Compositions and methods for non-targeted activation of endogenous genes

A eukaryotic cell in vitro comprising a vector, said vector comprising (i) a first promoter operably linked to a nucleotide sequence encoding a selectable marker, wherein said nucleotide sequence lacks a functional polyadenylation signal, and (ii) a second promoter operably linked to an unpaired splice donor, wherein said vector is non-homologously integrated into the genome of said eukaryotic cell in such a way that a fusion transcript comprising the nucleotide sequence encoding the selectable marker and/or the unpaired splice donor and one or more exons of an endogenous gene is expressed under the control of said first or said second promoter and wherein said unpaired splice donor is spliced to a splice acceptor of said endogenous gene to produce said fusion transcript, and coding sequence in said endogenous gene is translated.

A eukaryotic cell in vitro comprising a vector, said vector comprising (i) a first promoter operably linked to a nucleotide sequence encoding a selectable marker, wherein said nucleotide sequence lacks a functional polyadenylation signal, and (ii) a second promoter operably linked to an unpaired splice donor, wherein said vector is non-homologously integrated into the genome of said eukaryotic cell in such a way that a fusion transcript comprising the nucleotide sequence encoding the selectable marker and/or the unpaired splice donor and one or more exons of an endogenous gene is expressed under the control of said first or said second promoter, and coding sequence in said endogenous gene is translated.

A vector comprising (i) a first promoter that functions in a eukaryotic cell operably linked to a nucleotide sequence encoding a selectable marker, wherein said nucleotide sequence lacks a functional polyadenylation signal, and (ii) a second promoter operably linked to an unpaired splice donor, said vector further comprising one or more transposition signals.

A vector comprising (i) a first promoter that functions in a eukaryotic cell operably linked to a nucleotide sequence encoding a selectable marker, wherein said nucleotide sequence lacks a functional polyadenylation signal, and (ii) a second promoter operably linked to an unpaired splice donor, said vector further comprising one or more viral origins of replication.

A vector comprising (i) a first promoter operably linked to a nucleotide sequence encoding a selectable marker, wherein said nucleotide sequence lacks a functional polyadenylation signal, and (ii) a second promoter operably linked to an unpaired splice donor, said vector further comprising one or more viral replication factor genes.

A vector comprising (i) a first promoter operably linked to a nucleotide sequence encoding a selectable marker, wherein said nucleotide sequence lacks a functional polyadenylation signal, and (ii) a second promoter operably linked to an unpaired splice donor, said vector further comprising genomic DNA.

A library of eukaryotic cells in vitro comprising a vector, said vector comprising (i) a first promoter operably linked to a nucleotide sequence encoding a selectable marker, wherein said nucleotide sequence lacks a functional polyadenylation signal, and (ii) a second promoter operably linked to an unpaired splice donor, wherein said vector is non-homologously integrated into the genome of said eukaryotic cell in such a way that a fusion transcript comprising the nucleotide sequences encoding the selectable marker and/or the unpaired splice donor and one or more exons of an endogenous gene is expressed under the control of said first or said second promoter and wherein said unpaired splice donor is spliced to a splice acceptor of said endogenous gene to produce said fusion transcript, and coding sequence in said endogenous gene is translated.

A method for increasing protein expression of an endogenous gene in a eukaryotic cell in vitro, said method comprising introducing a vector into said eukaryotic cell, said vector comprising (i) a first promoter operably linked to a nucleotide sequence encoding a selectable marker, wherein said nucleotide sequence lacks a functional polyadenylation signal, and (ii) a second promoter operably linked to an unpaired splice donor, wherein said vector is non-homologously integrated into the genome of said eukaryotic cell in such a way that a fusion transcript comprising the nucleotide sequence encoding the selectable marker and/or the unpaired splice donor and one or more exons of an endogenous gene is expressed under the control of said first or said second promoter and wherein said unpaired splice donor is spliced to a splice acceptor of said endogenous gene to produce said fusion transcript, and coding sequence in said endogenous gene is translated.

A method for increasing protein expression of an endogenous gene in a eukaryotic cell in vitro, said method comprising introducing a vector into said eukaryotic cell, said vector comprising (i) a first promoter operably linked to a nucleotide sequence encoding a selectable marker, wherein said nucleotide sequence lacks a functional polyadenylation signal, and (ii) a second promoter operably linked to an unpaired splice donor into said cell, wherein said vector is non-homologously integrated into the genome of said eukaryotic cell in such a way that a fusion transcript comprising the nucleotide sequence encoding the selectable marker and/or the unpaired splice donor and one or more exons of an endogenous gene is expressed under the control of said first or said second promoter, and coding sequence in said endogenous gene is translated.