earticle

영어

Severe combined immune deficiency (SCID) pig is the important animal model for translational medical research, such as the development of humanized tissues and organs for transplantation and long-term evaluation of transplanted human cancer or stem cells. FOXN1 gene encodes a transcription factor essential for the development and function of thymic epithelial cells (TECs), the primary lymphoid organ that supports T-cell development and selection. This study was performed to produce the FOXN1 knockout (KO) SCID pigs’ embryos using the Crispr/Cpf1 system. Porcine genomic DNA sequences were analyzed, and the target sequences were selected using a web tool, Benchling (https://benchling.com/). The designed T7-crDNA oligos were synthesized by the Oligonucleotide Synthesis Service (Macrogen Inc., Seoul, Korea). pTE4396 (#74- 041; Addgene, Cambridge, MA, USA) vector was used as a template for AsCpf1 RNA synthesis. MEGAshortscript™ T7 Transcription Kit (Ambion, Austin, TX, USA) was used for the pFOXN1 target-crRNA synthesis, and RiboMAX™ Large Scale RNA Production Systems (Promega, Madison, WI) was used for the AsCpf1 RNA synthesis. The RNAs were diluted in microinjection buffer (10 mM Tris-HCl and 1 mM EDTA). 50 ng/μL of each pFOXN1 target-crRNA and 100 ng/μL AsCpf1 mRNA were co-injected into the cytoplasm of pronuclear stage PA embryos. Data were analyzed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science) mean±SEM. As a result, the cleavage rates were no significant (p<0.05) difference between the control and microinjected groups (79.0±6.3 and 67.9±4.4), and there was no significant (p<0.05) difference in the development rate of blastocysts (60.6±12.1 and 49.3±6.6). These results suggest that there is no negative effect of embryo development by microinjection. To further analyze gene editing efficiency, it will be required to assay Indel and sequencing in the gDNA of a single blastocyst.