Vcam1^tm2Dmil

Sequential tag and exchange gene targeting was used to introduce a 6 nucleotide mutation in the 5NF-B binding site of the transcriptional promoter, and a 3 nucleotide mutation in the 5UTR of Vcam1 in exon 1. The first, "tag", targeting vector replaces exon 1 (encoding the signal peptide) and part of exon 2 (encoding immunoglobulin-like domain 1) with a pgk-neomycin/TK cassettes. The Tag construct is transfected into embryonic stems cells and a single correctly targeted clone is transfected with the second construct. The second "Exchange" targeting construct replaces the neo cassette and introduces a 6 nucleotide substitution 5' of the transcription start site resulting in the disruption of the 5'NFKB1 (NF-kappaB) binding site domain and a 3 nucleotide substitution downstream of the translation start site in the 5' UTR of exon 1. The 3 nt substitution is used to distinguish the mutant and wild-type mRNA and hnRNA. Western blot ananlysis demonstrates the presence of mutant protein in heart endothelial cells. (J:227109)