PLP Splicing in Vivo Analysis in a Mouse Model

  • Cambi, Franca (PI)

Grants and Contracts Details


Research plans at the U. of Kentucky. We plan to isolatethe "run" cloneof ES cells in which the vector sequences are removed by intracbromosomal recombination while the targeted deletion is retained. To isolate identify the run clones, we will grow the 5 recombinant ES cells in medium containing FIAU, which selects against TIc containing clones. Therefore, cells that contain vector sequences with the Tk promoter will not grow in this medium and will be selected out whereas clones that will grow in these conditions should not contain vector sequences. We will carry out experiments in duplicate plates, in one plate the medium will contain G418 and the second plate the medium will contain FIAU. Clones that do grow in FIAU (TIc-), but do not grow in G418 (Neo-) will be analyzed by PCRanalysis to verify the presence of the 19-bp deletion. Clones that still contain the 19-bp deletion will be further characterized by Southern blot with probes internal and external the targeted gene to verify the integrity of the targeted region. Clones in which internal rearrengements have occurred will be discarded. The clone that contains the targeted gene and retains the deletion of 19-bp intronic ebhancer will be sequenced and will be used to inject into the oocytes in order to generate the chimeras.
Effective start/end date6/1/0412/31/04


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