Promote system for regulatable gene expression in mammalian cells
The present invention is directed to a bidirectional human cytomegalovirus (hCMV) promoter that can be used to promote transcription on both strands of a double stranded DNA molecule. When used as part of a system that includes tet operator and the gene coding for the tet repressor, the promoter can be used to induce mammalian gene expression in a highly regulated way.
1. A bidirectional promoter system derived from the hCMV major immediate-early promoter and comprising the structure X-Y-Z or X-Z, wherein:
X is a sequence that is complementary to 29-130 consecutive nucleotides at the 3′ end of SEQ ID NO:2, and is located at the 5′ end of Y or Z but in a reverse orientation;
Y is a nucleotide linker sequence that is not critical to promoter activity and that is 1-200 nucleotides in length; and
Z comprises 500-604 contiguous nucleotides extending from the start site of transcription of the sequence shown as SEQ ID NO:2 in the 3′ to 5′ direction and is located at the 3′ end of Y or X, terminating at the start site of transcription.
2. The bidirectional promoter system of claim 1 , wherein Y is 1-100 nucleotides in length.
3. The bidirectional promoter system of claim 1 , wherein Y is 1-40 nucleotides in length.
4. The bidirectional promoter system of claim 1 , wherein X is a sequence that is complementary to 80-110 consecutive nucleotides at the 3′ end of SEQ ID NO:2, terminating at the start site of transcription.
5. The bidirectional promoter system of claim 1 , wherein X is a sequence that is complementary to 94-108 consecutive nucleotides at the 3′ end of SEQ ID NO:2, terminating at the start site of transcription.
6. The bidirectional promoter system of claim 1 , wherein said promoter is operably linked to a gene located 3′ to Z.
7. The bidirectional promoter system of claim 6 , wherein Y is 1-100 nucleotides in length.
8. The bidirectional promoter system of claim 6 , wherein Y is 1-40 nucleotides in length.
9. The bidirectional promoter system of claim 6 , wherein X is a sequence that is complementary to 80-110 consecutive nucleotides at the 3′ end of SEQ ID NO:2, terminating at the start site of transcription.
10. The bidirectional promoter system of claim 6 , wherein X is a sequence that is complementary to 94-108 consecutive nucleotides at the 3′ end of SEQ ID NO:2, terminating at the start site of transcription.