Bacterial Artificial Chromosome Mutagenesis Using Recombineering

Kumaran Narayanan,Qingwen Chen

doi:10.1155/2011/971296

Abstract

Gene expression from bacterial artificial chromosome (BAC) clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented a major obstacle for introducing modifications using conventional genetic engineering strategies. The development of in vivo homologous recombination strategies based on recombineering in E. coli has helped resolve this problem by enabling facile engineering of high molecular weight BAC DNA without dependence on suitably placed restriction enzymes or cloning steps. These techniques have considerably expanded the possibilities for studying functional genetics using BACs in vitro and in vivo.

Highlights

The information generated from the Genome Projects will be of the greatest value if it can be converted into functional data, if this increases our understanding of normal gene function and allows strategies to be developed for prevention and treatment of human disease
A number of techniques were developed in the late 1990s that were based on homologous recombination pathways, which meant they were not limited by the size of a bacterial artificial chromosome (BAC), permitting much more flexible engineering compared to conventional genetic engineering using restriction enzymes or site-specific recombination methods
To deal with the large size that is inherent of BACs, several novel methods were tested based on site-specific recombination systems, including the P1-derived Cre-lox [40, 41], the baker’s yeast Saccharomyces cerevisiae FlpFRT machineries [42], as well as traditional homologous recombination methods, but they have mostly had limited success (Table 1)

Summary

Introduction

The information generated from the Genome Projects will be of the greatest value if it can be converted into functional data, if this increases our understanding of normal gene function and allows strategies to be developed for prevention and treatment of human disease. Until about a decade ago, the large size of BACs has presented major hurdle for their precise manipulation to introduce specific changes such as mutations, reporter genes, and markers for functional studies in the mammalian environment [11,12,13] To address this problem, a number of techniques were developed in the late 1990s that were based on homologous recombination pathways, which meant they were not limited by the size of a BAC, permitting much more flexible engineering compared to conventional genetic engineering using restriction enzymes or site-specific recombination methods. This paper will focus on the rise of recombineering as the major homologous recombinationmediated approach for BAC modifications, as well as its novel applications and future prospects in the art of genetic tinkering

BAC Modifications Using Recombineering

Emergence of Recombineering as an Ideal BAC Modification Technique

Evolution of Recombineering Technologies

Applications of Recombineering

Findings

Conclusions and Perspectives