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US 2003/0194809 A1Published application
Method of controlling site-specific recombination
AssigneeE. I. Du Pont de Nemours and Company
InventorsNarendra S. Yadav, Jianjun Yang
Life of this patent
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Abstract
The present invention provides methods for the conditional or regulated expression of a site-specific recombinase using split intein-mediated protein splicing. This enhances the temporal and tissue-specificity of trait gene expression and allows for fine tuning of expression specificity.
Claims30
An inactive recombinase element selected from the group consisting of:
- a) a DNA construct having the general structure in a 5′ to 3′ orientation comprising:
- P1-ssrN-IntN, wherein:
- (i) P1 is a promoter;
- (ii) ssrN is the N-terminal portion of a site-specific recombinase;
- and
- (iii) IntN is the N-terminal portion of a split intein;
- wherein each of P1, ssrN, and IntN are operably linked such that activation of the promoter results in the expression of the ssrN-IntN fusion protein lacking recombinase activity;
- and
- b) a DNA construct having the general structure in a 5′ to 3′ orientation comprising:
- P2-IntC-ssrC, wherein:
- (i) P2 is a promoter;
- (ii) ssrC is the C-terminal portion of a site-specific recombinase;
- and
- (iii) IntC is the C-terminal portion of a split intein;
- wherein each of P2, ssrC, and IntC are operably linked such that activation of the promoter results in the expression of the IntC-ssrC fusion protein lacking recombinase activity, and wherein the inactive recombinase elements of (a) and (b) when present together in a cell will form an active recombinase protein by intein-mediated trans-protein splicing.
12 more elements
An intein-mediated site-specific recombination system comprising:
- a) an inactive recombinase element selected from the group consisting of:
- 1) a DNA construct having the general structure in a 5′ to 3 ′ orientation comprising:
- P1-ssrN-IntN, wherein:
- (i) P1 is a promoter;
- (ii) ssrN is the N-terminal portion of a site-specific recombinase;
- and
- (iii) IntN is the N-terminal portion of a split intein;
- wherein each of P1, ssrN, and IntN are operably linked such that activation of the promoter results in the expression of the ssrN-IntN fusion protein lacking recombinase activity;
- and
- 2) a DNA construct having the general structure in a 5′ to 3′ orientation comprising:
- P2-IntC-ssrC, wherein:
- (i) P2 is a promoter;
- (ii) ssrC is the C-terminal portion of a site-specific recombinase;
- and
- (iii) IntC is the C-terminal portion of a split intein;
- wherein each of P2, ssrC, and IntC are operably linked such that activation of the promoter results in the expression of the IntC-ssrC fusion protein lacking recombinase activity, and wherein the inactive recombinase elements of (a) and (b), when present together in a cell, will form an active recombinase protein by intein-mediated trans-protein splicing;
- and
- b) a site-specific recombinase DNA substrate, wherein said substrate undergoes site-specific recombination in the presence of the active recombinase.
15 more elements
A method for conditionally activating a trait gene in a hybrid plant comprising:
- a) providing an intein-mediated site-specific recombination system comprising:
- 1) an inactive recombinase element having the general structure P1-ssrN-IntN, wherein:
- (i) P1 is a promoter;
- (ii) ssrN is the N-terminal portion of a site-specific recombinase;
- and
- (iii) IntN is the N-terminal portion of a split intein;
- 2) an inactive recombinase element having the general structure P2-IntC-ssrC, wherein:
- (i) P2 is a promoter that may be the same or different than P1;
- (ii) ssrC is the C-terminal portion of a site-specific recombinase;
- and
- (iii) IntC is the C-terminal portion of a split intein;
- 3) a trait expression construct having the general structure in a 5′ to 3′ orientation:
- P3-RS-STP-RS-TG, wherein:
- (i) P3 is a promoter;
- (ii) TG is a trait gene;
- (iii) RS is a recombinase site responsive to the recombinase;
- and
- (iv) STP is a stop fragment;
- b) providing a first transgenic plant comprising the inactive recombinase element of (a)(1) and the trait expression construct of (a)(3);
- c) providing a second transgenic plant comprising the inactive recombinase element of (a)(2) and the trait expression construct of (a)(3);
- d) crossing the first and second plants such that:
- (i) co-expression of ssrN-intN and IntC-ssrC fusion proteins in the first generation results in intein-mediated protein splicing to generate a functional active recombinase;
- and
- (ii) excision of the stop fragment by the action of the active recombinase on the recombinase sites operably links P3 and the trait gene, resulting in the expression of the trait gene in the hybrid plant.
21 more elements
A method for conditionally removing a trait gene in a hybrid plant comprising:
- a) providing an intein-mediated site-specific recombination system comprising:
- 1) an inactive recombinase element having the general structure P1-ssrN-IntN, wherein:
- (i) P1 is a promoter;
- (ii) ssrN is the N-terminal portion of a site-specific recombinase;
- and
- (iii) IntN is the N-terminal portion of a split intein;
- 2) an inactive recombinase element having the general structure P1-IntC-ssrC, wherein:
- (i) P2 is a promoter that may be the same or different than P1;
- (ii) ssrC is the C-terminal portion of a site-specific recombinase;
- and
- (iii) IntC is the C-terminal portion of a split intein;
- and
- 3) a trait expression construct having a floxed trait gene:
- RS-TG-RS, wherein:
- (i) TG is a trait gene;
- and
- (ii) RS are directly repeated recombinase sites responsive to the recombinase.
- b) providing a first transgenic plant comprising the inactive recombinase element of (a)(1) and the trait expression construct of (a)(3);
- c) providing a second transgenic plant comprising the inactive recombinase element of (a)(2) and the trait expression construct of (a)(3);
- d) crossing the first and second plants such that:
- (i) co-expression of ssrN-intN and IntC-ssrC fusion proteins in the first generation results in intein-mediated protein splicing to generate a functional active recombinase;
- and
- (ii) excision of the trait gene by the action of the active recombinase.
20 more elements
Dependencies are inferred from the claim text.