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.

Recent 2 decades, including in vitro maturation (IVM), assisted reproductive technologies (ARTs) achieved noteworthy development. However the efficiency of ARTs with in vitro matured oocytes is still lower than that with in vivo oocytes. To overcome those limitations, many researchers attempted to adapt co-culture system during IVM and consequently maturation efficiency has been increased. The beneficial effects of applying co-culture system is contemplated base on communication and interaction between various somatic cells and oocytes, achievement of paracrine factors, and spatial effects of extracellular matrix (ECM) from somatic cell surface. The understanding of co-culture system can provide some information to narrow the gap between in vitro and in vivo. Here we will review current studies about issues for understanding cu-culture system with various somatic cells to improve in vitro maturation microenvironment and provide bird view and strategies for further studies.