title: Improved methods for the generation of human gene knockout and knockin cell lines.
abstract: Recent studies have demonstrated the utility of recombinant adeno-associated viral (rAAV) vectors in the generation of human knockout cell lines. The efficiency with which such cell lines can be generated using rAAV, in comparison with more extensively described plasmid-based approaches, has not been directly tested. In this report, we demonstrate that targeting constructs delivered by rAAV vectors were nearly 25-fold more efficient than transfected plasmids that target the same exon. In addition, we describe a novel vector configuration which we term the synthetic exon promoter trap (SEPT). This targeting element further improved the efficiency of knockout generation and uniquely facilitated the generation of knockin alterations. An rAAV-based SEPT targeting construct was used to transfer a mutant CTNNB1 allele, encoding an oncogenic form of beta-catenin, from one cell line to another. This versatile method was thus shown to facilitate the efficient integration of small, defined sequence alterations into the chromosomes of cultured human cells.
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
authors.forename: Ozlem
authors.initials: O
authors.lastname: Topaloglu
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Hurley
authors.forename: Ozlem
authors.initials: O
authors.lastname: Yildirim
authors.forename: Curt I
authors.initials: CI
authors.lastname: Civin
authors.forename: Fred
authors.initials: F
authors.lastname: Bunz
mesh_terms: D045744:Cell Line, Tumor; D003598:Cytoskeletal Proteins / Q000235:genetics; D000229:Dependovirus / Q000235:genetics; D005091:Exons; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D015534:Trans-Activators / Q000235:genetics; D051176:beta Catenin
chemical_list: C495270:CTNNB1 protein, human; D003598:Cytoskeletal Proteins; D015534:Trans-Activators; D051176:beta Catenin