Dec 04, 2024

Public workspaceGenome Edited (KO) Cell Line Generation –

The Journal of Cell Biology
DOI
dx.doi.org/10.17504/protocols.io.dm6gp9bp5vzp/v1
  • 1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 2Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 3Program in Cellular Neuroscience, Neurodegeneration and Repair;
  • 4Wu Tsai Institute Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
  • 6Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Amanda Bentley-DeSousa
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DOI: dx.doi.org/10.17504/protocols.io.dm6gp9bp5vzp/v1
Protocol Citation: Amanda Bentley-DeSousa, Shawn Ferguson 2024. Genome Edited (KO) Cell Line Generation –. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp9bp5vzp/v1
Manuscript citation:
https://doi.org/10.1101/2023.10.31.564602
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: December 02, 2024
Last Modified: December 04, 2024
Protocol Integer ID: 113413
Keywords: RAW 264.7, KO, Synthego
Funders Acknowledgements:
Aligning Science Across Parkinson’s Disease
Grant ID: ASAP-000580
Abstract
This protocol details steps taken to create KO cell lines in RAW 264.7 cells.
Materials
DMEM (Thermo Fisher Scientific, 11965-092)
FBS (Thermo Fisher Scientific, 16140-071)
Penicillin/Streptomycin (Thermo Fisher Scientific, 15140122)
CellStripper (Corning, 356230)
Synthego CRISPR Gene Knockout V2 Mouse Kits
Lipofectamine CRISPRiMAX (ThermoFisher Scientific, CMAX00003)
Cas9 (Synthego CRISPR Gene Knockout V2)
Opti-Mem (Thermo Fisher Scientific, 31985062)
Day 0
Day 0
Plate 2.5 x 10^5 RAW 264.7 cells per well in a 6-well dish.
Day 1
Day 1
Transfect cells using Lipofectamine CRISPRiMAX, gene specific sgRNAs and recombinant Cas9 (Synthego CRISPR Gene Knockout V2).
CRISPRiMAX protocol
In Tube 1 combine Amount62.5 µL Opti-MEM, Amount3.25 µL sgRNA (from 3 uM stock), Amount2.5 µL Cas9 (from 3 uM stock), and Amount2.5 µL Cas9+ then incubate for Duration00:05:00 . In Tube 2 combine Amount62.5 µL Opti-MEM and Amount3.75 µL CRISPRMAX then incubate for Duration00:05:00 . Combine tube 1 and tube 2. Incubate for Duration00:20:00 . Add drop-wise to 1 well containing Amount1.8 mL of media.

Day 3
Day 3
Change the media.
Daty 4
Daty 4
Plate single cells into 96-well dishes to obtain clonal cell lines. After subculturing, confirm cell lines via immunoblotting