Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2001-02-16
2004-10-26
Mckelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S183000, C435S320100
Reexamination Certificate
active
06808925
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to the field of biotechnology, and more specifically to the field of genomic modification. Disclosed herein are altered recombinases, including compositions thereof, expression vectors, and methods of use thereof, for the generation of transgenic cells, tissues, plants, and animals. The compositions, vectors, and methods of the present invention are also useful in gene therapy techniques.
BACKGROUND OF THE INVENTION
The current inability to perform efficient, site-specific integration of incoming DNA into the chromosomes of higher organisms is holding up advances in basic and applied biology. Recently strategies for chromosomal integration that take advantage of the high efficiency and tight sequence specificity of recombinase enzymes isolated from microorganisms have been described. In particular, a class of phage integrases that includes the &phgr;C31 integrase (Kuhstoss, S., and Rao, R. N., J. Mol. Biol. 222, 897-908 (1991); Rausch H., and Lehmann, M., Nucleic Acids Research 19, 5187-5189 (1991)) have been shown to function in mammalian cells (Groth, A. C., et al., Proc. Natl. Acad. Sci. USA 97, 5995-6000 (2000)).
Such site-specific recombinase enzymes have long DNA recognition sites that are typically not present even in the large genomes of mammalian cells. However, it has been recently demonstrated that recombinase pseudo sites, i.e. sites with a significant degree of identity to the wild-type binding site for the recombinase, are present in these genomes (Thyagarajan, B., et al., Gene 244, 47-54 (2000)).
The present disclosure teaches methods to alter the specificity of recombinases to provide altered recombinases that can be used more effectively in genetic engineering of the chromosomes of higher cells.
SUMMARY OF THE INVENTION
The present invention relates to the identification, isolation, cloning, expression, purification, and methods of use of altered recombinases. In one aspect, the present invention is directed to a method of site-specifically integrating a polynucleotide sequence of interest in a genome of a target cell using an altered recombinase of the present invention.
In one embodiment, the present invention is directed to a method for identifying an altered recombinase. In the method a population of cells is typically provided wherein cells of the population comprise a first plasmid (e.g., a resident plasmid). The first plasmid may comprise a transcriptional promoter region adjacent a first recombination site adjacent a transcription terminator adjacent a second recombination site adjacent a coding sequence of interest. The order of these components is promoter-first recombination site-transcription terminator-second recombination site-coding sequence of interest, wherein said first and second recombination sites act as substrates for a first recombinase and read-through transcription of the coding sequence of interest is minimized or essentially eliminated. The promoter is functional in the cell and an operable linkage of promoter and coding sequence of interest results from a recombination event between the two recombination sites (i.e., the transcription terminator is removed).
The population of cells is then transformed with a group (or population) of second plasmids (e.g., cloning plasmids). The group of plasmids comprises at least one second plasmid comprising a coding sequence for an altered recombinase operable linked to a promoter functional in the cell. Typically the group of plasmids represents a shuffled library of recombinases or mutagenized recombinases. Methods of generating such recombinase variants are described herein. The nucleic acid coding sequence for the altered recombinase typically differs from the coding sequence of the first recombinase(or parent recombinases) by at least one base pair, where that difference typically gives rise to at least one amino acid differences between the polypeptide coding sequences of the altered recombinase and the parent recombinase.
The cells are maintained under conditions that allow recombination to occur between the first and second recombination sites, wherein the recombination event is mediated by the altered recombinase. The population of transformed cells is then screened (or a genetic selection is applied) to identify a product encoded by the coding sequence of interest. Such a product may include, but is not limited to, a product identifiable by screening or selection, such as an RNA product or, ultimately, a polypeptide product. Cells producing the product encoded by the sequence of interest are then isolated and coding sequences of the altered recombinase, encoded by these cells, are isolated and identified. In a preferred embodiment, the altered recombinase provides an improved recombination frequency between the first and second recombination sites relative to the recombination frequency between the first and second recombination sites mediated by the first recombinase. Identification of variants having reduced or similar recombination frequencies is also possible using the methods of the present invention.
The first or parent recombinase may be, for example, a wild-type phage recombinase such as &phgr;C31, TP901-1, and R4. Other recombinases may be used in the method of the present invention as the first recombinase, including altered recombinases identified by previous cycles of screening using the methods of the present invention. Further, altered recombinases may be obtained using more than one “first” recombinase (e.g., in a family shuffling method).
The recombination sites used as substrates in the method of the present invention include, but are not limited to, wild-type attB, wild-type attP, pseudo-attB and pseudo-attP. Typically, at least one of the recombination sites provide a substrate for the first recombinase. Pseudo-sites may be identified, using methods described herein, in the genome of essentially any target cell, including, but not limited to, human and rodent cells.
The method of identifying an altered recombinase can be carried out in a number of cell types as described herein. In a preferred embodiment the method is carried out in bacterial cells.
The coding sequence of interest can encode a product that can be identified by a screen or selection, including, but not limited to, polypeptide products such as beta-galactosidase. Other reporter markers are described herein, as well as selectable markers.
In another aspect the present invention relates to an altered recombinase produced by the methods of the present invention. Typically the altered recombinase comprises a polypeptide wherein at least one amino acid is different from a wild-type sequence of the first recombinase, wherein the altered recombinase has improved recombination efficiency towards wild-type or pseudo att site sequences relative to the first recombinase. As discussed above, altered recombinases identified by the methods of the present invention may have increased, decreased, or similar recombination efficiencies related to the parent recombinases. Exemplary altered recombinases identified by the methods of the present invention include SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. The present invention also includes nucleic acid sequences encoding the polypeptide sequences of the altered recombinases.
In yet another aspect, the present invention is directed to a method of site-specifically integrating a polynucleotide sequence of interest in a genome of a cell. The method comprises introducing (i) a circular targeting construct, comprising a first recombination site and the polynucleotide sequence of interest, and (ii) an altered recombinase into the cell, wherein the genome of the cell comprises a second recombination site native to the genome and recombination between the first and second recombination sites is facilitated by the site-specific recombinase. The cell is maintained under conditions that allow recombination between the first and second recombination sites and the recombination is mediated by the site-specific recombinas
Bret E. Field, Bozicevic, Field & Francis
Mckelvey Terry
The Board of Trustees of the Leland Stanford Junior University
Vogel Nancy
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