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A targeting vector containing a self-excising loxP-flanked neomycin/sperm-specific Cre cassette was used to target a loxP site and a I-SceI endonuclease site upstream of the S-mu switching region and downstream of the E-mu enhancer. The construct was electroporated into albino C57BL/6 embryonic stem (ES) cells. Correctly targeted ES clones were injected into recipient blastocysts. The resulting male founder had self-excision of the neomycin/Cre cassette, leaving the loxP and I-SceI sites in the germline. Correct targeting was confirmed by Southern blotting. B cell development and numbers were normal in heterozygote mice. (J:145691)