| First Author | Horii T | Year | 2017 |
| Journal | Sci Rep | Volume | 7 |
| Issue | 1 | Pages | 7891 |
| PubMed ID | 28801621 | Mgi Jnum | J:287496 |
| Mgi Id | MGI:6407613 | Doi | 10.1038/s41598-017-08496-8 |
| Citation | Horii T, et al. (2017) Efficient generation of conditional knockout mice via sequential introduction of lox sites. Sci Rep 7(1):7891 |
| abstractText | Conditional knockout using Cre/lox is essential for functional analysis of genes. CRISPR/Cas in combination with two sets of guide RNAs and a single-stranded oligonucleotide enables simultaneous insertion of two lox sequences. However, this method induces double-strand breaks at two sites on the same chromosome, which causes an undesirable chromosomal deletion and reduces the flanked lox (flox) rate. To solve this problem, we investigated a method that sequentially introduces each lox sequence at the 1-cell and 2-cell embryonic stages, respectively. The sequential method was applied to both microinjection and electroporation systems. Sequential electroporation improved the flox efficiency compared with ordinary simultaneous microinjection, leading to a high yield of offspring with floxed alleles. Finally, we directly produced Cre/lox mice containing both the Cre transgene and floxed allele via sequential electroporation using Cre zygotes, which accelerated the generation of conditional knockout mice compared with the ordinary method. |
