title: Efficient PRNP gene targeting in bovine fibroblasts by adeno-associated virus vectors.
abstract: Gene-targeted livestock can be created by combining ex vivo manipulation of cultured nuclear donor cells with cloning by nuclear transfer. However, this process can be limited by the low gene targeting frequencies obtained by transfection methods, and the limited ex vivo life span of the normal nuclear donor cells. We have developed an alternative gene targeting method based on the delivery of linear, single-stranded DNA molecules by adeno-associated virus (AAV) vectors, which can be used to introduce a variety of different mutations at single copy loci in normal human cells. Here we show that AAV vectors can efficiently target the PRNP gene encoding the prion protein PrP in bovine fetal fibroblasts, which can be used as nuclear donors to clone cattle. Cattle with both PRNP genes disrupted should be resistant to bovine spongiform encephalopathy.
authors.affiliation: Department of Medicine, University of Washington, Seattle, Washington 98195-7720, USA.
authors.forename: Roli K
authors.initials: RK
authors.lastname: Hirata
authors.forename: Cong
authors.initials: C
authors.lastname: Xu
authors.forename: Rong
authors.initials: R
authors.lastname: Dong
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Miller
authors.forename: Stacy
authors.initials: S
authors.lastname: Ferguson
authors.forename: David W
authors.initials: DW
authors.lastname: Russell
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D019976:Cloning, Organism; D000229:Dependovirus / Q000235:genetics; D016643:Encephalopathy, Bovine Spongiform / Q000517:prevention & control; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D009419:Nerve Tissue Proteins / Q000235:genetics*; D053652:Nuclear Transfer Techniques; D011328:Prions / Q000235:genetics*
chemical_list: D009419:Nerve Tissue Proteins; D011328:Prions