Grants and Contracts Details
Description
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.
Status | Finished |
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Effective start/end date | 6/1/04 → 12/31/04 |
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