sgRNAs for elimination of mouse Irsp53, Irtks, Mtss2, Mtss2, Abi1, Abi2, and mouse or rat Myo10 were ligated into expression plasmid pSpCas9(BB)-2A-Puro(PX459)V2.0 (67 (link)). The targeting construct for the Raph1 gene has been reported (35 (link)). After transfection and selection with puromycin of B16-F1, NIH 3T3, Rat2, and derived cells, routinely cultivated at 37 °C and 5% CO2 in high-glucose DMEM culture medium supplemented with 1% penicillin-streptomycin, 10% FBS, and 2 mM UltraGlutamine, clonal cell lines were validated by the TIDE sequence trace decomposition web tool (40 (link)) and immunoblotting. Detailed information is described in SI Appendix, Materials and Methods .
Protocol detail
CRISPR Knockout of Mouse Cytoskeleton Regulators
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Abi1
Abi2
Cell lines
Cells
Clonal
Culture medium
Glucose
Irsp53
Mouse
Nih 3t3
Penicillin
Plasmid
Puromycin
Streptomycin
Transfection
Corresponding Organization :
Other organizations : Medizinische Hochschule Hannover, FIRC Institute of Molecular Oncology, Italian Association for Cancer Research, University of Milan, Helmholtz Centre for Infection Research, Technische Universität Braunschweig
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Variable analysis
independent variables
- SgRNAs for elimination of mouse Irsp53, Irtks, Mtss2, Mtss2, Abi1, Abi2, and mouse or rat Myo10
- Targeting construct for the Raph1 gene
- Cell lines validated by the TIDE sequence trace decomposition web tool
- Protein levels validated by immunoblotting
- B16-F1, NIH 3T3, Rat2, and derived cells
- Cells routinely cultivated at 37 °C and 5% CO2 in high-glucose DMEM culture medium supplemented with 1% penicillin-streptomycin, 10% FBS, and 2 mM UltraGlutamine
- Positive controls not explicitly mentioned
- Negative controls not explicitly mentioned
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