Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR
Summary: Gene functions can be assessed in mouse embryonic stem (ES) cells and in mutant mice derived from mutant ES cells. Here, we describe an approach for efficient isolation of the ES clones carrying deletion mutations at the target genes by CRISPR-Cas9. Two sgRNAs against a target gene are co-e...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-06-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166722003161 |
| _version_ | 1828814739324534784 |
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| author | Yuhan Liu Qian Chen Chenglin Song Zhen Xu Shuting Yang Xiajun Li |
| author_facet | Yuhan Liu Qian Chen Chenglin Song Zhen Xu Shuting Yang Xiajun Li |
| author_sort | Yuhan Liu |
| collection | DOAJ |
| description | Summary: Gene functions can be assessed in mouse embryonic stem (ES) cells and in mutant mice derived from mutant ES cells. Here, we describe an approach for efficient isolation of the ES clones carrying deletion mutations at the target genes by CRISPR-Cas9. Two sgRNAs against a target gene are co-expressed with puromycin-resistant gene in ES cells through co-transfection followed by transient puromycin selection. Deletion mutations are identified by PCR from individual ES clones that are picked from puromycin-selected ES cells. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-12-12T10:31:14Z |
| format | Article |
| id | doaj.art-93adf71d3993471b8aadb419e6d7c319 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-12-12T10:31:14Z |
| publishDate | 2022-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-93adf71d3993471b8aadb419e6d7c3192022-12-22T00:27:22ZengElsevierSTAR Protocols2666-16672022-06-0132101436Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPRYuhan Liu0Qian Chen1Chenglin Song2Zhen Xu3Shuting Yang4Xiajun Li5School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, ChinaSchool of Life Science and Technology, ShanghaiTech University, Shanghai 201210, ChinaSchool of Life Science and Technology, ShanghaiTech University, Shanghai 201210, ChinaSchool of Life Science and Technology, ShanghaiTech University, Shanghai 201210, ChinaSchool of Life Science and Technology, ShanghaiTech University, Shanghai 201210, ChinaSchool of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; Genome Editing Center, ShanghaiTech University, Shanghai 201210, China; Corresponding authorSummary: Gene functions can be assessed in mouse embryonic stem (ES) cells and in mutant mice derived from mutant ES cells. Here, we describe an approach for efficient isolation of the ES clones carrying deletion mutations at the target genes by CRISPR-Cas9. Two sgRNAs against a target gene are co-expressed with puromycin-resistant gene in ES cells through co-transfection followed by transient puromycin selection. Deletion mutations are identified by PCR from individual ES clones that are picked from puromycin-selected ES cells. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166722003161GeneticsMolecular BiologyCRISPRStem Cells |
| spellingShingle | Yuhan Liu Qian Chen Chenglin Song Zhen Xu Shuting Yang Xiajun Li Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR STAR Protocols Genetics Molecular Biology CRISPR Stem Cells |
| title | Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR |
| title_full | Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR |
| title_fullStr | Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR |
| title_full_unstemmed | Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR |
| title_short | Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR |
| title_sort | efficient isolation of mouse deletion mutant embryonic stem cells by crispr |
| topic | Genetics Molecular Biology CRISPR Stem Cells |
| url | http://www.sciencedirect.com/science/article/pii/S2666166722003161 |
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