Patent ID: 8043611

Claim:
An isolated mammalian cell for packaging a retroviral vector, wherein the mammalian cell comprises a regulated gene expression system comprising three nucleic acid constructs, wherein three nucleic acid constructs comprise: (i) a first nucleic acid construct comprising: (1) a first inducible promoter that regulates the expression (transcription) of a first message (transcript), wherein the first inducible promoter is auto-regulated such that expression of the first message is repressed by the protein encoded by the first message, wherein the first inducible promoter is TRE and the first message is tTA, (2) a constitutively active promoter that regulates the expression (transcription) of a first selection marker, and (3) a second inducible promoter that regulates the expression (transcription) of a second message (transcript) and a third message (transcript), wherein the first message (transcript) comprises a fusion protein-encoding message, and the fusion protein comprises: a first protein domain that can activate transcription of the first and the second inducible promoters, and a second protein domain that can bind a tetracycline, such that when the second protein domain is in a tetracycline-bound form the transcription-activating domain of the first domain cannot activate transcription of the first and the second inducible promoters, and the second message (transcript) comprises a Rev RNA/RRE binding protein-encoding message (transcript), or a message encoding a chimeric protein comprising a Rev RNA/RRE binding protein, and the third message (transcript) comprises a tat protein-encoding message (transcript), or a message encoding chimeric protein comprising a tat protein; (ii) a second nucleic acid construct comprising: (1) a nucleotide sequence encoding a chimeric U3/TAR2 inducible promoter upregulated by the tat protein (of the third message), wherein the U3/TAR2 inducible promoter regulates expression (transcription) of a fourth message (transcript) comprising a retroviral protein-coding message, and (2) a constitutively active promoter regulating the expression (transcription) of a second selection marker; and, (iii) a third nucleic acid construct comprising: (1) a constitutively active promoter regulating the expression (transcription) of an RRE-regulated viral envelope message (transcript), and (2) a second constitutively active promoter that regulates expression of (operatively linked to) a third selectable marker; wherein the Rev RNA binding protein regulates expression of RRE regulated viral envelope-encoding message of the third nucleic acid construct.