NR AOWR
AU McLoughlin,V.; Head,M.W.; Tate,R.J.; Baybutt,H.N.; Connolly,J.G.
TI Expression of Wild type Human and Mouse PrPc in Xenopus oocytes
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - DG-14
PT Konferenz-Poster
AB
Previously, we have shown that epitope tagged mouse and hamster PrPc can be expressed in Xenopus oocytes (Connolly et al., 2002, NeuroReport, 13, 1229-1233). Also in that study, RT PCR analysis showed that no endogenous Xenopus laevis PrPc mRNA is present in Xenopus oocytes. We have now extended this work to show that wild type human and mouse PrPc can be expressed and detected within Xenopus oocytes non-isotopically. mRNAs were transcribed in vitro from template DNAs encoding wild type Mouse and Hamster PrPc and injected into Xenopus oocytes. Oocytes were incubated for 2 days at 18 C (Class 3 containment) before homogenisation. Crude membrane fractions were extracted and then separated on SDS denaturing gels. Expression of PrPc was assayed by Western Blotting. No PrPc expression was detected in extracts from uninjected oocytes. However in oocytes injected with RNA encoding human PrPc, PrPc was detected with both 3F4 and 6H4 antibodies. As expected, the wild type mouse PrPc expressed in oocytes was not recognised by the 3F4 antibody, but was recognised by 6H4. Both forms of PrPc were similar in size and abundance to PrPc protein from human brain and recombinant hamster PrPc. Non-glycosylated, mono-glycosylated and di-glycosylated forms of the protein appear to be expressed; and an additional, higher molecular glycoform was also seen in some oocyte samples.
This work demonstrates that with appropriate safety precautions, Xenopus oocytes are a very versatile system for studying the properties of various glycosylated prion protein isoforms from a variety of species.
This work was supported by the Department of Health.
AD Victoria McLoughlin, Roth J. Tate, John G. Connolly, University of Strathclyde, UK; Mark W. Head, University of Edinburgh, UK; Herbert Baybutt, Institiute for Animal Health, UK
SP englisch
PO Deutschland