The Cytogenetics Core will interact with Projects II, III and IV providing tradition-banded chromosome analysis and fluorescence in situ hybridization (FISH). This Core will provide four essential services. DETECTION OF CHIMERISM IN YAC's: Yeast DNA will be labeled by nick translation with bio-tin-labeled nucleotides, denatured and preannealed with total yeast DNA. Hybridization will be carried out on denatured metaphase chromosome spreads overnight a appropriate temperature and formamide concentration. This will be followed by a post-hybridization wash and the probe will be detected wit avidin-fluorescein. Metaphase spreads will be photographed and evaluated from information on copy number will be provided to the primary investigator. The sensitivity of this test varies both with the size of the chimeric portion and the percent of the YAC that it represents. In general, chimeric segments of 50-100kb can be visualized from the YAC's 500-700kb, the average size to be used in this project. MAPPING OF YAC INTEGRATION SITES IN ES LINES: Probe DNA will be handled as above. Slides will be G-banded prior to hybridization and metaphase spreads will be photographed and coordinates recorded. Slides will be destained and used in the FISH procedure as described above. Identical spreads will be re-photographed and by comparing the bright field and fluorescent analysis, the integration site can be identified. Information will be provided to the investigator. For the yeast chromosome integration sites will also be assessed using. EVALUATION OF ES LINES PRIOR TO INTRODUCTION OF BLASTOCYTES: The modal chromosome number in ES lines will be determined from twenty unbanded metaphase spreads in order to determine ploidy. CHROMOSOME ANALYSIS OF G-BANDED METAPHASE CHROMOSOMES FROM MOUSE: Traditional banded chromosome analysis will involve preparing metaphase spreads from mitotic cultures and treatment of slides with heat and trypsin prior to standing with Giemsa. Fore each line twenty metaphase spreads will be examined, five spreads will be completely analyzed and two karyotypes will be prepared.
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