We have developed a bacterial F-factor based system for cloning large fragments of mammalian DNA in E coli (the BAC system). The primary aims of this proposal are to construct a genomic library of the mouse in the BAC vector and to use these clones for physical mapping of the mouse genome. Previous work with human genome BAC vectors indicates that the large human inserts are not chimeric, they are stable, representative, and are relatively easy to manipulate. The mouse library will consist of arrayed bacterial clones with average insert sizes of approximately 175kb, and will represent 4-5 times coverage of the mouse genome. The clones will be rendered onto high density filters for colony hybridization, and will be pooled to permit library screening by PCR. We will use a number of different approaches to identify clones corresponding to established physical and genetic landmarks. Initial mapping experiments will focus on regions of the mouse chromosome that are replete with markers. We will use the library to isolate clones originating from the proximal region of mouse chromosome 17, and establish """"""""contigs"""""""", or groups of overlapping clones. We will also focus on other regions that have high densities of both genetically and physically mapped markers. These experiments will provide us with the opportunity to quickly verify the extent to which the library is representative and to demonstrate generalizable procedures for using BACs to generate detailed maps of any region in the genome. In addition, we will isolate BAC clones which contain the STSs developed by Eric Lander's group and by other groups which will provide virtually complete coverage of the mouse genome in BACs. We will also apply the techniques of fluorescent in situ hybridization to analysis of the mouse genome. We will carry out BAC hybridizations to metaphase chromosomes to map BACs and confirm mapping data established by other approaches.
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