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Immunology and Infection

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基因敲击由CRISPR/Cas9和细胞排序在巨噬细胞和T细胞线

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Immunology and Infection
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JoVE Journal Immunology and Infection

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

Please note that all translations are automatically generated. Click here for the English version.

基因敲击由CRISPR/Cas9和细胞排序在巨噬细胞和T细胞线

DOI:

10.3791/62328-v

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11:32 min

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November 13, 2021

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Lichen Zhang, Rong Huang, Liaoxun Lu, Rui Fu, Guo Guo, Yanrong Gu, Zhuangzhuang Liu, Le He, Marie Malissen, Yinming Liang

¹The Laboratory of Genetic Regulators in the Immune System, School of Laboratory Medicine,Xinxiang Medical University, ²Centre d’Immunologie de Marseille-Luminy,Aix Marseille Université

Chapters

00:05Introduction
00:32Design and Plasmid Construction of sgRNAs Targeting Rosa26 Locus
02:28Design and Construction of Targeting Vector as Homologous Recombination Template
03:46Electroporation of Macrophage and T Cell Lines
07:19Cell Sorting to Isolate Putative Knock-in Cells
08:58Screening and Validation of Positive Knock-in Cells
10:16Representative Results
10:52Conclusion

Summary

Automatic Translation

该协议使用荧光记者和细胞分拣来简化巨噬细胞和T细胞系的敲击实验。两个质粒用于这些简化的敲击实验，即CRISPR/Cas9-和DsRed2表达质粒和同源重组供体质粒表达EBFP2，这是永久整合在 Rosa26 点在免疫细胞。

Tags

CRISPR/Cas9 Gene Knock-in Macrophage T Cell Lines ROSA26 Locus SgRNA Design Homologous Recombination Targeting Vector Fluorescent Reporter Cell Sorting

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