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JoVE Journal
Immunology and Infection
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Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines
 

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Article DOI: 10.3791/62328-v 11:32 min November 13th, 2021
November 13th, 2021

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Summary

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Tags

Gene Knock-in CRISPR/Cas9 Cell Sorting Macrophage T Cell Lines Fluorescent Reporters ROSA26 Locus Transgenes Plasmid Construction SgRNAs Mouse Rosa26 Locus Genomic Sequence CRISPOR PAM Motif Specificity Score Off Targets Knock-in Efficiency Annealed Phosphorated Cloning Linearized Vector
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