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Optimized CRISPR-Cas9-based strategy for complex gene targeting in murine embryonic stem cells for germline transmission.

Bio Protoc. 12:e4423 (2022)
Postprint DOI
Open Access Green
Although CRISPR-Cas9 genome editing can be performed directly in single-cell mouse zygotes, the targeting efficiency for more complex modifications such as the insertion of two loxP sites, multiple mutations in cis, or the precise insertion or deletion of longer DNA sequences often remains low (Cohen, 2016). Thus, targeting and validation of correct genomic modification in murine embryonic stem cells (ESCs) with subsequent injection into early-stage mouse embryos may still be preferable, allowing for large-scale screening in vitro before transfer of thoroughly characterized and genetically defined ESC clones into the germline. This procedure can result in a reduction of animal numbers with cost effectiveness and compliance with the 3R principle of animal welfare regulations. Here, we demonstrate that after transfection of homology templates and PX458 CRISPR-Cas9 plasmids, EGFP-positive ESCs can be sorted with a flow cytometer for the enrichment of CRISPR-Cas9-expressing cells. Cell sorting obviates antibiotic selection and therefore allows for more gentle culture conditions and faster outgrowth of ESC clones, which are then screened by qPCR for correct genomic modifications. qPCR screening is more convenient and less time-consuming compared to analyzing PCR samples on agarose gels. Positive ESC clones are validated by PCR analysis and sequencing and can serve for injection into early-stage mouse embryos for the generation of chimeric mice with germline transmission. Therefore, we describe here a simple and straightforward protocol for CRISPR-Cas9-directed gene targeting in ESCs.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Schlagwörter Crispr-cas9 ; Gemms ; Gene Targeting ; Genetically Engineered Mouse Mutants ; Mesc ; Murine Embryonic Stem Cells ; Mutagenesis
e-ISSN 2331-8325
Zeitschrift Bio-Protocol
Quellenangaben Band: 12, Heft: 10, Seiten: , Artikelnummer: e4423 Supplement: ,
Verlag bio-protocol.org
Verlagsort Sunnyvale, CA
Begutachtungsstatus Peer reviewed
Förderungen Deutsche Krebshilfe
Deutsche Forschungsgemeinschaft