Production and analysis of transgenic mice containing yeast artificial chromosomes.

Abstract

Until the advent of transgenic mouse technology, studies of mammalian gene expression and regulation were largely limited to cell lines transfected with constructs containing limited genetic information. Developmental studies were not possible since cell lines are generally locked into one ontogenic stage. Transgenic mice provided two improvements and facilitated the study of gene function in development and disease. First, transgene expression in mice allowed assessment of phenotypes. Second, gene expression could be studied throughout development and tissue-specific regulatory elements could be analyzed. However, expression of transgenes was erratic due to position effects and copy number-independent expression. In many instances these problems were due to the nature of the transgene constructs, which typically were limited in their size due to constraints on how large a DNA fragment could be isolated without degradation and introduced into the mouse. Thus, deletion of cis sequences was necessary during the design of transgenes to be injected. Many large genes with exons spanning several hundred kilobases or multigenic loci could not be used as transgenes. cDNAs were substituted for large genes or individual genes from a multigene cluster were utilized in place of an entire locus. However, expression from these constructs was subject to the effects of surrounding chromatin into which they were integrated. Some improvement resulted when other cis-acting elements, such as enhancers, introns or polyadenylation signals were included. These additional sequences assisted studies in which the main goal was to express the transgene, but these truncated constructs lacked their natural regulatory elements and thus developmental studies were not necessarily indicative of how the native gene might be regulated. Ideally, a system in which large genes or loci could be successfully used in the generation of transgenic mice might improve the utility of transgenic studies. Inclusion of distal regulatory elements within the native locus might validate developmental studies and insulate the construct from position effects. With this in mind several lab groups successfully implemented the use of large DNA constructs, in particular yeast artificial chromosomes (YACs), as transgenes (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21).