Identification and expression of cosmids with an allelic variant of class I alcohol dehydrogenase in transgenic mice

The mouse Adh1 gene exhibits tissue-specific regulation, is de developmenta lly regulated, and is androgen regulated in kidney and adrenal tissue. To s tudy this complex regulation phenotype a transgenic mouse approach has been used to investigate regulatory regions of the gene necessary for proper ti ssue expression and hormonal control. Transgenic mice have been produced wi th an Adh1 minigene as a reporter behind either 2.5- or 10 kb of 5'-flankin g sequence [1]. Complete androgen regulation in kidney requires a region be tween - 2.5 and - 10 kb. A sequence extending to - 10 kb does not confer li ver expression in this minigene construct. B6.S mice express an electrophor etically Variant protein resulting from a known nucleotide substitution res ulting in a restriction endonuclease length polymorphism. Transgenic mice h arboring B6.S cosmids can be studied for expression analysis at both protei n and mRNA levels, identification of transgenic founders and inheritance st udies are greatly facilitated by a PCR-restriction endonuclease cleavage ap proach, the entire mouse gene is used as a reporter, and the formation of h eterodimeric enzyme molecules can be used to infer expression of the transg ene in the proper cell types within a given tissue. Expression of a B6.S co smid containing the entire Adh1 gene and 6 kb of 5'- and 21 kb of 3'-flanki ng region occurs in transgenic mice in a copy number dependent manner in a number of tissues, but expression in liver does not occur. The ability to a nalyze expression at the protein and mRNA levels has been confirmed using t his system. Future directions will involve the use of large BAC clones modi fied by RARE cleavage to identify the liver specific elements necessary for expression. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.