CORE--CYTOGENETICS

Johns Hopkins University, Baltimore MD

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Abstract

The Cytogenetics Core will interact with the subprojects and the Animal Core, providing banded chromosome analysis and fluorescence in situ hybridization (FISH). The Core will provide five essential services. DETECTION OF CHIMERISM in YACs: FISH happing will be carried out on human and mouse metaphase chromosomes and information on the number and location of hybridization sites will be provided. Chromosome bands will be identified using reverse DAPI images captured at the time of the FISH mapping. The sensitivity of this test varies with the size of the chimeric portion with the YAC. In general, chimeric segments representing 20-50kb can be detected. Mapping of YAC, PAC and PLASMID INTEGRATION SITES IN ES LINES: FISH methods will be used and chromosomal integrates sites will be identified using reverse DAPI images. Molecular FISH, described below, will be used to determine co-integration of different constructs into one site. EVALUATION OF ES LINES; Prior to injection into blastocysts, ES lines will be evaluated for chromosome number and single integration site. Metaphase preparations will be made from mitotic cultures and the model chromosome number will be determined. CHROMOSOME ANALYSIS: Human karyotyping will be available (Projects V) using a giemsa-trypsin banding method (G-banding). Blood samples from the T(16;17)65Dn mouse and controls will be routinely checked for the presence or absence of the small accessory chromosome, Ts65Dn, in order to monitor the Hopkins mouse colony maintained by the Animal Core. With both G-banding and Fish techniques, available, newly engineered EG and ES lines will be evaluated for chromosomal rearrangements (duplications/deficiencies) and breakpoint locations (projects I and II). MAPPING by MULTICOLOR- FISH to METAPHASE CHROMOSOMES and STRETCHED CHROMATIN (fiber-FISH): Simultaneous hybridization of up to three probes, each labeled with a different fluorochrome, can be detected on mouse or human chromosomes. This facilitates rapid screening and ordering of probes on a chromosome and will be especially helpful in the analysis of chromosomal rearrangements (Projects I and II).