earticle

영어

As a disease model, rats have similar physiological characteristics to humans and are used in various research methods. However, a protocol to generate genome-edited rats has not been well established. Since its development as a genome editing tool, the CRISPR/ Cas9 system has been widely adapted as an efficient tool to generate knockout (KO) or transgenic rats. In general, microinjection technique, which involves direct injection of CRISPR/ Cas9 into one-cell embryos were utilized for generating KO rodents. However, this technique has several disadvantages: 1. User has to be well-trained to manipulate single zygotes; 2. User can only inject one cell at a time (long and laborious); 3. Low viability after microinjection is observed. To overcome these, we adapted electroporation system which is relatively easy to handle and up to 100 zygotes can be electroporated at once. In this study, female Sprague Dawley (SD) rats at 8-week-old were superovualted and mated with mature male SD rat. Zygotes were obtained the following day and eletroporation was performed (Genome Editor, BEX CO, Japan) to introduce sgRNA and Cas9 protein targeting slc16a2 gene. Zygotes were cultured in a incubator and only two-cell stage embryos were transferred into the recipients. After gestation periods, genome edited founder (F0) offspring were born and confirmed by genomic PCR. Homozygous KO pups (F1) were delivered by mating founder rats. While higher levels of serum triiodothyronine were detected in homozygous KO (1.30±0.26 ng/mL, n=3) than control group (1.13±0.21 ng/mL, n=3), thyroxine levels were lower in the homozygous KO F1 (3.13±0.50 μg/dL, n=3) compared to the control group (6.53±1.19 μg/dL, n=3). In addition, there was a significant difference in body weight between homozygous KO F1 and the control group (homozygous KO F1 : 287.23± 8.26 g vs control group : 339.03+52.26 g, p<0.05, t-test, SPSS). Using this method, KO rats were produced with high efficiency. In the future, electroporation-based genome editing technology can provide simple and efficient methods to produce KO rat models.