Improved methods for the generation of human gene knockout and knockin cell lines

Ozlem Topaloglu, Paula Hurley, Ozlem Yildirim, Curt I. Civin, Fred Bunz

Research output: Contribution to journalArticle

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 β-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.

Original languageEnglish (US)
Pages (from-to)1-7
Number of pages7
JournalNucleic Acids Research
Volume33
Issue number18
DOIs
Publication statusPublished - 2005

    Fingerprint

ASJC Scopus subject areas

  • Genetics

Cite this