Floxing by Electroporating Single-Cell Embryos with Two CRISPR RNPs and Two ssODNs.
Methods Mol Biol
; 2631: 231-252, 2023.
Article
em En
| MEDLINE
| ID: mdl-36995670
ABSTRACT
Floxed alleles and Cre drivers are two components of most conditional knockout mouse models, which are not only important for studying a given gene in a tissue-specific manner, but also useful for functional analysis of various sized genomic regions. With the increased demand for floxed mouse models in biomedical research, reliable and economical creation of floxed alleles is clearly highly valuable yet remains challenging. Here we provide technical details on the method consisting of electroporating single-cell embryos with CRISPR RNPs and ssODNs, next-generation sequencing (NGS)-based genotyping, an in vitro Cre assay (recombination followed by PCR) for loxP phasing determination, and optional second round targeting of an indel in cis with one loxP insertion in embryos obtained via in vitro fertilization (IVF). As importantly, we present protocols for validation of gRNAs and ssODNs before electroporation of embryos, to confirm phasing of loxP and the indel to be retargeted in individual blastocysts and an alternative strategy to insert loxP sites sequentially. Together, we hope to help researchers reliably obtain floxed alleles in a predictable and timely manner.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Eletroporação
/
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Estados Unidos