Aug 26, 2023

Public workspaceshRNA knockdown

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvwdjj7lmk/v1
  • wusj 1,
  • Nancy C. Hernandez Villegas2,
  • schekman 1
  • 1Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States;
  • 2Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, United States
Nancy C. Hernandez Villegas
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvwdjj7lmk/v1
Protocol Citation: wusj , Nancy C. Hernandez Villegas, schekman 2023. shRNA knockdown. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdjj7lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 04, 2023
Last Modified: August 26, 2023
Protocol Integer ID: 85962
Keywords: dnajc5 knockdown, shrna knockdown this protocol, shrna knockdown, using shrna, stable cell line
Funders Acknowledgements:
Nancy C. Hernandez Villegas
Grant ID: NIH training program T32GM139780
Abstract
This protocol describes a standard procedure to generate a stable cell line for a DNAJC5 knockdown using shRNA.


Materials
Reagents


ItemCatalog numberManufacturer
HEK 293T cellsCell Culture Facility, UC Berkeley
plKO.1-Hygroplasmids
DNAJC5-shRNA
pMD2.G plasmidAddgene
PsPAX2 plasmidAddgene
Lenti-X Concentrator631231Takara Bio
SH-SY5Y cellsCell Culture Facility, UC Berkeley
Hygromycin B (50 mg/mL)10687010Thermo Scientific

Troubleshooting
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.