shRNA knockdown
Nancy C. Hernandez Villegas, schekman, wusj
Abstract
This protocol describes a standard procedure to generate a stable cell line for a DNAJC5 knockdown using shRNA.
Steps
shRNA knockdown
HEK293T cells were plated at a density such that on the day of transfection they are no more than 50% confluent.
plKO.1-Hygroplasmids-containing shRNA targeting DNAJC5 (ccggGCAACCTCAGATGACATTAAACTCGAGTTTAATGTCATCTGAGGTTGCTTTTTG) together with pMD2.G and PsPAX2 were transfected into HEK293T cells to produce lentiviral particles for 72 hr.
Lentivirus particles were concentrated with Lenti-X Concentrator follow manufacturer protocol (Takara Bio).
SH-SY5Y cells were transduced by lentivirus before differentiation.
Three days post transduction, cells were selected with 250 μg/ml hygromycin for 10 days.
The selected cells were differentiated, and the knockdown was verified with immunoblot.
