Chromosome Engineering by Homologous Recombination

Basic Sciences

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Abstract

Recently, we have developed an in vivo cloning and gene modification system using lambda-mediated homologous recombination (Red). This system eliminates the need to cut DNA with restriction enzymes or join DNA fragments with DNA ligase. This extremely efficient recombination system will we believe, revolutionize the way in which recombinant DNA work is done. Both ssDNA oligos (as short as 20 bases) and linear dsDNA (as short as 30 bp) can be used to generate recombinants on the bacterial chromosome, on plasmids, or on genomic BAC library clones. ssDNA oligos have been used to create point mutations, to repair mutations, to create deletions, and to create small insertions on the chromosome and in BAC clones. Recombination of ssDNA requires only the lambda phage Beta protein which binds the ssDNA oligonucleotide that is electroporated into cells and anneals that nucleotide to its target homology in the cell. Annealing occurs to the ssDNA intermediates that form during replication. Recombination of long dsDNA (>1000bp) requires lambda phage functions Exo and Gam in addition to Beta. Exo degrades 5' ends of dsDNA generating ssDNA overhangs for Beta to bind. Gam inhibits host nucleases which destroy the entering linear DNA. Various bacterial vectors have been developed to ensure efficient and accurate genetic engineering via this in vivo recombineering technology.