Advanced Gene-Targeting Methods to Generate Cell Line Models that Preserve Native Regulatory Elements for Efficient High-Throughput Drug Screenings

摘要

Many genes were known to cause Parkinsonism. One of these genes is the SNCA which codes for a-synuclein. Elevated levels of misfolded a-synuclein cause dopaminergic neuronal loss in PD. Misfolded ot-synuclein clearance and HADC inhibitors treatment were neuroprotective to dopaminergic neurons in PD animal and cell models suggesting that manipulating transcriptional gene expression can be used to prevent dopaminergic neuronal death in PD patients. However, there is no cell line model that has the entire SNCA regulatory elements for identifying compounds that act on the SNCA regulatory elements. We aimed to produce cell lines that express either a-synuclein-luciferase or a-synuclein-GFP for high-throughput drug screenings for compounds that regulate SNCA transcriptional functions. We constructed a pair of ZFN-Fokl and donor plasmids consisting of a GFP or Luciferase gene flanked by ~ 800 bp sequences up- and downstream of the ZFN-Fokl cleaved site of the SNCA gene. The ZFN-Fokl and donor plasmids were cotransfected into growing SH-SY5Y cells, selected by 10 ug/ml puromycin, and confirmed by RT-PCR, Western blots, and chemical treatments. Two SH-SY5Y cell lines expressing a-synuclein-luciferase (Luc6B) or oe-synuclein-GFP (GFP12) were generated. RT-PCR confirmed that the reporter gene was located at the desired site. Western blots using anti-GFP, anti-a-synuclein, and anti-luciferase antibodies confirmed that both cell lines produced the desired a-synuclein fusion proteins. Valproic acid treatments of the Luc6B and GFP12 cell lines significantly increased the expression of levels a-synuclein luciferase and a-synuclein-GFP. In conclusion, we have used the ZFN method- successfully to generate cell lines which will be useful in high-throughput drug screenings to identify compounds that can inhibit the elevation or expression of a-synuclein. These cell lines provide unique tools for drug screens as they include human SNCA regulatory control regions in promoters, introns, and even distant sites that potentially interact through chromatin loops.