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 developmentally regulated, and is androgen regulated in kidney and adrenal tissue. To study this complex regulation phenotype a transgenic mouse approach has been used to investigate regulatory regions of the gene necessary for proper tissue expression and hormonal control. Transgenic mice have been produced with an Adh1 minigene as a reporter behind either 2.5- or 10 kb of 5'-flanking sequence [1]. Complete androgen regulation in kidney requires a region between -2.5 and -10 kb. A sequence extending to -10 kb does not confer liver expression in this minigene construct. B6.S mice express an electrophoretically variant protein resulting from a known nucleotide substitution resulting in a restriction endonuclease length polymorphism. Transgenic mice harboring B6.S cosmids can be studied for expression analysis at both protein and mRNA levels, identification of transgenic founders and inheritance studies are greatly facilitated by a PCR-restriction endonuclease cleavage approach, the entire mouse gene is used as a reporter, and the formation of heterodimeric enzyme molecules can be used to infer expression of the transgene in the proper cell types within a given tissue. Expression of a B6.S cosmid containing the entire Adh1 gene and 6 kb of 5'- and 21 kb of 3'-flanking 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 analyze expression at the protein and mRNA levels has been confirmed using this system. Future directions will involve the use of large BAC clones modified by RARE cleavage to identify the liver specific elements necessary for expression.
Szalai
G
G
Department of Biological Sciences, University of South Carolina, 29208, Columbia, SC, USA.
Ceci
J
J
Dewey
M
M
Felder
M
M
D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D000483:Alleles; D000818:Animals; D003360:Cosmids / Q000235:genetics*; D015870:Gene Expression; D005809:Genes, Regulator; D014644:Genetic Variation; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010641:Phenotype; D012150:Polymorphism, Restriction Fragment Length; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution; D014162:Transfection
D012333:RNA, Messenger; D000426:Alcohol Dehydrogenase