Introduction: Forkhead box protein N1 (FoxN1) regulates development, differentiation, and function of thymus epithelial cells (TECs), both in the prenatal and postnatal thymus. In mice, knockout of the FoxN1 results in two well-known defects as hairlessness and athymia. However, knockout of FoxN1 in other species has not been discovered yet. RNA-guided endonucleases (RGENs), derived from the prokaryotic type II CRISPR-Cas system, enable targeted specific genes. We successfully targeted FoxN1 in pig by using CRISPR-Cas system.
Materials and methods: RGEN design: We designed specific single-guide RNA (sgRNAs) which targets exon 2 of porcine FoxN1, and constructed Cas9 vector system.
Transfection and Microinjection: Cas9-FoxN1 vectors were transfected intoporcine fibroblastusing lipofectamin 2000 reagent according to manufacturers protocol. Also to induce parthenogenesis, Cas9-FoxN1 vectors (4, 8, and 16 ng/μl) were microinjected into the porcineoocytes and these were activated by BTX.
T7 endonuclease I assay: The region of DNA containing the target site was amplified by PCR using the specific primer set. The PCR products were hybridized to form heteroduplex DNA, and digested by T7 endonuclease I for 1 h at 37 °C. The DNA was analyzed by gel electrophoresis using 2% agarose gel.
Results and discussion: To confirm the knockout of FoxN1, we analyzed the DNA cleavage activities by T7 endonuclease I assay both fibroblasts and oocytes. As a result, there was no significantly difference in knockout efficiency among injected DNA concentrations, 4 ng (20.7%), 8 ng (15.4%) and 16 ng (20%), respectively. However, we checked that Cas9-FoxN1 could successfully knockout the porcine FoxN1. This study suggests that knockout pig is also producible using Cas9-system by DNA microinjection into zygotes, directly.
02 - 04 Sep 2014
World Congress of Reproductive Biology