Abstract
[Introduction] Modern techniques that require costly equipment such as microinjection and electroporation are generally used to produce the mutant porcine embryos. Our previous report showed that a lipofection-mediated gene-editing system without specialized equipment could be performed in zona pellucida (ZP)-free oocytes and embryos. However, the factors that affect the system efficiency need to be further examined. In the present study, we evaluated two factors affecting lipofection transfection efficiency involved in the timing of subject CRISPR/Cas9 system into ZP-free zygotes and the duration of lipofection treatment. [Materials and Methods] To examine adequate introduction timing, the zygotes collected at 5 h, 10 h, and 15 h post-in vitro fertilization (IVF) were incubated with lipofection reagent, guide RNA, and Cas9 for 5 h. Next, to optimize the exposure duration, the 10 h post-IVF-zygotes were incubated with lipofection reagent for 0 h, 2.5 h, 5 h, 10 h, and 20 h. The developmental capacity of treated embryos and the genotypes of the resulting blastocysts were evaluated. [Results] The lipofection treatment could be performed in 10 h, 15 h post-IVF zygotes for 5 h of incubation to generate the mutant blastocysts, and there were no detrimental effects on the developmental capacity among the treated zygotes. However, in the exposure duration treatment, the blastocysts formation rates and mutation rates of the resulting blastocysts decreased as the duration increased from 2.5 h to 20 h. In conclusion, a lipofection-mediated gene transfection system is feasible, particularly when the zygotes derived from 10 h post-IVF were treated with lipofection for 2.5 h to generate mutant blastocysts.
