NR AOUX
AU Klose,R.; Schwengler,F.; Zakhartchenko,V.; Lü,L.; Wenigerkind,H.; Wolf,E.
TI Efficient targeting of PRNP in bovine primary fibroblasts - basis for the generation of PrPc-deficient cattle for prion research
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - BR-66
PT Konferenz-Poster
AB
Cattle lacking PrPc expression are required for the evaluation of the physiological functions of PrPc in bovine as well as for clarifying the role of PrPc in the pathogenesis of BSE. If PrPc-deficient cattle proof to be resistant to BSE as is expected from studies in Prnp-/- mice (Büeler et al., 1993), such cattle are very important for animal biotechnology, providing a source of biopharmaceutical products devoid the risk for BSE. Due to the lack of germ-line competent embryonic stem cells in livestock species, nuclear transfer using targeted donor cells is so far the only possibility to perform gene knock-outs in livestock species.
We constructed a targeting vector for bovine PRNP in which the entire coding sequence for PrPc is replaced by a promoter-less neomycin resistance gene and optimized the transfection and selection strategy in primary fetal fibroblasts for efficient recovery of targeted clones. We achieved efficient targeting of the PRNP gene in bovine fibroblasts (3.5 x 10-6 to 9.7 x 10-5). So far, 3 clones have been successfully used for nuclear transfer, and blastocysts have been transferred to recipients. One pregnancy was diagnosed by ultrasound.
From PRNP+/- fetuses fibroblasts are established in which the second PRNP allele is targeted using a promoter-less hygromycin resistance gene. PRNP-/- cells are then used for nuclear transfer to produce PrPc-deficient offspring.
AD R. Klose, F. Schwengler, V. Zakhartchenko, L. Lü, E. Wolf, Department of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, Munich, Germany; H. Wenigerkind, E. Wolf, Bavarian Research Center for Biology of Reproduction (BFZF), Oberschleissheim, Germany
SP englisch
PO Deutschland