahl N, Borchelt D R & Prusiner S B. Differential release of cellular and scrapie prion protein from cellular membranes by phosphatidylinositol-specific phospholipase C [J]. Biochemistry, 1990, 29: Kretzschmar H A, Prusiner S B, Stowring L E, et al. Scrapie prion proteins are synthesized in neurons [J]. Am J Pathol, 1986, 122: 1 5. Moser M, Colello R J, Pott U, et al. Developmental expression of the prion protein gene in glial cells [J]. Neuron, 1995, 14: Brown D R. Prion protein peptide neurotoxicity can be mediated by astrocytes [J].J Neurochem, 1999, 73: Brown D R, Besinger A, Herms J W, et al. Microglial expression of the prion protein [J]. Neuroreport, 1998,9: Diomede L, Sozzani S, Luini W, et al. Activation effects of a prion protein fragment [PrP-( )] on human leucocytes [J]. Biochem J, 1996, 320: Brown D R, Schmidt B, Groschup M H, et al. Prion protein expression in muscle cells and toxicity of a prion protein fragment [J]. Eur J Cell Biol, 1998, 75(1): [10]Prusiner S B. Prions. Proc Natl Acta Sci USA, 1998, 95: Brandner S, Isenmann S, Raeber A. Normal host prion protein necessary for scrapie-induced neurotoxicity [J]. Nature, 1996, 379: Kimberlin R H, Walker C A. Pathogenesis of scrapie (strain 263K) in hamsters infected intracerebrally, intraperitoneally or intraocularly [J].J Comp Pathol, 1986,67: Kimberlin R H, Walker C A. Incubation periods in six models of intraperitoneally injected scrapie depend mainly on the dynamics of agent replication within the nervous system and not the lymphoreticular system [J]. J Gen Virol, 1988, 69: Ales Tichpad, Michael W P faffl, Andrea Didier. Tissue-specific expression pattern of bovine prion gene: quantification using real-time RT-PCR [J]. Molecular and Cellular Probes, 2003, 17:5-10 Aucouturier P, Carp R I, Carnaud C, et al. Prion diseases and the immune system [J]. Clin Immunol, 2000, 96: Cashman N R, Loertscher R, Nalbantoglu J, et al. Cellular isoform of the scrapie agent protein participates in lymphocyte activation [J]. Cell, 1990, 61: Giulietti A, Overbergh L, Valckx D, et al. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression [J].Methods, 2001, 25(4): Walker N J. Real-time and quantitative PCR: applications to mechanism-based toxicology [J]. J Biochem Mol Toxicol, 2001, 15(3): Seung-Il choi, Won-Kyu Ju, Eun-Kyoung Choi Jin Kim, et al. Mitochondrial dysfunction induced by oxidative stress in the brain of hamsters infected with the 263K scrapie agent [J].Acta Neuropathol, 1998, 96: [J],,,. 139A [J]., Robakis N K, Sawh P R, Wolfe G C, et al. Isolation of a cdna clone encoding the leader peptide of prion protein and expression of the homologous gene in various tissues [J]. Proc Natl Acad Sci USA, 1986, 83:

8 Collinge J, Whittington M A, Sidle K C, et al. Prion protein is necessary for normal synaptic function [J]. Nature, 1994, 370: The Study on Prion Gene Tissue-specific Expression of Golden Hamster* NING Zhang-yong, ZHAO De-ming, YANG Jian-min, CUI Ya-li, MENG Li-ping, WU Chang-de, QIN Xiiu-hui, MA Li-ying (National Animal Transmissible Spongiform Encephalopathies Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing , china) Abstract: Golden hamster is one of the favored experimental models for studying mechanisms of transmissible spongiform encephalopathies (TSE). We use the real-time fluorescent RT-PCR technique, constructing the standard plasmid and curve to quantify the PrP gene expression. Total RNA was isolated from four different regions of the brain and six peripheral organs to quantify the gene expression after the Reverse Transcript. The result showed that high mrna levels were present in all four regions of the brain examined. In peripheral organs examined, inguinal lymph node showed high level of the expression similar to that in overall brain, spleen, heart, liver and lung showed moderate levels of the expression and kidney showed the lowest expression. Our result provided the basic data of the function of the PrP c and the effection of different organs in prion diseases. Key words: Golden Hamster; Prion; Gene expression