In the pUCmini-iCAP capsid plasmid, the components tTA (or rrTA, tetracycline-controlled transactivator) and the TRE (tetracycline response element), which contains TetO (Tet operator) sequences, are key elements of a Tet-On inducible gene expression system designed to regulate and amplify the production of adeno-associated virus (AAV) capsids.
Here’s a breakdown of their functions:
- tTA (or rrTA): This is a fusion protein consisting of a tetracycline repressor (TetR) from bacteria fused to a transcriptional activation domain from the herpes simplex virus (VP16).
- In the pUCmini-iCAP plasmid, the tTA/TRE system is employed as a Tet-Off system which implies that tTA binds to the TetO sequences on the TRE, activating downstream gene expression in the absence of doxycycline.
- TetO (Tet operator) and TRE (tetracycline response element): The TRE is a promoter containing multiple repeats of the Tet operator (TetO) DNA sequence.
- In the absence of doxycycline, tTA (or rrTA) binds to the TetO sequences within the TRE, activating transcription of the downstream gene, which in this case would be the AAV capsid genes.
- This amplification loop, as referred to in depositor comments, boosts capsid expression and AAV production, potentially increasing titers by 1.5-5 fold according to unpublished data.
In essence, the pUCmini-iCAP plasmid utilizes the tTA/TRE amplification loop to drive robust and controllable AAV capsid expression without the need for additional doxycycline, making it a “tet-off” system that works like other rep-cap plasmids while offering enhanced AAV production. However, it’s crucial to ensure that the packaged rAAV genome doesn’t contain a Tet-responsive element that might interfere with cell health due to altered protein expression.