Tetracycline-inducible expression systems
AssigneeDna Twopointo Inc.
InventorJeremy Minshull
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Abstract
The invention provides inducible promoter systems and their components incorporating components of a tetracycline operon. By coordinating expression of different transcriptional units in these systems as a result of selection of promoters and/or linking the units into the same DNA molecule, these systems can achieve higher levels of expression of coding segments of interest, increased differential levels of expression between on- and off-states, and/or greater responsiveness to inducing agents than conventional systems.
Claims33
A nucleic acid comprising a transcriptional unit comprising a promoter having a sequence comprising SEQ ID NO:24, and no more than ten contiguous nucleotides on the 5′ side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with which SEQ ID NO:24 is naturally associated, in operable linkage with a heterologous coding segment.
A cell in vitro or a nonhuman transgenic animal having a genome comprising (a) a promoter having a sequence comprising SEQ ID NO:24, and no more than ten contiguous nucleotides on the 5′ side of the SEQ ID NO:24 from a natural chimpanzee CMV promoter with which SEQ ID NO:24 is naturally associated, operably linked to at least two tet operators and a coding segment, and (b) a promoter operably linked to a tet-repressor or modified tet-repressor fused to transcriptional activation domain, wherein expression of the coding segment can be regulated by supplying tetracycline or doxycycline, or other tetracycline analog to the cell or nonhuman transgenic animal, or (a) a promoter having a sequence comprising SEQ ID NO:24, and no more than ten contiguous nucleotides on the 5′ side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with which SEQ ID NO:24 is naturally associated, operably linked to at least one cumate operators and a coding segment, and (b) a promoter operably linked to a cumate repressor or modified cumate repressor fused to transcriptional activation domain, wherein expression of the coding segment can be regulated by supplying cumate to the cell or nonhuman transgenic animal.
A method for inducible expression of a coding segment comprising;
- providing a first transcriptional unit comprising in operable linkage with a least two tet-operators, a promoter having a nucleotide sequence comprising SEQ ID NO:24, and no more than ten contiguous nucleotides on the 5′ side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with which SEQ ID NO:24 is naturally associated, and a coding segment, and a second transcriptional unit comprising in operable linkage a promoter, and a segment encoding a tet-repressor or modified tet-repressor fused to a transcriptional activator, wherein the tet-repressor fused to the transcriptional activator is expressed and in the absence of tetracycline or doxycycline the tet-repressor binds to the at least two tet-operators and expression of the coding segment is increased, or the modified tet-repressor fused to the transcriptional activator is expressed and in the presence of tetracycline or doxycycline the modified tet-repressor binds to the at least two tet-operators and expression of the coding segment is increased, or providing a first transcriptional unit comprising in operable linkage with a least one cumate operators, a promoter having a nucleotide sequence comprising SEQ ID NO:24, and no more than ten contiguous nucleotides on the 5′ side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with which SEQ ID NO:24 is naturally associated, and a coding segment, and a second transcriptional unit comprising in operable linkage a promoter, and a segment encoding a cumate repressor or modified cumate repressor fused to a transcriptional activator, wherein the cumate repressor fused to the transcriptional activator is expressed and in the absence of cumate, the cumate repressor binds to the at least one cumate operator and expression of the coding segment is increased, or the modified cumate repressor fused to the transcriptional activator is expressed and in the presence of cumate the modified cumate repressor binds to the at least one cumate operators and expression of the coding segment is increased.
Dependencies are inferred from the claim text.