Site-specific serine recombinases and methods of their use

a serine recombinase and site-specific technology, applied in the field of site-specific serine recombinases and methods of their use, can solve the problems of high integration frequency, unidirectional integration, unsatisfactory chromosomal rearrangement, etc., and achieve site-specific recombination stability

Inactive Publication Date: 2008-01-24
PADIDAM MALLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] The ability of phage recombinases to specifically and efficiently direct recombination between DNA sequences in living cells makes them potentially useful in a variety of genetic engineering applications. Such applications include integration, excision, inversion, translocation and cassette exchange of polynucleotide sequences.

Problems solved by technology

This site-specific recombination system, however, only allows the induction of a reduced number of recombination events in the same genome.
Since each recombination reaction leaves a target sequence for the recombinase in the genome at the crossover site, and because recombinases (e.g. Cre and Flp) can catalyze intermolecular recombination, the whole process may lead to undesired chromosomal rearrangements.
As little or no recombination between any combination of sites other than attP or attB has been observed, the integrations are unidirectional and there is a high integration frequency.

Method used

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  • Site-specific serine recombinases and methods of their use
  • Site-specific serine recombinases and methods of their use
  • Site-specific serine recombinases and methods of their use

Examples

Experimental program
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example 1

Design, Synthesis, and Cloning of Recombinase Genes and Intramolecular Recombination Assay Plasmids

[0195] After analyzing the published literature and sequences available in Genbank, numerous site-specific recombinases were selected and assayed for DNA integration, excision, inversion, and replacement in mammalian and plant cells. The amino acid sequences for large site-specific recombinases of serine family (Smith, M. C. and H. M. Thorpe 2000 Diversity in the serine recombinases. Mol. Microbiol., 44:299-307) were obtained from GenBank and reverse translated to DNA. Since the sources of recombinases were from bacteria or bacterial viruses, we optimized the DNA sequence for recombinase expression in mammalian cells without changing the encoded amino acid sequence. The genes were totally synthesized using the codons for high-level human and mouse expression and with convenient restriction enzyme sites for cloning. In addition, regions of very high (>80%) or very low (<30%) GC content...

example 2

Transient Intramolecular Recombination Assays

[0215] In order to determine the activity of the recombinases in mammalian and plant cells, a transient assay was developed. Briefly, the assay consisted of cloning the recombinase gene into an expression plasmid, making the corresponding intramolecular recombination assay plasmid, introducing both plasmid DNAs into cells by transfection, and assaying for luciferase enzyme activity. The recombinase assay plasmids contained CMV Promoter-attP:STOP:attB-Luciferase Reporter gene-Terminator sequences. The STOP sequence is a transcription termination signal sequence. In the absence of recombination, expression of the luciferase reporter gene is prevented by the STOP sequence present between the promoter and reporter gene. Recombination between the attP and attB sites due to the introduced recombinase results in deletion of the STOP sequence and activation of reporter gene. This assay is sensitive and robust because it is an OFF to ON format an...

example 3

Stable Integration of Plasmid DNA Containing attP or attB Sequence into HEK293 Chromosome Containing the attB or attP Site

[0231] Assay for the integration of plasmid DNA at attP or attB site on the chromosome was done in a two-step process. In the first step, a stable cell line containing a single copy of attP or attB site of each enzyme was generated and characterized. In the second step, a plasmid containing the attP or attB site was integrated at the chromosomal attB or attP, respectively, in the presence of the recombinase expression plasmid.

Generation of Stable HEK293 Clones with attP or attB Sequence in the Chromosome

[0232] A single copy of attP or attB sequence of each recombinase (SEQ ID Numbers 11, 13-21) was introduced at the FRT locus in Flp-In™-293 cells obtained from Invitrogen [Carlsbad, Calif. (catalog #R750-07)] following the procedure recommended by the manufacturer. The FRT locus in Flp-In™-293 cells has a CMV promoter, FRT integration site for Flp recombinase,...

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Abstract

The present invention provides a method for obtaining site-specific recombination in a eukaryotic cell, the method comprising providing a eukaryotic cell that comprises a first recombination attachment site and a second recombination attachment site; contacting the first and second recombination attachment sites with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination attachment sites, wherein the recombinase polypeptide can mediate recombination between the first and second recombination attachment sites, the first recombination attachment site is a phage genomic recombination attachment site (attP) or a bacterial genomic recombination attachment site (attB), the second recombination site is attB or attP, and the recombinase is selected from the group consisting of a Listeria monocytogenes phage recombinase, a Streptococcus pyogenes phage recombinase, a Bacillus subtilis phage recombinase, a Mycobacterium tuberculosis phage recombinase and a Mycobacterium smegmatis phage recombinase, provided that when the first recombination attachment site is attB, the second recombination attachment site is attP and when the first recombination attachment site is attP, the second recombination attachment site is attB. The invention also describes compositions, 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 applications.

Description

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0001] This invention was made with government support under Grant No. 70NANB1H3062, awarded by National Institute of Standards and Technology, Advance Technology Program. The Government may have certain rights in the invention.FIELD OF THE INVENTION [0002] The present invention relates to the field of genetic engineering. Specifically the invention relates to compositions and methods for site-specifically integrating, deleting, inverting, exchanging, and translocating a polynucleotide into the genome of a cell. The invention also relates to enzyme, polynucleotides, polypeptides, and vector constructs. BACKGROUND OF THE INVENTION [0003] Many bacteriophage and integrative plasmids encode site-specific recombination systems that enable the stable incorporation of their genome into those of their hosts and excision of the genome from the host genome. In these systems, the minimal requirements for the recombination reaction are a recombin...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/09C07H21/00C12N15/63C12N5/06
CPCC12N9/00C12N9/22C12N15/52C12Y301/22A61P19/02A61P21/04A61P29/00A61P3/04A61P31/04A61P31/06A61P31/12A61P31/16A61P31/18A61P31/22A61P33/02A61P33/06A61P35/00A61P35/02A61P7/04A61P7/06A61P9/00C12N15/85C07H21/04C12N15/09C12N15/907C12N15/79
Inventor PADIDAM, MALLA
Owner PADIDAM MALLA
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