Cultured cell sublines highly susceptible to prion infection
Cultured cell sublines highly susceptible to prion infection
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DOI：
10.1128/jvi.74.9.4377-4386.2000
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发表时间：
2000-05-01
影响因子：
5.4
通讯作者：
Prusiner, SB
中科院分区：
文献类型：
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作者：
Bosque, PJ;Prusiner, SB
感染朊病毒的培养细胞系产生朊病毒蛋白的异常同种型（PrPSc）。为了获得用于大多数研究的产生足够量的PrPSc的细胞系，有必要亚克隆感染的培养物并选择产生最大量的PrPSc的亚克隆。由于感染后克隆可以引入感染和未感染细胞系之间的差异，我们寻求一种方法来产生朊病毒感染的细胞系，将避免克隆假象。使用一种改进的细胞印迹技术，它允许敏感和快速比较PrPSc水平在多个独立的细胞培养，我们发现显着的异质性方面的事先，在肿瘤细胞亚系的易感性。我们利用这种异质性来获得对朊病毒感染高度敏感的亚系，并使用这些细胞来产生朊病毒感染的细胞系，而无需进一步亚克隆。这些感染的亚系可以与同源的未感染的培养物进行比较，而不受克隆人工制品的干扰。我们还使用易感细胞系和我们的改良细胞印迹程序开发了一种敏感的和可重复的定量细胞培养生物测定朊病毒，我们发现，亚系对RML株朊病毒的敏感性至少是ME7株朊病毒的100倍。羊瘙痒症易感细胞系和抗羊瘙痒症细胞系之间的比较可能揭示调节朊病毒繁殖的因素。
Cultured cell lines infected with prions produce an abnormal isoform of the prion protein (PrPSc), In order to derive cell lines producing sufficient quantities of PrPSc for most studies, it has been necessary to subclone infected cultures and select the subclones producing the largest amounts of PrPSc. Since postinfection cloning can introduce differences between infected and uninfected cell lines, we sought an approach to generate prion-infected cell lines that would avoid clonal artifacts. Using an improved cell blot technique, which permits sensitive and rapid comparison of PrPSc levels in multiple independent cell cultures, we discovered marked heterogeneity with regard to prior, susceptibility in tumor cell sublines. We exploited this heterogeneity to derive sublines which are highly susceptible to prion infection and used these cells to generate prion-infected lines without further subcloning. These infected sublines can be compared to the cognate uninfected cultures without interference from cloning artifacts. We also used susceptible cell lines and our modified cell blot procedure to develop a sensitive and reproducible quantitative cell culture bioassay for prions, We found that the sublines were at least 100-fold more susceptible to strain RML prions than to strain ME7 prions, Comparisons between scrapie-susceptible and -resistant cell lines may reveal factors that modulate prion propagation.