In vitro analysis of prion proliferation : Modified PMCA method using PrP-gene deficient cell line

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In this study, protein misfolding cyclic amplification (PMCA) was performed using prion protein (PrP)-gene deficient cell line as the source of cellular PrP. Mouse and hamster scrapie and bovine spongiform encephalopathy (BSE) prion were used for seed. In 2008, cell lysate was prepared from prion protein (PrP)-gene deficient cell re-introduced with mouse, hamster, and bovine PrP-gene. In 2009, we compared the in vitro amplification efficiency in PMCA among hamster scrapie 263K, mouse scrapie Obihiro, mouse Chandler scrapie prions. As the results, PrPres could be amplified from brain homogenates infected with 263K and Chandler prion under the condition of PBS containing 1% TritonX-100, 4mM EDTA (40 cycles), whereas Obihiro prion could not be amplified. In addition, PMCA using cell lysate required different conditions for PrPres amplification compared to brain homogenate. Therefore, we concluded that the adaptation of PMCA conditions were required for efficient PrPres proliferation depending on prion strains and PrPC sources.