Efficient in vitro amplification of a mouse-adapted scrapie prion protein.
Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie prion protein (PrP(Sc)), a major protein component of the infectious agents associated with prion diseases. Although exponential in vitro amplification of hamster scrapie PrP(Sc) has been established, the PMCA used was unsuccessful in achieving good amplification of PrP(Sc) from other animals. Here, we have investigated the cause of the insufficient PrP(Sc) amplification in mice and have developed an improved method suitable for amplification of the PrP(Sc) of the mouse-adapted scrapie prion strain Chandler. Mouse PrP(C), the cellular form of the prion protein, tends to become resistant to proteases during incubation independent of sonication. By adding digitonin to the reaction buffer as a lipid detergent, accumulation of the protease-resistant PrP(C) was inhibited; hence, mouse PrP(Sc) could be amplified to infinite levels. The present study is the first report describing effective amplification of PrP(Sc) of the mouse-adapted scrapie prion and this improved PMCA technique will contribute to prion research that uses mice as experimental animals.
Murayama
Yuichi
Y
Prion Disease Research Center, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. ymura@affrc.go.jp
Yoshioka
Miyako
M
Yokoyama
Takashi
T
Iwamaru
Yoshifumi
Y
Imamura
Morikazu
M
Masujin
Kentaro
K
Yoshiba
Sachiko
S
Mohri
Shirou
S
D000818:Animals; D051379:Mice; D008813:Mice, Inbred ICR; D018620:PrPSc Proteins / Q000737:chemistry / Q000378:metabolism*; D011328:Prions / Q000378:metabolism*; D017510:Protein Folding; D012608:Scrapie / Q000378:metabolism
D018620:PrPSc Proteins; D011328:Prions