Gene Targeting Vector Construction

PTL  generates straight knockout (KO), conditional knockout (CKO), knock-in (KI), and point-mutation (PM)  targeting vectors. All PTL gene targeting vectors carry a Neo positive selection cassette and a DTA (Diphtheria Toxin Alpha chain) negative selection cassette. 10 ug linearized targeting vector should be used for each round of electroporation. 250 ug/ml effective G418 should be used during drug selection. DTA cassette is an autonomous negative selection cassette which doesn’t require additional drug for negative selection to work during gene targeting in ES cells .

The timeline for gene targeting vector construction:

  • Knockout (KO) Vector
         4 weeks after the design is  finalized.
  • Conditional Knockout (CKO) Vector
         4 weeks after the design is  finalized.
  • Knock-in (KI) Vector
         6 weeks after the design is  finalized.
  • Point Mutation (PM) Vector
         6 weeks after the design is  finalized.

PTL provides electronic sequence of the gene targeting vector. Clients may design a Southern screen or a PCR screen based on the sequence.