Journal
BMC BIOLOGY
Volume 20, Issue 1, Pages -Publisher
BMC
DOI: 10.1186/s12915-021-01223-w
Keywords
Mouse models; Gene editing; Conditional knockout mice; CRISPR; Floxing; Functional genomics
Categories
Funding
- NCI Cancer Center Support Grant [P30 CA091842]
- NIH/NIDDK grant [2P01 DK096990-06A1]
- Rheumatic Disease Research Resource-Based Center at Washington University
- HIH/NIAMS [P30AR073752]
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In this study, we propose an improved method for floxing genomic regions of any desired size using electroporation of fertilized eggs, which is highly reliable and predictable in terms of timeline.
Background Floxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. However, an efficient and reliable strategy to flox genomic regions of any desired size is still lacking. Results Here, we demonstrate that the method combining electroporation of fertilized eggs with gRNA/Cas9 complexes and single-stranded oligodeoxynucleotides (ssODNs), assessing phasing of loxP insertions in founders using an in vitro Cre assay and an optional, highly specific and efficient second-round targeting ensures the generation of floxed F1 animals in roughly five months for a wide range of sequence lengths (448 bp to 160 kb reported here). Conclusions Floxed alleles can be reliably obtained in a predictable timeline using the improved method of electroporation of two gRNA/Cas9 ribonucleoprotein particles (RNPs) and two ssODNs.
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