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High Efficiency Single Stranded Homologous Recombination in Host Cells Deficient for Mismatch Repair
Donald L. Court et al. (NCI); PCT Application No. PCT/US03/14657 filed 09 May 2003 (DHHS Reference No. E-038-2003/0-PCT-01); Licensing Contact: Norbert Pontzer; 301/435-5502; firstname.lastname@example.org.
Homologous recombination is the process of exchanging DNA between two molecules through regions of identical sequence. Homologous recombination provides an alternative to using restriction endonucleases and ligases for producing recombinant DNA. However, the background level of homologous recombination in E. coli is very low even with long homology arms. Previous improvements have provided methods of using bacterophage lambda Red recombination functions to greatly increase the recombination frequency of endogenous single- and double-stranded DNA with relatively short homology arms. This type of genetic engineering has been named “recombineering,” a convenient term to describe homology-dependent, recombination-mediated, genetic engineering. Recombination with endogenous linear single-stranded DNA (ssDNA) is likely to occur by annealing with transiently single-stranded regions of the chromosome such as the replication fork. We show that only the Beta component of the Red function is required for this activity. (Published PCT Application WO00/21449; Nat. Rev. Genet. 2001, 2:769-779.)
When the ssDNA used for recombineering introduces change(s) near the DNA replication fork, the change(s) may trigger mismatch repair (MMR), which in turn can reduce the level of recombination. In the present invention, altering MMR function achieves a 10-to 100-fold increase in Red recombination. This increase raises the number of recombinants to 25 to 30 percent of treated cells surviving electroporation of the oligo. Methods of transiently inhibiting MMR and bacterial strains deficient for the production of MMR genes are also provided. (Annu. Rev. Genet. 2002, 36:361-88.)Start Signature
Dated: November 7, 2003.
Steven M. Ferguson,
Director, Division of Technology Development and Transfer, Office of Technology Transfer, National Institutes of Health.
[FR Doc. 03-28657 Filed 11-14-03; 8:45 am]
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