Obtained Transfection Cell Line for iPSCs Isolation from Bactrian Camels (Camelus bactrianus) via CRISPR/Cas9
LI Zong-Shuai1, SHEN Pei-Lei1, LIU Lei-Lei2, YANG Yang2, LI Hai-Jiang2, ZHANG Quan-Wei1,GONG Ji-Shang2, ZHAO Xing-Xu1,2, ZHANG Yong1,2,*
1 College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China; 2 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
Abstract：Bactrian camels (Camelus bactrianus) is a endangered species in China's northwest region. It has special physiological characteristics and has widely uses. Induced pluripotent stem cells (iPSCs) has important application value in protecting the germplasm resources of this species. However, the traditional methods for the isolation and identification of iPSCs are complicated and cumbersome. For the rapid and efficient isolation and identification of iPSCs, the CRISPR/Cas9 system was used in this experiment. The single guide RNA (sgRNA) was designed near the stop codon in reference to the Nanog gene expressed only in the embryonic stem cells (ESCs) stage of Bactrian camel, and the cleavage vector was successfully constructed and screened; the whole camel genome was used as a template to obtain upstream and downstream homology arms, and two reporter genes of green fluorescent protein gene (GFP) and neomycin resistance gene (Neo) were inserted between the two homologous arms, the expression vector was successfully constructed. At the same time, the minimum lethal dose of geneticin (G418) of Bactrian camel fibroblasts was obtained by the minimum lethal dose experiment. After sequencing and verification of the above two vectors,the Bactrian camel fibroblasts were co-transfected with the expression vector and the cleavage vector and cultured for 48 h, and then cultured for two weeks in a selection medium containing G418; then, the normal culture medium was used to expand the cells after the drug screening, the highly viable stable transfected cell line was successfully obtained. This experiment will provide biomaterials for the correct screening of iPSCs by sequencing the reporter gene after the Nanog gene.
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