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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
Published on: June 16, 2017
Generating stable cell lines with quantifiable protein production using CRISPR/Cas9-mediated knock-in.
Chiu-An Lo1,2, Alexander W Greben1,2, Brian Edwin Chen1,2
1Centre for Research in Neuroscience, Research Institute of the McGill University Health Centre.
Biotechniques
|April 14, 2017
Summary
This study presents an efficient method for creating stable cell lines that produce recombinant proteins consistently. Using CRISPR/Cas9 gene editing and a novel reporter system ensures predictable and quantifiable protein expression levels.
Area of Science:
- Biotechnology
- Molecular Biology
- Genetic Engineering
Background:
- Cell lines are crucial for recombinant protein production.
- Current methods for generating these cell lines are time-consuming and yield inconsistent expression levels.
Purpose of the Study:
- To develop an efficient and reliable method for creating stable cell lines for recombinant protein expression.
- To achieve controllable and quantifiable protein production.
Main Methods:
- Utilized CRISPR/Cas9 to integrate transgenes into specific genomic loci.
- Employed endogenous promoters to drive transgene expression for consistency.
- Incorporated a protein quantitation reporter (PQR) for simultaneous production of endogenous protein, recombinant protein, and a fluorescent reporter.
Main Results:
- Achieved stable cell lines with predictable recombinant protein expression.
- Demonstrated controllable expression levels by selecting specific endogenous promoters.
- Enabled continuous monitoring of both endogenous and recombinant protein levels via fluorescence.
Conclusions:
- The described method offers an efficient approach for generating stable recombinant protein-expressing cell lines.
- This technique ensures consistent, quantifiable, and controllable protein production.
- The integrated reporter system facilitates real-time monitoring of protein expression.
