The Study On CRISPR/Cas9 Mediated Green Fluorescent Protein Gene Targeting At Bovine KRT18 Locus

Cytokeratin,a member of scleroprotein family,is an ectodermal structure protein.Researches have reported that keratin plays a vital role in the reproductive process,especially in early embryo development,and cytokeratin 18(KRT18)has been used as a trophectoderm marker in bovine.Keratin is primarily expressed in epidermal cells and simple epithelial cells.Due to its highly specific in the type of epithelial cell and tissue differentiation,keratin can be used as a marker of trophoblast epithelial cells.KRT18 belongs to type I(isoelectric point from 4.9 to 5.4)acidic keratin,and performs a key function not only in stabilizing cell structure,morphology,maintaining integrity of cell and functionality,but also in the intracellular physiological processes,Ssuch as cell-cycle,intracellular signaling pathway,apoptosis,mitosis and early embryonic implantation.To integrate the GFP gene at bovine KRT18 locus,a CRISPR/Cas9 vector was constructed according the bovine KRT18 gene,and a targeting vector was constructed from a plasmid that contains neomycin resistance gene(Neor).We modifieed basic framework of vector by deleting CAG promoter(named pGlrkt-2),then we constructed targeting vector by inserting two homologous arms into the vector,which is targeting to bovine KRT18 CRISPR/Cas9 cleavage site,and named it as pGlrkt.The exogenous gene is humanized EGFP,in the upstream of EGFP is 5'homologous arm at the size of 995 bp,in the downstream of expressing framework(Neor-1)is a 3' homologous arm at the size of 946 bp.The results of restriction enzyme digestion and sequencing analyses confirmed that the vector containing the correct KRT18 homologous fragments and exogenous gene.The expression of GFP was confirmed by liposome transfection of the vector into bovine uterine epithelial cells.In order to integrate of EGFP gene into bovine KRT18 locus,we transfected CRISPR/Cas9 and pGlrkt plasmids into the bovine fetal fibroblast cells by electroporation.After G418 screening,47 single cell colonies were obtained by flow cytometry sorting and mouth pipetting.The cells were expanded and the targeting events were tested by cross homology arm PCR screening.Finally,3 exogenous gene site-specific integration cell lines were obtained and targeting efficiency is 6%,which will be used as an important tool to study the KRT18 role in bovine early embryo development.