NR AXPQ
AU Kang,S.G.; Lee,S.I.; Lee,D.Y.; Kang,M.L.; Kim,Y.S.; Kim,B.H.
TI Reconstitution of N2a Cells to be Expressed Bovine PrPc and Suppressed Endogenous Mouse PrPc by Lentivector-mediated RNAi
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Natural and Experimental Strains P02.13
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
It has been cleared in evidences that there are several strains of the causative agents of prion disease which can be distinguished by the disease characteristics, in particular the incubation periods and neuropathological profiles. However, the most prion researches have been investigated through mouse and hamster model as experimental animals. Although knockout strategies have been classically applied to reduce the gene expression, these are not practically applicable for therapeutic issues, and an intergenic splicing event brought an ectopic gene expression which caused cebellar Purkinje cell degeneration in some case of Prnp knockout mice. The use of RNA interference (RNAi) which is a highly conserved and sequence-specific posttranscriptional gene-silencing mechanism was recently shown to result in high and specific inhibition of targeted gene expression.
To establish in vitro BSE-specific model, we reconstructed N2a cell to be expressed bovine PrPc by transfection of bovine Prnp, on the other hand, suppressed endogenous mouse PrPc by lentivector mediated RNAi. The synthetic siRNAs and the lentivector-mediated shRNAs targeting N- and C- terminus of mouse Prnp were designed and applied to N2a and NbP cell which is the reconstructed N2a cell expressing bovine PrPc. Both siRNA-1 and -2 effectively decreased the expression of prion protein by more than 80%. This down-regulation was associated with a concomitant reduction of the mRNA level. For the tetracycline (Tet)-on regulation of shRNAs, N2a and NbP cells were viral-transduced with Lenti6/TR and then with the designed Lenti4-shRNAs. The expression of prion protein was efficiently blocked when the plasmids carrying shRNA-1 and -2 were viral-transduced simultaneously and the transcription was turned on through adding 2 µg/ml of Tet. The reconstructed N2a and NbP with shRNA1 and 2, which are inducible Prnp knockdown cell lines using Tet-on regulatory system might be considered as a profitable tool for development of the therapeutics and basic prion researches as well as BSE.
This study was supported by BK21 Program for Veterinary Science, KRF 2006-005J502901 and the Research Institute of Veterinary Science, Seoul National University, Korea.
Reference:
1. Aguzzi, A. & Heikenwalder, M. (2006) Nat Rev Microbiol 4, 765-775.
2. Cullen, B. R. (2006) Nat Methods 3, 67.
AD S.-G. Kang, S.I. Lee, D.-Y. Lee, M.L. Kang, College of Veterinary Medicine, Seoul National University, Department of Infectious Diseases, Korea; Y.-S. Kim, B.-H. Kim, Ilsong Institute of Life Science, College of Medicine, Hallym University, Department of Microbiology, Korea
SP englisch
PO Schottland
EA pdf-Datei und Poster (Autorenliste reduziert um D.Y Lee und ergänzt um H.S. Yoo)