Enhanced homologous recombination mediated by lambda recombination proteins

Abstract

Disclosed herein are methods for generating recombinant DNA molecules in cells using homologous recombination mediated by recombinases and similar proteins. The methods promote high efficiency homologous recombination in bacterial cells, and in eukaryotic cells such as mammalian cells. The methods are useful for cloning, the generation of transgenic and knockout animals, and gene replacement. The methods are also useful for subcloning large DNA fragments without the need for restriction enzymes. The methods are also useful for repairing single or multiple base mutations to wild type or creating specific mutations in the genome. Also disclosed are bacterial strains and vectors which are useful for high-efficiency homologous recombination.

Description

PRIORITY CLAIM[0001] This application is a continuation of U.S. patent application Ser. No. 10 / 366,044, filed Feb. 12, 2003, which claims priority to International Application No. PCT / US01 / 25507, filed Aug. 14, 2001, which claims the benefit of U.S. Provisional Application No. 60 / 225,164, filed Aug. 14, 2000, and claims the benefit of U.S. Provisional Application No. 60 / 271,632, filed Feb. 26, 2001. All of the prior applications are incorporated by reference herein in their entirety.FIELD[0002] The present disclosure relates to methods to enhance homologous recombination in bacteria and eukaryotic cells using recombination proteins, such as those derived from bacteriophage lambda. It also relates to methods for modifying genomic DNA in bacterial artificial chromosomes (BACs) and to subcloning of genomic DNA from BACs into multicopy plasmids.[0003] Concerted use of restriction endonucleases and DNA ligases allows in vitro recombination of DNA sequences. The recombinant DNA generated ...

AI Technical Summary

Benefits of technology

The present patent provides methods for cloning DNA molecules in cells using a defective prophage containing a pL promoter. The methods involve inducing homologous recombination between nucleic acids in the cell by introducing a temperature-sensitive repressor, cI857, which is repressed at low temperatures and derepressed at high temperatures. The methods also involve introducing single-stranded DNA molecules and a single-stranded DNA binding polypeptide to promote homologous recombination. The patent also describes the use of these methods for bacterial cells and for modifying eukaryotic cells. The patent also includes a diagram showing the structure of the defective prophage and a strategy for BAC engineering. Overall, the patent provides new methods for efficient cloning and modification of DNA molecules in cells.

Problems solved by technology

However, the restriction-ligation approach has two practical limitations: first, DNA molecules can be precisely combined only if convenient restriction sites are available; second, because useful restriction sites often repeat in a long stretch of DNA, the size of DNA fragments that can be manipulated are limited, usually to less than about 25 kilobases.

Such short linear DNA segments can transform yeast, but subsequent manipulation of recombinant DNA in yeast is laborious.

It is generally easier to work in bacteria, but linear DNA fragments do not readily transform bacteria (due in part to degradation by bacterial exonucleases).

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