US 5,859,309 A · Vector for integration site independent gene expression in mammalian host cells

Abstract

A vector for the integration of a gene into the genetic material of a mammalian host cell such that the gene may be expressed by the host cell. The vector comprises a promoter and the gene and an immunoglobulin dominant control region derived from the mouse .lambda. immunoglobulin gene locus capable of eliciting host cell-type restricted, integration site independent, copy number dependent expression of the said gene. The DNaseI super hypersensitive site exemplified are i) about 2.35 kb upstream of the CAP site of the rearranged .lambda..sub.1 gene, ii) about 2.5 kb upstream of the genomic V.lambda..sub.2 segment or iii) about 30 kb downstream of the rearranged .lambda..sub.1 gene. Mammalian host cells transformed with the vector are disclosed as are transgenic mammals transformed with the vector and a method of producing a polypeptide comprising culturing a transformed mammalian cell. A method of gene therapy comprising the steps of i) removing stem cells from the body of a mammal, ii) optionally killing stem cells remaining in the body, iii) transforming the removed stem cells with the containing a gene deficient or absent in the body, and iv) replacing the transformed stem cells in the body is also disclosed. Also disclosed is functional mouse immunoglobulin .lambda..sub.1 enhancer consisting of a DNA sequence comprising all or a functional part of the DNA sequence between the EcoRI site 3.8 kb downstream of the Xho I site in the rearranged mouse .lambda..sub.1 gene and the SnaBI site 10 kb downstream of this Xho I site. The functional mouse immunoglobulin .lambda..sub.1 enhancer may comprise all or a functional part of i) the 1.3 kb first HindIII to HindII DNA fragment downstream of the EcoRI site 3.8 kb downstream of the Xho I site in the rearranged mouse .lambda..sub.1 gene, ii) the 3.3 kb HindII to HindII DNA fragment downstream of the EcoRI site 3.8 kb downstream of the Xho I site in the rearranged mouse .lambda..sub.1 gene and spanning the SnaBI site 10 kb downstream of this Xho I site.

Claims18

Dependent claims

An isolated DNA molecule, comprising

(i) a dominant control region that is functional in a mammalian cell-type which permits immunoglobulin gene expression,
(ii) a promoter that is functional in said mammalian cell-type, and
(iii) a gene operatively linked to said promoter, wherein
the region in said DNA consisting of:
(i), (ii) and (iii) and DNA therebetween has a nucleotide sequence different from that of naturally occurring DNA, and
said dominant control region being characterized in that in naturally occurring DNA:
(I) it is associated with a naturally occurring immunoglobulin gene locus;
and
(II) it is locatable in naturally occurring DNA by association with a DNase I super hypersensitive site;
wherein said dominant control region is characterized in that it stimulates expression of said gene when said DNA molecule is integrated into a genome of a host cell which permits expression of said naturally occurring immunoglobulin gene locus, such that said expression:
(a) is dependent on the number of copies of said gene that are integrated into said genome in that said expression increases as said number of copies of said gene increases;
and
(b) is independent of the integration site of said DNA molecule in said genome.

10 more elements

A non-human transgenic mammal comprising, integrated into its genome, a recombinant DNA molecule comprising:

(i) a dominant control region that is functional in a mammalian cell-type which permits immunoglobulin gene expression,
(ii) a promoter that is functional in said mammalian cell-type, and
(iii) a recombinant gene operatively linked to said promoter, wherein the region in said DNA consisting of (i), (ii) and (iii) and DNA therebetween has a nucleotide sequence different from that of a naturally occurring DNA sequence, and said dominant control region being characterized in that:
(I) it is associated in naturally occurring DNA with a naturally occurring immunoglobulin gene locus;
and
(II) it is locatable in naturally occurring DNA by association with a DNase I super hypersensitive site;
wherein said recombinant DNA is integrated into a genomic locus that is different from the immunoglobulin gene locus of the dominant control region in naturally occurring DNA and wherein said dominant control region stimulates expression of said recombinant gene when said recombinant DNA molecule is integrated into a genome of a host cell of said cell-type, such that said expression:
(a) is dependent on the number of copies of said recombinant gene that are integrated into said genome in that said expression increases as said number of copies of said recombinant increases;
(b) is independent of the integration site of said recombinant DNA molecule in said genome;
and
(c) results in production in said host cell of said cell type of a detectable amount of product encoded by said recombinant gene.

8 more elements

A non-human transgenic mammal of a first species comprising, integrated into its genome, a recombinant DNA comprising:

(i) a dominant control region from a second species of mammal that is specific for a mammalian cell-type which permits immunoglobulin gene expression,
(ii) a promoter that is functional in said mammalian cell-type, and
(iii) a recombinant gene operatively linked to said promoter, when the region in said DNA consisting of (i), (ii) and (iii) and DNA therebetween has a nucleotide sequence different from that of a naturally occurring DNA sequence, and said dominant control region being characterized in that:
(I) it is associated in naturally occurring DNA with a naturally occurring immunoglobulin gene locus;
and
(II) it is locatable in naturally occurring DNA by association wit a DNase I super hypersensitive site;
wherein said dominant control region stimulates expression of said recombinant gene when said recombinant DNA molecule is integrated into a genome of a host cell of said cell-type, such that said expression:
(a) is dependent on the number of copies of said recombinant gene that are integrated into said genome in that said expression increases as said number of copies of sad recombinant gene increases;
(b) is independent of the integration site of said DNA molecule in said genome;
and
(c) results in production in said host cell of said cell-type of a detectable amount of product encoded by said recombinant gene.

8 more elements

Dependencies are inferred from the claim text.

Vector for integration site independent gene expression in mammalian host cells

Life of this patent

Vector for integration site independent gene expression in mammalian host cells

Life of this patent

Abstract

Claims18