Removal of loxPFlanked Short Genomic Segment in ES Cells by Transient Expression of Cre Recombinase

1. Electroporate the correctly targeted ES cell line with 50 |Jg/mL of circular plas-mid containing the Cre recombinase gene driven by an ES cell transcriptionally active promoter, for example, the pBS185 plasmid, which contains Cre under the control of the hCMV promoter (35).

2. After electroporation, plate the cells very sparsely (approx 1000 cells/10-cm dish) onto gelatinized 10 cm plates each containing 10 mL of DMEM+.

3. Change media daily until the colonies have attained a size that is ready to pick (usually takes 7 d).

4. Pick colonies into 96-well plates, and expand into two or more plates. One plate is frozen, and the others are used for PCR or Southern screening to detect the required recombination event. A subset of the colonies will be mosaic for the Cre-mediated excision, therefore requiring further subcloning for the derivation of pure lines (see Notes 19 and 20).

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