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 a transcriptional regulatory sequence that is operably linked to an exon comprising a translational start codon, a secretion signal sequence, and an epitope tag sequence, said exon defined at the 3′ end by an unpaired splice donor site;
• (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 transcriptional regulatory sequence is operably 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 gene in a cell comprising:

• (a) introducing into at least one eukaryotic cell in vitro a vector construct comprising a transcriptional regulatory sequence that is operably linked to an exon comprising a translational start codon, a secretion signal sequence, and an epitope tag sequence, said exon defined at the 3′ end by an unpaired splice donor site;
• (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 transcriptional regulatory sequence is operably linked to said endogenous cellular gene;
• (c) maintaining said cell produced in step (b) under conditions suitable for over-expressing an endogenous gene or a 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 gene, said method comprising:

• (a) introducing into at least one eukaryotic cell in vitro a vector construct comprising a transcriptional regulatory sequence that is operably linked to an exon comprising a translational start codon, a secretion signal sequence, and an epitope tag sequence, said exon defined at the 3′ end by an unpaired splice donor site;
• (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 transcriptional regulatory sequence is operably linked to said endogenous gene;
• (c) culturing said cell in serum-free medium under conditions that allow over-expression of said endogenous gene or a portion thereof in said cell by up-regulation of said endogenous 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 endogenous gene or portion thereof, thereby identifying said protein product.

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 cell in vitro said genomic DNA and a vector, said vector comprising a selectable marker and a transcriptional regulatory sequence that is operably linked to an exon comprising a translational start codon, a secretion signal sequence, and an epitope tag sequence, said exon being defined at the 3′ end by an unpaired splice donor site;
• (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 on said vector construct and said gene or portion thereof.

A method for activating expression from an endogenous gene comprising:

• (a) introducing into a eukaryoptic cell in vitro a vector construct comprising a transcriptional regulatory sequence that is operably linked to an exon comprising a translational start codon, a secretion signal sequence, and an epitope tag sequence, said exon defined at the 3′ end by an unpaired splice donor site;
• (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 vector construct and said endogenous gene, whereby said endogenous gene is activated by one or more vector construct-encoded nucleotide sequences.