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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
Published on: June 16, 2017
Genetic knockouts and knockins in human somatic cells
Carlo Rago1, Bert Vogelstein, Fred Bunz
1Sidney Kimmel Comprehensive Cancer Center, Cellular and Molecular Medicine Program, Johns Hopkins University School of Medicine, 1550 Orleans Street CRB2-453, Baltimore, Maryland 21231, USA.
Nature Protocols
|November 17, 2007
Summary
This study details a method for gene targeting in human cells using recombinant adeno-associated viruses (rAAVs). This efficient technique allows for the precise modification of mammalian gene function for research purposes.
Area of Science:
- Molecular Biology
- Genetics
- Mammalian Cell Culture
Background:
- Gene targeting is crucial for understanding mammalian gene function.
- Traditional methods have been adapted for cultured human cells.
- Recombinant adeno-associated viruses (rAAVs) represent a significant innovation in gene targeting.
Purpose of the Study:
- To describe a protocol for gene targeting in cultured human cells using rAAVs.
- To enable the generation of cell lines with specific gene alterations for functional analysis.
Main Methods:
- Design and construction of DNA targeting vectors.
- Production of infectious rAAV stocks.
- Generation and screening of cell clones for homologous recombinants.
- Iterative targeting of multiple gene alleles.
Main Results:
- A protocol enabling the generation of a cell line with a single altered allele in 3 months.
- The capability to target a second allele of the same gene in an additional 3 months.
Conclusions:
- rAAV-mediated gene targeting provides an efficient method for analyzing mammalian gene function in human cells.
- This protocol facilitates the rapid generation of genetically modified cell lines for research.
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