Figure 1. Generation and characterization of the ovinized SH-SY5Y cell lines.
(A) Generation of SH-SY5YΔPRNP cell lines expressing the ovine VRQ PrPC variant and its subsequent infection with the PG127 strain of sheep-derived prions passaged in tg338 mice. HindIII and EcoRI restriction sites were used to clone the ovine PRNP construct. Ticks in the plasmid map correspond to increments of 1,000 base pairs. hSP, human signal peptide (purple); oSP, ovine signal peptide (blue); hovS, monoclonal ovSH-SY5Y; povS, polyclonal ovSH-SY5Y; ovSH-SY5Y, SH-SY5YΔPRNP transfected with a plasmid harboring the sequence for ovine PRNP (ovPRNP). (B) Western blot analysis comparing the expression levels of PrPC in hovS and povS with those in wt SH-SY5Y and in the human cell lines U251-MG and LN229. HovS and povS showed similar PrPC expression levels as U251-MG and LN229, whereas the levels in wt SH-SY5Y were slightly lower. SH-SY5YΔPRNP cells were used as negative control and actin as loading control. The anti-PrP antibody POM2 was used for detection. (C) Confocal imaging to detect cell surface exposed PrPC on hovS and povS. hovS and a subpopulation of povS showed a strong signal for cell surface exposed PrPC, whereas no detectable signal was visible for SH-SY5YΔPRNP. LN229 cells were used as positive control. The anti-PrP antibody POM1 (here and henceforth) was used for detection of PrP.
Source data are available for this figure.
