Tyrosine recombinase for genetic engineering
AssigneesEuropean Molecular Biology Laboratory, l 'Universite catholique de Louvain
InventorsA. Francis Stewart, Youming Zhang, Bernard Hallet
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Abstract
The present invention is directed to methods and compositions for TnpI-mediated genetic engineering using the Bacillus thuringiensis recombinase TnpI and the TnpI recombination substrates TRT or TRT′, and variants thereof. In particular, the invention relates to TRT or TRT′ sequences, and variants thereof, vectors, cells and kits useful for TnpI-mediated genetic recombination, as well as methods for the use of TRT or TRT′ sequences for TnpI-mediated genetic engineering.
Claims52
A composition comprising an isolated DNA molecule comprising one or more copies of TRT (SEQ ID NO:3), or a functional variant thereof, with the proviso that the DNA molecule does not comprise the entire sequence of TRT″ (SEQ ID NO:4).
A composition comprising an isolated DNA molecule comprising one or more copies of TRT′ (SEQ ID NO:2), or a functional variant thereof, with the proviso that the DNA molecule does not comprise the entire sequence of TRT″ (SEQ ID NO:1).
A cell transformed with a DNA molecule, said DNA molecule comprising one or more copies of TRT (SEQ ID NO:3), or a functional variant thereof, with the proviso that the DNA molecule does not comprise the entire sequence of TRT″ (SEQ ID NO:4).
A cell transformed with a DNA molecule, said DNA molecule comprising one or more copies of TRT′ (SEQ ID NO:2), or a functional variant thereof, with the proviso that the DNA molecule does not comprise the entire sequence of TRT″ (SEQ ID NO:4).
A eukaryotic cell transformed with a DNA molecule integrated into its chromosome, said DNA molecule comprising one or more copies of TRT (SEQ ID NO:3) or a functional variant thereof, or TRT′ (SEQ ID NO:2), or a functional variant thereof.
A kit comprising in one or more containers:
- a) an isolated DNA molecule comprising one or more copies of TRT (SEQ ID NO:3) or a functional variant thereof;
- and
- b) an isolated TnpI protein, a TnpI expression vector or a cell capable of expressing TnpI.
A kit comprising in one or more containers:
- a) an isolated DNA molecule comprising one or more copies of TRT′ (SEQ ID NO:2) or a functional variant thereof;
- and
- b) an isolated TnpI protein, a TnpI expression vector or a cell capable of expressing TnpI.
A method for effecting TnpI-mediated site-specific recombination comprising exposing a first TnpI recombination target site and a second TnpI recombination target site with TnpI protein, under sufficient conditions and in an amount sufficient to mediate site-specific recombination between the first and second TnpI recombination target sites, wherein the first and second TnpI recombination target sites are selected from the group consisting of TRT (SEQ ID NO:3) or a functional variant thereof, and TRT′ (SEQ ID NO:2) or a functional variant thereof, with the proviso that the DNA molecule does not comprise the entire sequence of TRT″ (SEQ ID NO:4).
A method for effecting site-specific recombination in a non-Bacillus thuringiensis cell comprising exposing a first TnpI recombination target site and a second TnpI recombination target site to TnpI protein, under sufficient conditions and in an amount sufficient to mediate site-specific recombination between the first TnpI recombination target site and second TnpI recombination target site, wherein the first and second TnpI recombination target sites are selected from the group consisting of TRT (SEQ ID NO:3) or a functional variant thereof, and TRT′ (SEQ ID NO:2) or a functional variant thereof.
A method of producing a circular DNA vaccine comprising:
- a) introducing a DNA molecule into a non-Bacillus thuringiensis cell, said DNA molecule comprising, in the following order, from 5′ to 3′, a DNA sequence encoding an antigen of interest, a first TRT′ site or functional variant thereof, an origin of replication and, optionally, one or more selectable markers, and a second TRT′ site or functional variant thereof;
- and
- b) contacting said cell with TnpI protein under sufficient conditions and in an amount sufficient to mediate site-specific recombination between the first and second TRT′ sites or functional variants thereof, such that recombination between the first and the second TRT′ sites or functional variants thereof results in deletion of the origin of replication and the optional one or more selectable markers from the DNA molecule,
- such that a circular DNA vaccine encoding an antigen of interest is produced.
Dependencies are inferred from the claim text.