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.
| Thomason, Lynn C; Oppenheim, Amos B; Court, Donald L (2009) Modifying bacteriophage lambda with recombineering. Methods Mol Biol 501:239-51 |
| Luo, Xiao; Hsiao, He-Hsuan; Bubunenko, Mikhail et al. (2008) Structural and functional analysis of the E. coli NusB-S10 transcription antitermination complex. Mol Cell 32:791-802 |
| Gan, Jianhua; Shaw, Gary; Tropea, Joseph E et al. (2008) A stepwise model for double-stranded RNA processing by ribonuclease III. Mol Microbiol 67:143-54 |
| Korepanov, Alexey P; Gongadze, George M; Garber, Maria B et al. (2007) Importance of the 5 S rRNA-binding ribosomal proteins for cell viability and translation in Escherichia coli. J Mol Biol 366:1199-208 |
| Court, Donald L; Oppenheim, Amos B; Adhya, Sankar L (2007) A new look at bacteriophage lambda genetic networks. J Bacteriol 189:298-304 |
| Chan, Waiin; Costantino, Nina; Li, Ruixue et al. (2007) A recombineering based approach for high-throughput conditional knockout targeting vector construction. Nucleic Acids Res 35:e64 |
| Sawitzke, James A; Thomason, Lynn C; Costantino, Nina et al. (2007) Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond. Methods Enzymol 421:171-99 |
| Thomason, Lynn C; Costantino, Nina; Shaw, Dana V et al. (2007) Multicopy plasmid modification with phage lambda Red recombineering. Plasmid 58:148-58 |
| Bubunenko, Mikhail; Korepanov, Alexey; Court, Donald L et al. (2006) 30S ribosomal subunits can be assembled in vivo without primary binding ribosomal protein S15. RNA 12:1229-39 |
| Datta, Simanti; Costantino, Nina; Court, Donald L (2006) A set of recombineering plasmids for gram-negative bacteria. Gene 379:109-15 |
Showing the most recent 10 out of 20 publications
