An frt flanked PGK-neomycin resistance cassette was inserted downstream of exon 5. In addition, exon 5 and the inserted neomycin gene were flanked by 2 loxP sites. Successfully targeted ES cells were transiently transfected with flp-recombinase to remove the neomycin gene. Neither the loxP sequences nor the remaining frt sequence had an adverse affect on gene expression as determined by Western blot analysis. (J:86626)