Engineering a murine cell line for the stable propagation of hamster prions

Abstract

Edited by Ursula Jakob Prions are infectious protein aggregates that cause several fatal neurodegenerative diseases. Prion research has been hindered by a lack of cellular paradigms for studying the replication of prions from different species. Although hamster prions have been widely used to study prion replication in animals and within in vitro amplification systems, they have proved challenging to propagate in cultured cells. Because the murine catecholaminergic cell line CAD5 is susceptible to a diverse range of mouse prion strains, we hypothesized that it might also be capable of propagating nonmouse prions. Here, using CRISPR/ Cas9-mediated genome engineering, we demonstrate that CAD5 cells lacking endogenous mouse PrP expression (CAD5-PrP ؊/؊ cells) can be chronically infected with hamster prions following stable expression of hamster PrP. When exposed to the 263K, HY, or 139H hamster prion strains, these cells stably propagated high levels of protease-resistant PrP. Hamster prion replication required absence of mouse PrP, and hamster PrP inhibited the propagation of mouse prions. Cellular homogenates from 263K-infected cells exhibited prion seeding activity in the RT-QuIC assay and were infectious to naïve cells expressing hamster PrP. Interestingly, murine N2a neuroblastoma cells ablated for endogenous PrP expression were susceptible to mouse prions, but not hamster prions upon expression of cognate PrP, suggesting that CAD5 cells either possess cellular factors that enhance or lack factors that restrict the diversity of prion strains that can be propagated. We conclude that transfected CAD5-PrP ؊/؊ cells may be a useful tool for assessing the biology of prion strains and dissecting the mechanism of prion replication.

Gene-edited murine cell lines for propagation of chronic wasting disease prions

“…Even though “promiscuous” PrPs like bank vole PrP are suitable for a range of prions, they may not necessarily provide optimum infectivity as was observed here with MEF cells. Very recently, the successful engineering of CAD5 cells for the stable propagation of hamster prions was shown 54 .…”

An astrocyte cell line that differentially propagates murine prions

“…Interestingly, these were mostly established cell lines that allowed persistent propagation of mouse-adapted scrapie prion strains like 22L, RML, or ME7 ( 24 , 25 ). By using genetically engineered cells, it seems possible to expand the range of prions which can be propagated in such cell lines ( 26 – 28 ). There still is no cell line that can persistently propagate human or bovine prions ( 25 ).…”

From Cell Culture to Organoids-Model Systems for Investigating Prion Strain Characteristics

“…These bvCAD5 cells could also be infected with 22L prions [ 33 ]. Additionally, Bourkas et al, demonstrated that CAD5 cells expressing hamster PrP could propagate the 263K, hyper (HY) and 139H strains of hamster-adapted prions, but not the drowsy (DY) strain [ 32 ]. Thus, CRISPR has proven to be a useful tool for investigating species barriers.…”