Production of genome-edited mice by visualization of nucleases introduced into the embryos using electroporation

Abstract

Genome editing technology contributes to the quick and highly efficient production of genetically engineered animals. These animals are helpful in clarifying the mechanism of human disease. Recently, a new electroporation technique (TAKE: Technique for animal knockout system by electroporation) was developed to produce genome-edited animals by introducing nucleases into intact embryos using electroporation instead of the microinjection method. The aim of this study was to increase the efficiency of production of genome-edited animals using the TAKE method. In the conventional protocol, it was difficult to confirm the introduction of nucleases into embryos and energization during operation. Using only embryos that introduced nucleases for embryo transfer, it will lead to increased efficiency in the production of genome-edited animals. This study examined the visualization in the introduction of nucleases into the embryos by using nucleases fluorescent labeled with ATTO-550. The embryos were transfected with Cas9 protein and fluorescent labeled dual guide RNA (mixture with crRNA and tracrRNA with ATTO-550) targeted tyrosinase gene by the TAKE method. All embryos that survived after electroporation showed fluorescence. Of these embryos with fluorescence, 43.7% developed to morphologically normal offspring. In addition, 91.7% of offspring were edited by the tyrosinase gene. This study is the first to demonstrate that the introduction of nucleases into embryos by the TAKE method could be visualized using fluorescent-labeled nucleases. This improved TAKE method can be used to produce genome-edited animals and confirm energization during operation.

Successful pseudopregnancy of rats by short period artificial stimulation using sonic vibration

“…The pronuclear and two-cell embryos that were transferred into the oviducts of these pseudopregnant female rats developed into normal offspring. This protocol is expected to be useful for the production of genome-edited animals 26 – 28 , production of generations from cryopreserved gametes 29 , 30 , and specific pathogen-free (SPF) animal colonies. Furthermore, this improved procedure could reduce the need for large breeding spaces and pain during treatment to induce pseudopregnancy and contribute to animal welfare and 3Rs.…”

Amount of Cas9 protein introduced into mouse embryos via electroporation affects the genome-editing rate

“…It is possible to discriminate the results of genome editing from the eye color of offspring derived from C57BL/6 × ICR embryos without genetic analysis by knocking out the tyrosinase gene. The nuclease solution for embryo electroporation contained 200 ng/μl Cas9-GFP, 15 μM crRNA, 15 μM tracrRNA or a mixture solution with 7.5 μM tracrRNA and 7.5 μM tracrRNA-ATTO550 in Opti-MEM (Thermo Fisher Scientific Inc., MA, USA) [ 8 ] was prepared just before electroporation.…”

“…The fluorescence of electroporated embryos was observed using an inverted microscope (Figs 1 and 3). The fluorescence intensity inside each embryo was measured using the ImageJ software (https://imagej.nih.gov/ij/) (Fig 1) [8]. The mean gray values for each embryo were plotted and compared (Figs 2B and 4B).…”

“…However, it is difficult to confirm nuclease delivery into embryos after electroporation using the conventional TAKE method. We have previously demonstrated the successful visualization of nucleases in embryos following electroporation using fluorescently-labeled tracrRNA as part of the guide RNA [ 8 ]. In that study, the genome-editing rate had significantly increased with increasing pulse length; however, no significant differences were observed in the average fluorescence intensities at the different pulse lengths.…”

The iodide transporter Slc26a7 impacts thyroid function more strongly than Slc26a4 in mice

“…RiboNucleoProtein (RNP) complexes with Alt-R S.p. Cas9 nuclease V3 (IDT) were transferred to C57BL/6N fertilized oocytes as previously described 17 . F0 mice with the desired mutations were crossed with wild-type (WT) mice to obtain heterozygous F1 mice.…”