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6 targeted ES cells were transfected with 20 μg of supercoiled pMC-CreN plasmid (a gift of H. Gu) that was optimized for efficient nuclear localization. After 10 days, resistant clones were screened by PCR. Cre-mediated recombination led to a small (Fig. 1D) and a large (Fig. 1E) deletion. The latter event was preferred with a ratio of 9:1 to the small deletion encompassing only the selection cassette. Correct recombination was confirmed by Southern blot analysis (Fig. 1, H and I). To confirm correct mutation, the mutant and wild-type alleles were recloned by PCR and sequenced. Mutated clones of both types were injected into blastocysts, and germline transmission was obtained for both. The traits were bred to homozygosity, yielding the KVKΔtail and ΔM1M2 lines.
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Animal experiments were conducted in accordance with guidelines provided by the German law on experimentation with live animals. We thank K. Rajewsky and M. Reth for the exchange of data before publication, C. Westphal and S. Meier for blastocyst injection, H. Mittelstädt for technical assistance, P. Yu for the ε germline locus DNA, and R. Carsetti for reading the manuscript and stimulating discussions. G.A. was supported by a stipend of the Erwin-Schrödinger-Foundation.
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