Robust, synergistic regulation of human gene expression using TALE activators.
Maeder ML, Linder SJ, Reyon D, Angstman JF, Fu Y, Sander JD, Joung JK
Nat Methods. 2013 Feb 10. doi: 10.1038/nmeth.2366.
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PubMed
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Article
TALE Transcriptional Activator Expression Vectors
DNA fragments encoding TAL effector repeat arrays (assembled using the REAL, REAL-Fast or FLASH methods) can be cloned into these vectors for expression as TALE transcriptional activators. The Joung Lab has recently shown that TALE activators made on the framework encoded in these plasmids robustly yield highly active artificial transcription factors. All eight expression vectors have the EF1α promoter for efficient mammalian cell expression, encode one of the four 0.5 TALE repeats, and encode either a VP64 or a NF-κB p65 transcriptional activation domain at the carboxy-terminus of the protein. TALE activators expressed from these vectors also have a nuclear localization signal encoded at their amino-terminus.
The REAL, REAL-Fast, and FLASH assembly methods yield TAL effector repeat arrays with identical DNA and amino acid sequences (i.e. only the details of the assembly are different among the three methods, the final products are the same).
The publicly available, web-based ZiFiT Targeter (Link opens in a new window) software program has been updated to enable identification of potential TALE activator binding sites in any given sequence of interest. The program generates graphical guides that facilitate construction of TALE repeat arrays by REAL or REAL-Fast assembly or names of plasmids required for FLASH assembly.
Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 43884 | MLM3367 | Activation Domain: VP64; RVD of C-terminal 0.5 TALE repeat: NI (binds A) |
| 43885 | MLM3585 | Activation Domain: VP64; RVD of C-terminal 0.5 TALE repeat: HD (binds C) |
| 43886 | MLM3587 | Activation Domain: VP64; RVD of C-terminal 0.5 TALE repeat: NN (binds G) |
| 43887 | MLM3583 | Activation Domain: VP64; RVD of C-terminal 0.5 TALE repeat: NG (binds T) |
| 43888 | MLM2581 | Activation Domain: NF-kB; RVD of C-terminal 0.5 TALE repeat: NI (binds A) |
| 43889 | MLM2583 | Activation Domain: NF-kB; RVD of C-terminal 0.5 TALE repeat: HD (binds C) |
| 43890 | MLM2585 | Activation Domain: NF-kB; RVD of C-terminal 0.5 TALE repeat: NN (binds G) |
| 43891 | MLM2579 | Activation Domain: NF-kB; RVD of C-terminal 0.5 TALE repeat: NG (binds T) |