IP Library › Granted Patent US 12,351,813
Granted Patent B2
US 12,351,813 · App. 18/067,686 · Granted Jul 8, 2025

Tetracycline-inducible expression system

Inventor: Jeremy Minshull (Los Altos, CA)
Assignee: DNA TWOPOINTO INC.
C12N15/86A01K67/0275A61K48/0066C12N5/0682C12N15/8509A01K2217/05A01K2217/203A01K2227/105C12N2710/16142C12N2800/90C12N2830/003C12N2830/50
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Quick Facts
Patent No.
US 12,351,813
App. No.
18/067,686
Granted
Jul 8, 2025
Kind
B2
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.

Claims (30)

1. 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.

2. The nucleic acid of claim 1 , further comprising at least first and second tet-operators in operable linkage with the promoter and wherein the first and second tet-operators are situated 3′ to the promoter, downstream of a TATA box and upstream of the coding segment and its transcription start site.

3. The nucleic acid of claim 2 , wherein the promoter and tet-operators together form a sequence consisting of SEQ ID NO:11.

4. The nucleic acid of claim 1 , further comprising at least one cumate operator in operable linkage with the promoter and wherein each cumate operator is situated 3′ to the promoter, downstream of a TATA box and upstream of the coding segment and its transcription start site.

5. The nucleic acid of claim 4 , wherein each cumate operator has a sequence selected independently from any of SEQ ID NOS:156-158.

6. The nucleic acid of claim 1 , further comprising a second transcriptional unit comprising a second promoter operably linked to a segment encoding a tet-repressor effective to bind a tet-operator 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.

7. The nucleic acid of claim 6 , wherein the second promoter is a weaker promoter than a human CMV promoter.

8. The nucleic acid of claim 6 , wherein the second promoter is a eukaryotic translation elongation factor 2 (EEF2) or phosphoglycerate kinase (PGK) promoter.

9. The nucleic acid of claim 6 , wherein the second promoter has a sequence selected from any of SEQ ID NOS:17-21, 33 and 34.

10. The nucleic acid of claim 6 , wherein the tet-repressor has an amino acid sequence comprising SEQ ID NO:5.

11. The nucleic acid of claim 1 , further comprising a second transcriptional unit comprising a second promoter operably linked to a segment encoding a cumate repressor effective to bind a 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 operators, thereby increasing expression of the coding segment.

12. The nucleic acid of claim 11 , wherein the promoter of the second transcriptional unit is a weaker promoter than a human CMV promoter.

13. The nucleic acid of claim 11 , wherein the promoter of the second transcriptional unit is a eukaryotic translation elongation factor 2 (EEF2) or phosphoglycerate kinase (PGK) promoter.

14. The nucleic acid of claim 11 , wherein the promoter is selected from any of SEQ ID NOS:17-21, 33 and 34.

15. The nucleic acid of claim 11 , wherein the cumate repressor has an amino acid sequence comprising SEQ ID NO:170.

16. The nucleic acid of claim 1 , wherein the coding segment encodes a protein.

17. A transposon comprising the nucleic acid of claim 1 flanked by inverted repeats of the transposon such that the nucleic acid is integrated into a cell with a transposase.

18. The transposon of claim 17 , which is a piggyBac-like transposon.

19. An isolated cell transformed with the nucleic acid of claim 1 .

20. The isolated cell of claim 19 , which is mammalian.

21. A non-human animal transformed with the nucleic acid of claim 1 .

22. A cultured cell or non-human transgenic animal having a genome comprising the nucleic acid of claim 1 .

23. 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.

24. The method of claim 23 , wherein the first and second transcriptional units are components of the same contiguous DNA molecule.

25. The method of claim 24 , further comprising introducing the contiguous DNA molecule into the cell.

26. The method of claim 25 , wherein the cell is a mammalian cell, and wherein the first and second transcriptional units integrate into the genome of the cell.

27. The method of claim 23 , wherein the first and second transcriptional units are components of a transposon.

28. The method of claim 27 , wherein the transposon is a piggyBac-like transposon and wherein the transposon is integrated in the cell with a transposase.

29. 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.

Continuity (3)
Continuation 17700405 · Mar 21, 2022
Provisional Application 63165484 · Mar 24, 2021
Related Publication 20230323396A1 · Oct 12, 2023
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