Enhanced Homologous Recombination Mediated by Lambda Recombination Proteins
Description of Invention:
The present invention concerns methods to enhance homologous recombination in bacteria and eukaryotic cells using recombination proteins derived from bacteriophage lambda. It also concerns methods for promoting homologous recombination using other recombination proteins.
Concerted use of restriction endonucleases and DNA ligases allows in vitro recombination of DNA sequences. The recombinant DNA generated by restriction and ligation may be amplified in an appropriate microorganism such as E. coli, and used for diverse purposes including gene therapy. 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 20 kilobases.
Homologous recombination, generally defined as an exchange of homologous segments anywhere along a length of two DNA molecules, provides an alternative method for engineering DNA. In generating recombinant DNA with homologous recombination, a microorganism such as E. coli, or a eukaryotic cell such as a yeast or vertebrate cell, is transformed with an exogenous strand of DNA. The center of the exogenous DNA contains the desired transgene, whereas each flank contains a segment of homology with the cell's DNA. The exogenous DNA is introduced into the cell with standard techniques such as electroporation or calcium phosphate-mediated transfection, and recombines into the cell's DNA, for example with the assistance of recombination-promoting proteins in the cell.
In generating recombinant DNA by homologous recombination, it is often advantageous to work with short linear segments of DNA. For example, a mutation may be introduced into a linear segment of DNA using polymerase chain reaction (PCR) techniques. Under proper circumstances, the mutation may then be introduced into cellular DNA by homologous recombination. 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). Accordingly, recombinants are rare, require special poorly-growing strains (such as RecBCD- strains) and generally require thousands of base pairs of homology. This invention teaches an improved method of promoting homologous recombination in bacteria.
In eukaryotic cells, targeted homologous recombination provides a basis for targeting and altering essentially any desired sequence in a duplex DNA molecule, such as targeting a DNA sequence in a chromosome for replacement by another sequence. This invention teaches methods useful for treating human genetic diseases, the creation of transgenic animals, or modifying the germline of other organisms.
Inventors:
Donald L. Court et al. (NCI)
Patent Status:
DHHS Reference No. E-177-2000/0 --
U.S. Provisional Application No. 60/255,164 filed 14 Aug 2000
DHHS Reference No. E-177-2000/1 --
PCT Application No. PCT/US01/25507 filed 14 Aug 2001, which published as WO 02/14495 on 21 Feb 2002
DHHS Reference No. E-177-2000/2 --
U.S. Patent No. 7,144,734 issued 05 Dec 2006
U.S. Patent Application No. 10/692,553 filed 23 Oct 2003
Portfolios: Gene Based Therapies
Gene Based Therapies -Therapeutics-Gene Therapy-Vectors-Control sequences/genes Gene Based Therapies -Therapeutics-Gene Therapy-Vectors Gene Based Therapies -Therapeutics-Gene Therapy Gene Based Therapies -Therapeutics
For Additional Information Please Contact: Charlene A. Sydnor Ph.D.
NIH Office of Technology Transfer
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Rockville, MD 20852-3804
Phone: 301/435-4689
Email: sydnorc@mail.nih.gov
Fax: 301/402-0220
