Methods and compositions for genomic modification
View Patent ↗The present invention provides methods of site-specifically integrating a polynucleotide sequence of interest in a genome of a eucaryotic cell, as well as, enzymes, polypeptides, and a variety of vector constructs useful therefore. In the method, a targeting construct comprises, for example, (i) a first recombination site and a polynucleotide sequence of interest, and (ii) a site-specific recombinase, which are introduced into the cell. The genome of the cell comprises a second recombination site. Recombination between the first and second recombination sites is facilitated by the site-specific recombinase. The invention 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 techniques.
1. A method of integrating a nucleic acid into a genome of a fibroblast cell in vitro, comprising:
introducing into said fibroblast cell in vitro (i) an expression cassette comprising a polynucleotide encoding a φC31 phage recombinase; and (ii) a targeting vector comprising a nucleic acid and a single φC31 vector attachment site; and
maintaining said cell under conditions sufficient for said targeting vector to integrate into an endogenous target site of said genome by a recombination event mediated by said φC31 phage recombinase.
2. The method according to claim 1 , wherein said fibroblast cell is derived from a human subject.
3. The method according to claim 1 , wherein said nucleic acid comprises a eukaryotic coding sequence.
4. The method according to claim 3 , wherein said coding sequence encodes a polypeptide.
5. The method according to claim 3 , wherein said coding sequence encodes an RNA molecule.
6. The method according to claim 3 , wherein said coding sequence is present in an expression cassette.
7. A method comprising:
introducing into a genome of a fibroblast cell in vitro (i) an expression cassette comprising a polynucleotide encoding a φC31 phage recombinase; and (ii) a targeting vector comprising a nucleic acid and a single φC31 vector attachment site;
maintaining said cell under conditions sufficient for said targeting vector to integrate into an endogenous target site of said genome by a recombination event mediated by said φC31 phage recombinase; and
locally administering said cell to a human subject following integration of said nucleic acid into said genome of said cell.
8. The method according to claim 1 , wherein said vector attachment site is a bacterial genomic recombination site (attB), a phage genomic recombination site (attP), a pseudo bacterial genomic recombination site (pseudo-attB), or a pseudo phage genomic recombination site (pseudo-attP).