Difference between revisions of "Part:BBa K747100"

Revision as of 20:59, 15 June 2015

pTALEN

Precise Gene Knockout:

TALENs are a very powerful tool for efficient gene knockout. In order to prove that our TALEN construct was functional, we decided to simply knock out a destabilized GFP gene on a plasmid, which we co-transfected with our TALEN plasmids into HEK cells. Moreover, we also transfected our cells with an mCherry vector to normalize for transfection efficiency. TAL constructs were designed to bind to opposite strands of the target plasmid in a way that the FokI monomers of each TALEN construct would be able to dimerize in the spacer region between the TALEN binding sites. 48 hours after transfection, gene knock-out efficiency was evaluated by FACS analysis.

The FokI-nuclease was inserted into the Plug and Play Effecor Cassette of the Eukaryotic TAL expression vector via golden gate cloning.

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Sequence and Features