Compositions and methods for non-targeted activation of endogenous genes

A method for producing an expression product of an endogenous cellular gene or portion thereof comprising:

• (a) introducing into a eukaryotic cell in vitro a vector construct comprising in operable linkage:
• (1) a transcriptional regulatory sequence,
• (2) an exon comprising a nucleic acid sequence encoding a partial signal sequence that can complement a partial signal sequence in an endogenous cellular gene to produce a functional signal sequence, and
• (3) an unpaired splice donor site that defines the 3′ end of the exon in (2);
• (b) maintaining said cell under conditions suitable for integrating said vector construct into the genome of said cell by non-homologous recombination such that the sequences of (1), (2) and (3) are opetably linked to said endogenous cellular gene;
• and
• (c) maintaining said cell produced in step (b) under conditions suitable for over-expressing, by means of said integrated transcriptional regulatory sequence, said endogenous cellular gene, or portion thereof, in said cell, thereby producing said expression product.

A method for producing an expression product of an endogenous cellular gene or portion thereof in a cell comprising:

• (a) introducing into at least one eukaryotic cell in vitro a vector construct comprising in operable linkage:
• (1) a transcriptional regulatory sequence,
• (2) an exon comprising a nucleic acid sequence encoding a partial signal sequence that can complement a partial signal sequence in an endogenous cellular gene to produce a functional signal sequence, and
• (3) an unpaired splice donor site that defines the 3′ end of the exon in (2);
• (b) maintaining said cell under conditions suitable for integrating said vector construct into the genome of said cell by non-homologous recombination such that the sequences of (1), (2) and (3) are operably linked to said endogenous cellular gene;
• (c) maintaining said cell produced in step (b) under conditions suitable for over-expressing said endogenous cellular gene or portion thereof in said cell by up-regulation of said gene in said cell by said transcriptional regulatory sequence;
• and
• (d) culturing said cell in serum-free medium, resulting in production of said expression product.

A method of identifying a protein product over-expressed by an endogenous cellular gene or portion thereof, said method comprising:

• (a) introducing into at least one eukaryotic cell in vitro a vector construct comprising in operable linkage:
• (1) a transcriptional regulatory sequence,
• (2) an exon comprising a nucleic acid sequence encoding a partial signal sequence that can complement a partial signal sequence in an endogenous cellular gene to produce a functional signal sequence, and
• (3) an unpaired splice donor site that defines the 3′ end of the exon in (2);
• (b) maintaining said cell under conditions suitable for integrating said vector construct into the genome of said cell by non-homologous recombination such that the sequences of (1), (2) and (3) are operably linked to said endogenous cellular gene;
• (c) culturing said cell in serum-free medium under conditions that allow over-expression of said endogenous cellular gene or a portion thereof in said cell by up-regulation of said gene in said cell by said transcriptional regulatory sequence, thereby producing cell-conditioned media;
• and
• (d) screening said cell-conditioned media for the presence of the protein product of over-expression of said gene or portion thereof, thereby identifying said protein product.

A method for activation of an endogenous cellular gene in a cell comprising:

• (a) transfecting a eukaryotic cell in vitro with a vector construct comprising in operable linkage:
• (1) a transcriptional regulatory sequence,
• (2) an exon comprising a nucleic acid sequence encoding a partial signal sequence that can complement a partial signal sequence in an endogenous cellular gene to produce a functional signal sequence, and
• (3) an unpaired splice donor site that defines the 3′ end of the exon in (2);
• and
• (b) culturing said cell under conditions suitable for non-homologous integration of said vector construct into the genome of said cell, wherein said integration results in the activation of said endogenous cellular gene in the genome of said cell by means of said transcriptional regulatory sequence.

A method for identifying an endogenous cellular gene containing an exon by identifying a cDNA made from an RNA expression product of that gene, said method comprising:

• (a) transfecting a plurality of eukaryotic cells in vitro with a vector construct comprising in operable linkage:
• (1) a transcriptional regulatory sequences,
• (2) an exon comprising a nucleic acid sequence encoding a partial signal sequence that can complement a partial signal sequence in an endogenous cellular gene to produce a functional signal sequence, and
• (3) an unpaired splice donor site that defines the 3′ end of the exon in (2), wherein said vector construct does not contain a targeting sequence;
• (b) culturing said cells under conditions suitable for integration of the vector construct into the genome of the host cell;
• (c) selecting for cells in which said vector construct has integrated into the genomes of said cells;
• (d) isolating RNA from said selected cells;
• (e) producing cDNA from said isolated RNA;
• and
• (f) identifying cDNA that contains both vector and exon sequence, thereby identifying said gene containing said exon.

A method for activating expression from an endogenous cellular gene comprising:

• (a) introducing into a eukaryotic cell in vitro a vector construct comprising in operable linkage:
• (1) a transcriptional regulatory sequence,
• (2) an exon comprising a nucleic acid sequence encoding a partial signal sequence that can complement a partial signal sequence in an endogenous cellular gene to produce a functional signal sequence, and
• (3) an unpaired splice donor site that defines the 3′ end of the exon in (2);
• (b) introducing DNA breaks in the chromosome of said cell prior to or following introduction of said vector construct;
• and
• (c) maintaining said cell under conditions suitable for integrating said vector construct, by non-homologous recombination, into said chromosome so as to result in the formation of an operable linkage between said transcriptional regulatory sequence on said vector construct and said endogenous cellular gene, whereby said endogenous cellular gene is activated by said transcriptional regulatory sequence on said vector construct.

A method for expressing a gene or portion thereof, comprising:

• (a) obtaining genomic DNA, comprising one or more genes, or a portion of said one or more genes from one or more eukaryotic cells;
• (b) introducing into a eukaryotic cell in vitro said genomic DNA and a vector construct, said vector construct comprising a transcriptional regulatory sequence that is operably linked to an exon encoding a partial signal sequence that can complement a partial signal sequence in said genomic DNA and an unpaired splice donor site that defines the 3′ end of said exon;
• (c) selecting for a cell containing said genomic DNA and said vector;
• and
• (d) culturing said cell under conditions suitable for expression of said gene or portion thereof, wherein said gene or portion thereof is present on said genomic DNA and said expression results from the operable linkage between said transcriptional regulatory sequence in said vector construct and said gene or portion thereof.