Severe combined immune deficiency (SCID) pig is very important research model for biomedical research, such as the development of humanized tissues and organs for transplantation and long-term evaluation of transplanted cancer or stem cell of human origin. 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. In this study, we are going to produce the FOXN1 KO SCID pigs using the Crispr/Cpf1 method. Porcine genomic DNA sequences were analyzed and the target sequences were selected using a web tool, Benchling (https://benchling.com/). The designed crDNA oligos was synthesized by the Oligonucleotide Synthesis Service (Macrogen Inc., Seoul, Korea). To generate the AsCpf1-mCherry-Puro construct, pTE4396 (#74041; Addgene, Cambridge, MA, USA) was modified by removing the NeoR/KanR sequence using BstBI and SmaI. Then, the mCherry-Puro sequence from pSicoR-Ef1a-mCh-Puro (#31845; Addgene, Cambridge, MA, USA) digested with the same restriction enzymes was inserted into the aforementioned NeoR/KanR-deleted vector. The crDNA #1 or crDNA #2 was inserted into the pTE4396 and AsCpf1-mCherry-Puro vectors in the U6 promoter region using BsmBI enzyme, respectively. The two vectors were transfected with lipofectamine 3000 (Life Technologies, Grand Island, NY, USA) and selected with puromycin and G-418 antibiotics. As a result, we established a cell line into which two vectors (pTE4396+crFOXN1#2 and AsCpf1- mCherry-Puro+ crFOXN1#1) and were inserted. Further studies are needed to characterize FOXN1 KO cell lines.

The use of pigs in neuroscience has increased over the past years because the pigs are closely related to humans in terms of anatomy and physiology. Especially, the blood-brain barrier (BBB) maintains the homeostatic microenvironment in the central nervous system (CNS) and they can provide a valuable tool for studying the neurobiology. However, only a few putative blood-brain barrier (BBB) models have been generated by co-culture of porcine primary cells. The fundamental problem is that they lose some of their phenotypes when maintained in vitro for long-term culture. To establish improved in vitro porcine BBB models, we differentiated novel brain microvascular endothelial cells (BMECs) from porcine induced pluripotent stem cells (iPSCs) using a modified human-based protocol. Briefly, the dissociated single cells from iPSCs were seeded in Geltrex. For differentiation, cells were maintained for 3 days of expansion and then switched to unconditioned medium (UM) lacking bFGF for 6-7 days. Then, we subcultured cells onto collagen/fibronectin coated plates and changed BMEC medium for 2-3 weeks. About two weeks later, we observed a cluster of round cells surrounded by spindle shaped adherent cells termed as colony-forming units (CFU) of putative BMECs. Over time, the cluster of cells disappears and remained adherent spindle-shaped cells showed properties of endothelial cells. Although further studies will be needed, this study would be a great comparative analysis of the porcine and human in vitro BBB model.