Tetracycline-inducible expression system
AssigneeDna Twopointo Inc.
InventorJeremy Minshull
Life of this patent
Estimating the term…
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.
Claims29
A nucleic acid comprising a transcriptional unit comprising a hybrid mouse-human CMV promoter having a sequence comprising SEQ ID NO:10 in operable linkage with a coding segment.
A method for inducible expression of a coding segment, the method comprising, (i) culturing a cell comprising a first transcriptional unit comprising in operable linkage with at least two tet-operators, a hybrid mouse-human CMV promoter having a sequence comprising SEQ ID NO:10 and a coding segment, and a second transcriptional unit comprising in operable linkage a promoter, and a segment encoding a tet-repressor, (a) in the absence of tetracycline, doxycycline, or other tetracycline analog, wherein the tet-repressor is expressed and binds to the at least two tet-operators, thereby inhibiting expression of the coding segment, and (b) in the presence of tetracycline, doxycycline or other tetracycline analog, wherein the tet-repressor binds to the tetracycline, doxycycline or other tetracycline analog, which inhibits its binding to the tet-operators, thereby increasing expression of the coding segment,
- or (ii) culturing a cell comprising a first transcriptional unit comprising in operable linkage with at least one cumate operator, a hybrid mouse-human CMV promoter having a sequence comprising SEQ ID NO:10, and a coding segment, and a second transcriptional unit comprising in operable linkage a promoter, and a segment encoding a cumate repressor, (a) in the absence of cumate or an analog thereof, wherein the cumate repressor is expressed and binds to the at least one cumate operator, thereby inhibiting expression of the coding segment, and (b) in the presence of cumate or an analog thereof, wherein the cumate repressor binds to the cumate or analog thereof, which inhibits its binding to the cumate operator and expression of the coding segment is increased.
A system for inducible expression comprising (i) a first transcriptional unit comprising in operable linkage with at least two tet-operators, a hybrid mouse-human CMV promoter having a sequence comprising SEQ ID NO:10, and a coding segment, and a second transcriptional unit comprising in operable linkage a promoter, and a segment encoding a tet-repressor, wherein the tet-repressor is expressed and binds to the at least two tet-operators in the absence of tetracycline or doxycycline, thereby inhibiting expression of the coding segment, and in the presence of tetracycline or doxycycline, the tet-repressor binds to the tetracycline or doxycycline, which inhibits its binding to the tet-operators, thereby increasing expression of the coding segment, or (ii) a first transcriptional unit comprising in operable linkage with at least one cumate operator, a hybrid mouse-human CMV promoter comprising SEQ ID NO:10, and a coding segment, and a second transcriptional unit comprising in operable linkage a promoter, and a segment encoding a cumate repressor, wherein the cumate repressor is expressed and binds to the at least one cumate operator in the absence of cumate, thereby inhibiting expression of the coding segment, and in the presence of cumate, the cumate repressor binds to the cumate, which inhibits its binding to the cumate operator and expression of the coding segment is increased.
Dependencies are inferred from the claim text.