Efficient <i>PRNP</i> deletion in bovine genome using gene-editing technologies in bovine cells

<p>Even though prion (encoded by the <i>PRNP</i> gene) diseases like bovine spongiform encephalopathy (BSE) are fatal neurodegenerative diseases in cattle, their study via gene deletion has been limited due to the absence of cell lines or mutant models. In this study, we aim to develop an immortalized fibroblast cell line in which genome-engineering technology can be readily applied to create gene-modified clones for studies. To this end, this study is designed to 1) investigate the induction of primary fibroblasts to immortalization by introducing <i>Bmi-1</i> and <i>hTert</i> genes; 2) investigate the disruption of the <i>PRNP</i> in those cells; and 3) evaluate the gene expression and embryonic development using knockout (KO) cell lines. Primary cells from a male neonate were immortalized with <i>Bmi-1</i>and <i>hTert</i>. Immortalized cells were cultured for more than 180 days without any changes in their doubling time and morphology. Furthermore, to knockout the <i>PRNP</i> gene, plasmids that encode transcription activator-like effector nuclease (TALEN) pairs were transfected into the cells, and transfected single cells were propagated. Mutated clonal cell lines were confirmed by T7 endonuclease I assay and sequencing. Four knockout cell lines were used for somatic cell nuclear transfer (SCNT), and the resulting embryos were developed to the blastocyst stage. The genes (<i>CSNK2A1, FAM64A, MPG and PRND</i>) were affected after <i>PRNP</i> disruption in immortalized cells. In conclusion, we established immortalized cattle fibroblasts using <i>Bmi-1</i> and <i>hTert</i> genes, and used TALENs to knockout the <i>PRNP</i> gene in these immortalized cells. The efficient <i>PRNP</i> KO is expected to be a useful technology to develop our understanding of <i>in vitro</i> prion protein functions in cattle.</p