title: [Targeting Prnp in bovine fibroblasts by promoter-trap strategy].
abstract: Promoter-trap strategy for enriching targeted colonies has been usually used to elevate the gene targeting efficiency in somatic cells. Knocking out Prnp in animals by gene targeting can render them resistant to Prion diseases. We constructed a bovine Prnp promoter-less targeting vector BoPrPneo, then transfected the linearized vector into the bovine fetal fibroblasts BFF through electroporation. After selecting in cell culture medium with 250 microg/mL G418, we obtained 99 drug-resistant cell colonies, 4 of them were positive for targeted events after PCR screening, and the targeted colonies were further confirmed by sequencing and Southern blotting. This suggests that one allele of Prnp has been successfully knocked out in bovine fetal fibroblasts. This research supplies a simple, safe and effective method to targeting bovine Prnp.
authors.affiliation: School of Life Science and Technology, Tongji University, Shanghai 200092, China.
authors.forename: Caihong
authors.initials: C
authors.lastname: Zhu
authors.forename: Guohua
authors.initials: G
authors.lastname: Yu
authors.forename: Bei
authors.initials: B
authors.lastname: Li
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Xu
authors.forename: Huiqing
authors.initials: H
authors.lastname: Yu
authors.forename: Jianquan
authors.initials: J
authors.lastname: Chen
authors.forename: Min
authors.initials: M
authors.lastname: Qian
authors.forename: Guoxiang
authors.initials: G
authors.lastname: Cheng
mesh_terms: D000818:Animals; D002417:Cattle; D018274:Electroporation; D005333:Fetus; D005347:Fibroblasts / Q000378:metabolism*; D055786:Gene Knockout Techniques / Q000379:methods*; D017096:Prion Diseases / Q000235:genetics / Q000517:prevention & control; D011328:Prions / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014162:Transfection
chemical_list: D011328:Prions