The Targeted Genome Editing Service was created to provide the research community of Wisconsin with tools to edit the genomes of animal and plant models and cells. The short term goals are:
to design and construct Transcription Activator-Like Effector Nucleases (TALEN).
Services tailored for your projects
We strive to provide services that fit each lab’s individual needs. Users can choose different levels of services, from plasmids of engineered proteins to cells or cell lines harboring desired genomic changes. This service is fully functional with the transgenic animal facility to generate transgenic mouse and rat models.
Design of TALENs
After a user supplies the genomic loci need to be targeted, one exon will be chosen based on three criteria: shared by most splice variants, coding for critical domains for protein function, close to N terminal of the protein. Once the exon is selected, online TALEN targets selection software will be used to identify potential TALEN sites, and further selection among these targets will be based on specificity and length. The specificity of the TALEN targets will be assessed in silicon by two procedures: 1. Identify the potential off-targets by the online TALEN targets selection software. 2. BLAST all combinations of left and right TALEN target sites with 12-21 spacer in between against the genome. The majority of the genomic loci will yield at least one suitable target site by following this procedure. If not, more exons will be chosen and the design procedure will be repeated until a suitable site is identified.
Construction of TALENs
TALENs are constructed by ligating individual RVD repeat sequences together using a modified Golden Gate Cloning Protocol. Comparing to the commonly used Golden Gate Cloning strategy for TALENs, the modification includes: Truncated and changed N-term and C-term sequences of the TALENs; Cloning strategy suitable for making individual or a library of TALENs; 5bp instead of 4bp sticky ends on the individual TALEN building blocks/repeats for better ligation efficiency.