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CompoZr® Custom Zinc Finger Activators

Deliver Zinc Finger Activators to Cells to Up-Regulate Target Gene Expression

CompoZr Zinc Finger Activators (ZFAs or ZF-TFs) are engineered zinc finger transcription factors that facilitate highly specific, targeted transcriptional activation or repression by fusing zinc finger proteins to transcriptional activation or repression domains. These engineered ZF-TFs can upregulate or downregulate gene expression upon delivery into cells.  You can use ZFAs to upregulate genes encoding for transcription factors, including genes overexpressed for cellular reprogramming.

The new Sigma CompoZr ZFA products provide researchers with a custom designed Zinc Finger protein tethered to the NF-κB p65 activation domain. Gene activation varies with the different activation domains available, as these domains have a prominent effect on optimal transcriptional activation. In addition to the NF-κB p65 activation domain, we provide separate vectors with the following activation domains:  VP-16, VP-64 and 2X p65.  You have the ability to swap in the optimal activation domain for your cell line of interest.

ZFAs with different Activation Domains

Figure 1. ZFAs with different Activation Domains.


As an optional upgrade, Sigma will provide vectors containing the KRAB repressor domain, the G9A lysine methyltransferase domain, and the p300 histone acetlytransferase domain. The latter two domains are used to create epigenetic modifications.


ZFA with Optional Repressor and Epigenetic Modifier Domains

Figure 2. ZFA with Optional Repressor and Epigenetic Modifier Domains.

Features and Benefits

  • For Gene Expression and Gene Activation studies
  • CompoZr ZFAs will allow researchers to over-express endogenous genes with the advantage of having all splice variants in the appropriate ratios
  • Zinc finger proteins are custom designed to the promoter region within 1 kb of the transcriptional start site (TSS)
  • Custom design, assembly, and validation of ZFAs targeting your gene of interest (human and mouse only)
  • Also available as non-validated ZFAs for faster availability and lower cost
  • ZFA is a powerful, novel tool to modulate gene expression in human and mouse cells with low potential for off-targets

 

Product No. Description Pricing
CSTZFACT CompoZr Custom ZF Activators Plasmid Kit
Check local pricing
CSTZFACT + upgrade CompoZr Custom ZF Activators, Epigenetic Modifier and Repressor Plasmid Kit Email: ZFnops@sial.com
CSTZFACT Non-Validated Custom ZF Activators Plasmid Kit Email: ZFnops@sial.com
CZFA17184 CompoZr ZF Activator OCT4 Plasmid DNA Check local pricing
CZFA2136 CompoZr ZF Activator SOX2 Plasmid DNA Check local pricing
CZFA1504 CompoZr ZF Activator KLF4 Plasmid DNA Check local pricing
CZFA1041 CompoZr ZF Activator MYC Plasmid DNA Check local pricing

For questions and details on ordering, please send an email to ZFNOps@sial.com and one of our Technical Sales Specialist will contact you directly.

 

ZFA Plasmid Design and Cloning Options

ZF-TF molecules are delivered in plasmid format, in the vector backbone shown below (Figure 3).  Expression of ZF-p65 is driven by the CMV promoter, along with an upstream GFP-2A cDNA, allowing for visual selection of cells that contain your plasmid of interest. This in turn allows for more accurate expression data analysis for both in vivo and in vitro experiments.

Map of the ZF-TF cloning vector (with p65 effector domain)

Figure 3. Map of the ZF-TF cloning vector (with p65 effector domain).

General Description

Zinc finger nucleases (ZFNs) are a class of engineered DNA-binding proteins that facilitate targeted gene editing.  Now, Sigma-Aldrich provides ZF activators, repressors and epigenetic modifiers for gene expression studies. ZFAs, or similarly called ZF-TFs (zinc finger transcription factors) incorporate a transcriptional activation domain in place of the nuclease domain found in ZFNs and specifically activate coding and non-coding genes. The increase in specific gene expression is dependent on the target binding site and the effector domain selected. Native gene activation is through ZFAs binding to promoters and recruiting the transcriptional machinery. This approach for activation also has the benefit of expressing endogenous isoforms at the proper ratio.                 

*Please note all ZFA plasmids are made standard with the p65 activation domain. The additional activation, epigenetic or repressor domains provided with the kit can be subcloned by the end-user as needed.

Available Activation, Repression and Epigenetic Domains

Activation Domains:

  1. Transcription Factor p65, or the RelA NF-kappaB (nuclear factor kappaB) subunit,
  2. A 2xp65 consists of two repeating units of the p65 activation domain,
  3. VP16 is a trans-acting domain from the herpes simplex virus that induces early gene transcription,
  4. VP64 is a transactivating domain made up of a tetrameric repeat of VP16 minimal activation domain.

Repressor and Epigenetic modifiers

  1. Krab domain or the Krüppel-associated box, which acts as a transcriptional repressor.
  2. p300 is a histone acetyltransferase domain,
  3. G9A is a histone methyltransferase domain.

Optimizing ZF-TF Activity via Effector Domain Selection

The influence of each effector domain supplied on transcriptional activation, repression, or modification will vary by cell type and gene target. The end user can optimize targeted gene activation by fusing different effector domains to the zinc-finger domain in place of the NF-ĸB p65 activation subunit. In order to demonstrate the varied efficacy of each domain in the context of individual target gene activation, ZF-TFs targeting OCT4, SOX2, KLF4, and c-MYC were tested with p65, VP16, VP64 or 2Xp65 (two tandem copies of the p65 domain) activation domains (Figure 4).

Use of various effector domains with ZFPs in HEK293 cells to upregulate mRNA expression

Figure 4. Use of various effector domains with ZFPs in HEK293 cells to upregulate mRNA expression.


 References

  • Qi, J., Fischer J., Brown, C., Eastlund, E., Dvash, T., Zhong, B., Gerber, M., Lyons, I., Knight, S. and Kreader, C. (2014)  Engineered zinc-finger transcription factors activate OCT4 (POU5F1), SOX2, KLF4, c-MYC (MYC) and miR302/367. Nucleic Acids Res. (Epub May 3 2014). Abstract