Eight Journal abstracts demonstrating Gene Knock Theory

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Eight Journal abstracts demonstrating Gene Knock Theory

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

Ozlem Topaloglu, Paula J. Hurley, Ozlem Yildirim, Curt I. Civin1,2 and Fred Bunz* 

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 1Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine Baltimore, MD 21231, USA 2Department of Pediatrics, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine Baltimore, MD 21231, USA

Received August 11, 2005. Revised September 26, 2005. Accepted September 26, 2005.

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 ß-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. 

2.Insights into molecular mechanisms of contact hypersensitivity gained from gene knockout studies

Binghe Wang*, Claudio Feliciani, Irwin Freed*, Qinchao Cai* and Daniel N. Sauder* 

* Division of Dermatology, Sunnybrook and Women’s College Health Science Centre, University of Toronto, Ontario, Canada M4N 3M5, and
Department of Dermatology, University "G.d’Annunzio", Via dei Vestini Chieti, Italy

Contact hypersensitivity (CHS), a dendritic-cell (DC)-dependent, T-cell-mediated skin immune response to reactive haptens, has been a subject of intense research for many years. The molecular mechanisms underlying CHS are complicated and are not fully understood. During the past few years, varieties of gene-targeted knockout mice have been used in the study of CHS. Such studies have contributed significantly to our understanding of the mechanisms responsible for the initiation of CHS. This review focuses on insights into molecular requirements for CHS gained from knockout studies. 


3.Transposon-mediated generation of targeting vectors for the production of gene knockouts

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Chunfang Zhang1,*, Danny Kitsberg2, Hun Chy1, Qi Zhou2 and John R. Morrison1,3 

1 CopyRat Pty Ltd 27-31 Wright Street, Clayton, Victoria 3168, Australia 2 IngenKO Pty Ltd 27-31 Wright Street, Clayton, Victoria 3168, Australia 3 Monash Institute of Reproduction and Development, Monash University 27-31 Wright Street, Clayton, Victoria 3168, Australia

Received October 31, 2004. Revised December 22, 2004. Accepted December 22, 2004.

Vectors used for gene targeting experiments usually consist of a selectable marker flanked by two regions of homology to the targeted gene. In a homologous recombination event, the selectable marker replaces an essential element of the target gene rendering it inactive. Other applications of ...

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