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Updated: Aug 1, 2025
Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins
Published on: June 14, 2018
Protocol for casein kinase 1γ3 CSNK1G3 gene knockout and recombinant gene expression in cultured HeLa cells
1Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan; Department of Virology III, National Institute of Infectious Diseases, Musashimurayama City, Tokyo 208-0011, Japan.
Abstract:
Casein kinase 1γ is a subfamily of the casein kinase 1 family. Here, we present a protocol for gene knockout of CSNK1G3 in human cells and cloning and/or expression of CSNK1G1-3 cDNAs using a retroviral vector system. We first describe the genome editing procedures, including sgRNA design, introduction into HeLa cells, and verification of genome editing. Next, we describe the procedures for cloning human CSNK1G cDNAs, introduction into HeLa cells, and expression verification by western blot analysis. For complete information on the generation and use of this protocol, please refer to Goto et al.1.
Insights
This study details a protocol for gene knockout of CSNK1G3 and expression of CSNK1G1-3 in human cells using retroviral vectors. The methods cover genome editing, cDNA cloning, and expression verification for casein kinase 1γ research.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Casein kinase 1γ (CK1γ) is a subfamily within the larger casein kinase 1 family.
- Understanding the function of CK1γ members requires robust genetic manipulation tools.
Purpose of the Study:
- To establish a comprehensive protocol for gene knockout of CSNK1G3 in human cells.
- To describe methods for cloning and expressing CSNK1G1-3 cDNAs using a retroviral vector system.
Main Methods:
- Genome editing using sgRNA design, introduction into HeLa cells, and verification.
- Cloning of human CSNK1G cDNAs.
- Retroviral vector-mediated gene delivery into HeLa cells.
- Expression verification via Western blot analysis.
Main Results:
- Successful implementation of genome editing for CSNK1G3 knockout.
- Efficient cloning and expression of CSNK1G1-3 cDNAs in human cells.
- Demonstrated utility of the retroviral vector system for CK1γ subfamily studies.
Conclusions:
- The presented protocol provides a reliable method for genetic manipulation of the CK1γ subfamily in human cells.
- This toolkit facilitates further investigation into the biological roles of CSNK1G1, CSNK1G2, and CSNK1G3.
- The study offers a foundation for future research on CK1γ in various cellular contexts.
