US 5,801,030 A · Methods and vectors for site-specific recombination

Abstract

The present invention provides methods for site-specific recombination in a cell, as well as vectors which can be employed in such methods. The methods and vectors of the present invention can be used to obtain persistent gene expression in a cell and to modulate gene expression. One preferred method according to the invention comprises contacting a cell with a vector comprising an origin of replication functional in mammalian cells located between first and second recombining sites located in parallel. Another preferred method comprises, in part, contacting a cell with a vector comprising first and second recombining sites in antiparallel orientations such that the vector is internalized by the cell. In both methods, the cell is further provided with a site-specific recombinase that effects recombination between the first and second recombining sites of the vector.

Claims47

Dependent claims

A method of effecting site-specific recombination in a mammalian cell comprising:

(a) contacting said cell with a linear viral vector such that said vector is internalized by said cell, wherein said vector comprises (i) a first recombining site and a second recombining site in parallel orientation, between which are an origin of replication, which is functional in a mammalian cell, and a passenger gene, and (ii) viral encapsidation sequences in a region other than the region between said first and second recombining sites, and
(b) providing said cell with a site-specific recombinase that effects recombination between said first and second recombining sites of said vector.

A method of effecting site-specific recombination in a mammalian cell comprising:

(a) contacting said cell with a linear viral vector such that said vector is internalized by said cell, wherein said vector comprises (i) a first recombining site and a second recombining site in parallel orientation, between which are an origin of replication, which is functional in a mammalian cell, a first coding sequence, which is not operably linked to a promoter and which adjoins said first recombining site such that said coding sequence is oriented to be transcribed in the direction from said first recombining site to said second recombining site proceeding through said coding sequence, and a promoter, which adjoins said second recombining site such that said promoter is oriented to direct transcription in the direction from said first recombining site to said second recombining site proceeding through said promoter, and (ii) viral encapsidation sequences in a region other than the region between said first and second recombining sites, and
(b) providing said cell with a site-specific recombinase that effects recombination between said first and second recombining sites of said vector,
wherein, upon recombination, said first coding sequence is operably linked to said promoter.

A method of effecting site-specific recombination in a cell comprising:

(a) contacting said cell with a vector such that said vector is internalized by said cell, wherein said vector comprises:
(i) a first recombining site and a second recombining site in antiparallel orientation, between which is a promoter, which adjoins said second recombining site such that said promoter is oriented to direct transcription in the direction from said first recombining site to said second recombining site proceeding through said promoter,
(ii) a first coding sequence, which is located in a region other than the region between said first and second recombining sites comprising said promoter, adjoins said first recombining site such that said first coding sequence is oriented to be transcribed in the opposite direction from the direction of said first recombining site to said second recombining site proceeding through said promoter, and is not operably linked to a promoter, and
(iii) a second coding sequence, which is located in a region other than the region between said first and second recombining sites comprising said promoter, adjoins said second recombining site, and is operably linked to said promoter, and
(b) providing said cell with a site-specific recombinase that effects recombination between said first and second recombining sites of said vector,
wherein, upon recombination, said first coding sequence is operably linked to said promoter and said second coding sequence is not operably linked to said promoter.

3 more elements

A method of effecting site-specific recombination in a cell comprising:

(a) contacting said cell with a vector such that said vector is internalized by said cell, wherein said vector comprises:
(i) a first recombining site and a second recombining site in antiparallel orientation, between which are a first coding sequence, which adjoins said second recombining site such that said first coding sequence is oriented to be transcribed in the direction from said second recombining site to said first recombining site proceeding through said first coding sequence and which is not operably linked to a promoter, and a second coding sequence, which adjoins said first recombining site and is oriented to be transcribed in the direction from said first recombining site to said second recombining site proceeding through said second coding sequence, and
(ii) a promoter, which is located in a region other than the region between said first and second recombining sites comprising said first and second coding sequences, adjoins said first recombining site such that said promoter is oriented to direct transcription in the direction from said promoter to said second recombining site proceeding through said first recombining site and said second coding sequence, and is operably linked to said second coding sequence, and
(b) providing said cell with a site-specific recombinase that effects recombination between said first and second recombining sites of said vector,
wherein, upon recombination, said first coding sequence is operably linked to said promoter and said second coding sequence is not operably linked to said promoter.

2 more elements

A method of effecting site-specific recombination in a mammalian cell comprising:

(a) contacting said cell with a vector such that said vector is internalized by said cell, wherein said vector comprises (i) a first recombining site and a second recombining site in parallel orientation, between which are an origin of replication, which is functional in a mammalian cell, and a first promoter, which adjoins said first recombining site such that said first promoter is oriented to direct transcription in the opposite direction from the direction of said first recombining site to said second recombining site proceeding through said first promoter, (ii) a second promoter, which is located in a region other than the region between said first and second recombining sites comprising said origin and adjoins said first recombining site such that said second promoter is oriented to direct transcription in the direction from said second promoter to said second recombining site proceeding through said first recombining site, (iii) a first coding sequence, which is located in a region other than the region between said first and second recombining sites comprising said origin, adjoins said second recombining site such that said first coding sequence is oriented to be transcribed in the direction from said first recombining site to said second recombining site proceeding through said first promoter, and is not operably linked to a promoter, (iv) a second coding sequence, which is located in the region between said first and second recombining sites comprising said origin, adjoins said second recombining site such that said second coding sequence is oriented to be transcribed in the direction from said second recombining site to said first recombining site proceeding through said second coding sequence, and is not operably linked to a promoter, and (v) sequences that direct vector propagation in a mammalian cell located in a region other than the region between said first and second recombining sites comprising said origin, and
(b) providing said cell with a site-specific recombinase that effects recombination between said first and second recombining sites of said vector,
wherein, upon recombination, said second coding sequence is operably linked to said first promoter and said first coding sequence is operably linked to said second promoter.

A method of effecting site-specific recombination in a eukaryotic cell comprising:

(a) contacting said cell with a vector such that said vector is internalized by said cell, wherein said vector comprises a first recombining site and a second recombining site in parallel orientation, between which are (i) a first coding sequence, which adjoins said first recombining site such that said firs,t coding sequence is oriented to be transcribed in the direction from said first recombining site to said second recombining site proceeding through said first coding sequence, and which is not operably linked to a promoter, and (ii) a first promoter, which adjoins said second recombining site such that said first promoter is oriented to direct transcription in the direction from said first recombining site to said second recombining site proceeding through said first coding sequence, and
(b) providing said cell with a site-specific recombinase that effects recombination between said first and second recombining sites,
wherein, upon recombination, said first coding sequence is operably linked to said first promoter.

Dependencies are inferred from the claim text.

Methods and vectors for site-specific recombination

Life of this patent

Methods and vectors for site-specific recombination

Life of this patent

Abstract

Claims47