Lat^{tm8.1(CD2,-ECFP)Mal}

A construct consisting of IRES-human CD2 cassette, an inverted promoterless ECFP expression cassette, and a floxed neomycin-resistance cassette was inserted into the 3' UTR in exon 12 of the gene locus. An inverted loxP site was also inserted into intron 1. Correctly targeted ES cells were transiently transfected with cre-recombinase. This removed the neomycin-resistance cassette, disrupted the gene by inverting exons 2-11, placed the ECFP cassette under the control of the endogenous promoter, and left inverted loxP sites surrounding the modifications. No endogenous protein product was detected by immunoblot analysis of homozygote thymocytes. ECFP fluorescence was detected in Thy-1+ lymphocytes of both heterozygote and homozygote mice. Human CD2 expression was not detected due to its inverted nature relative to the locus. In the presence of cre-recombinase, exons 2-11 will re-orientate into the correct position while the ECFP cassette will flip into the non-transcriptional orientation. The human CD2 cassette will also move into the correct orientation and can serve as a marker for the reconfigured allele. (J:152149)