title: Efficient PRNP deletion in bovine genome using gene-editing technologies in bovine cells.
abstract: Even though prion (encoded by the PRNP 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 Bmi-1 and hTert genes; 2) investigate the disruption of the PRNP 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 Bmi-1and hTert. Immortalized cells were cultured for more than 180 days without any changes in their doubling time and morphology. Furthermore, to knockout the PRNP 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 (CSNK2A1, FAM64A, MPG and PRND) were affected after PRNP disruption in immortalized cells. In conclusion, we established immortalized cattle fibroblasts using Bmi-1 and hTert genes, and used TALENs to knockout the PRNP gene in these immortalized cells. The efficient PRNP KO is expected to be a useful technology to develop our understanding of in vitro prion protein functions in cattle.
authors.affiliation: a Laboratory of Theriogenology and Biotechnology; Department of Veterinary Clinical Science ; College of Veterinary Medicine and the Research Institute of Veterinary Science; Seoul National University ; Seoul , Republic of Korea ;
authors.forename: WooJae
authors.initials: W
authors.lastname: Choi
authors.forename: Eunji
authors.initials: E
authors.lastname: Kim
authors.forename: Soo-Young
authors.initials: SY
authors.lastname: Yum
authors.forename: ChoongIl
authors.initials: C
authors.lastname: Lee
authors.forename: JiHyun
authors.initials: J
authors.lastname: Lee
authors.forename: JoonHo
authors.initials: J
authors.lastname: Moon
authors.forename: Sisitha
authors.initials: S
authors.lastname: Ramachandra
authors.forename: Buddika Oshadi
authors.initials: BO
authors.lastname: Malaweera
authors.forename: JongKi
authors.initials: J
authors.lastname: Cho
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Kim
authors.forename: SeokJoong
authors.initials: S
authors.lastname: Kim
authors.forename: Goo
authors.initials: G
authors.lastname: Jang
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D016643:Encephalopathy, Bovine Spongiform / Q000235:genetics; D020022:Genetic Predisposition to Disease / Q000235:genetics; D008297:Male; D011328:Prions / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics*
chemical_list: D011328:Prions
keywords: PRNP; TALEN; cattle embryos; immortalization; knockout