Abstract: The subject invention provides a unidirectional site-specific integration system for integrating a nucleic acid into the genome of a target cell. The provided systems include a (1) a mutant, unidirectional site specific integrase, which can be provided by an integrase vector encoding the mutant integrase and (2) a targeting vector that includes: (a) a nucleic acid to be integrated; and (b) a vector attachment site, where the targeting vector attachment site serves as a substrate for the mutant, unidirectional site-specific integrase. In using the subject systems for site-specific integration, the targeting vector and integrase are introduced into the target cell and the cell is maintained under conditions sufficient to provide for site-specific integration of the nucleic acid into the target cell genome via a recombination event mediated by the site-specific integrase. Also provided are kits that include the subject systems.

Abstract: The present invention describes methods of identifying altered recombinases and compositions thereof, wherein at least one amino acid is different from a parent, wild-type recombinase and the altered recombinase has improved recombination efficiency towards wild-type and/or pseudo att site sequences relative to the parent, wild-type recombinase. The present invention also includes methods of modifying the genomes of cells using the altered recombinases, including methods of site-specifically integrating a polynucleotide sequence of interest in a genome of a eucaryotic cell.

Abstract: The present invention provides a method of treating an ocular disease in a subject. In a first step, a nucleic acid is introduced into cells or a tissue. The nucleic acid is introduced by electron avalanche transfection. With this technique, a high electric field induces a vapor bubble and plasma discharge between an electrode and the surrounding medium. The formation of a vapor bubble generates mechanical stress. Plasma discharge through the ionized vapor in the bubble enables connectivity between the electrode and the surrounding medium, so that mechanical stress and electric field are applied simultaneously, which results in permeabilization of the cells or tissue. This permeabilization in turn allows the nucleic acid to enter the cell or tissue. Cells or tissue containing the nucleic acid are then transplanted into an ocular region of the subject.

Abstract: The subject invention provides a unidirectional site-specific integration system for integrating a nucleic acid into the genome of a target cell. The provided system includes a site-specific integrating expression cassette (INTEC) vector, consisting of (a) a polynucleotide of interest operably linked to a promoter, (b) a single recombination site, and (c) a hybrid recombination site. In using the subject systems for site-specific integration, the INTEC vector and integrase are introduced into the target cell and the cell is maintained under conditions sufficient to provide for site-specific integration of the nucleic acid into the target cell genome via a recombination event mediated by the site-specific recombinase. Also provided are kits that include the subject systems. The subjects systems, methods and kits find use in a variety of different applications, several representative ones of which are described in detail as well.

Abstract: The subject invention provides a unidirectional site-specific integration system for integrating a nucleic acid into the genome of a target cell. The provided systems include a (1) a mutant, unidirectional site specific integrase, which can be provided by an integrase vector encoding the mutant integrase and (2) a targeting vector that includes: (a) a nucleic acid to be integrated; and (b) a vector attachment site, where the targeting vector attachment site serves as a substrate for the mutant, unidirectional site-specific integrase. In using the subject systems for site-specific integration, the targeting vector and integrase are introduced into the target cell and the cell is maintained under conditions sufficient to provide for site-specific integration of the nucleic acid into the target cell genome via a recombination event mediated by the site-specific integrase. Also provided are kits that include the subject systems.

Abstract: The present invention describes methods of identifying altered recombinases and compositions thereof, wherein at least one amino acid is different from a parent, wild-type recombinase and the altered recombinase has improved recombination efficiency towards wild-type and/or pseudo att site sequences relative to the parent, wild-type recombinase. The present invention also includes methods of modifying the genomes of cells using the altered recombinases, including methods of site-specifically integrating a polynucleotide sequence of interest in a genome of a eucaryotic cell.