Design and Plasmid Construction of sgRNAs Targeting Rosa26 Locus
2:28
Design and Construction of Targeting Vector as Homologous Recombination Template
3:46
Electroporation of Macrophage and T Cell Lines
7:19
Cell Sorting to Isolate Putative Knock-in Cells
8:58
Screening and Validation of Positive Knock-in Cells
10:16
Representative Results
10:52
Conclusion
副本
The goal of this protocol is to simplify CRISPR/Cas9 mediated knock-in experiment in macrophage and T-cell lines, with the aid of fluorescent reporters and affect cell sorting. ROSA26 Locus is known as a genomic safe harbor site for insertion of t
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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.
