TALEs were constructed using the Golden Gate TALEN kit (Addgene, Cambridge MA, USA) following previous published protocols (9 (link)). All TALEs were initially cloned into pTAL2 vector (Addgene) before subsequent insertion into a VP64-fused vector for luciferase confirmation assay. TALEs were further cloned into a phosphoglycerate kinase plasmid (pPGK) vector with KRAB-fused, FokI DD, or RR effector domains (3 (link)) (Addgene) for gene silencing or CAG collapse, respectively. Each TALE was designed for a unique 18 base pair sequence only found in either the promoter region of the huntingtin gene or the CAG expansion. For transcriptional repression of the mutant allele the TALE was designed to have the HD-associated SNP occurring in the first four repeat variable diresidues of the TALE plasmid.
Protocol detail
TALE-Mediated Targeting of Huntingtin
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Allele
Assay
Base pair
Collapse
Gene
Huntingtin
Luciferase
Phosphoglycerate kinase
Plasmid
Repression
Talen
Transcriptional
Vector
Corresponding Organization : University of California, Davis
Top 5 similar protocols
Variable analysis
independent variables
- TALE design
- Effector domains (KRAB-fused, FokI DD, or RR)
- Gene silencing
- CAG collapse
- Unique 18 base pair sequence found only in the promoter region of the huntingtin gene or the CAG expansion
- HD-associated SNP occurring in the first four repeat variable diresidues of the TALE plasmid for transcriptional repression of the mutant allele
- Positive control: Luciferase confirmation assay
- Negative control: Not explicitly mentioned
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!