The long term goal of Project II is to identify genes on mouse Chromosome 16 (Chr16) and human chromosome 21 (HC21) and to determine their contributions to maldevelopment when present at dosage imbalance. Functional analysis of large segments of these chromosomes will be effected by creating defined dosage imbalance in mice, using techniques developed through this program to isolate and modify yeast artificial chromosomes (YACs) and transfer them into murine embryonic stem (ES) cells. Modified ES cells will be analysed for integrity of the YAC- cloned segments. Appropriate lines will be used to make chimeras and thereby transmit YACs into the germ lines of mice, creating "YAC transgenic" animals with defined segmental aneuploidy. This approach will be used to refine mouse models to trisomy by creating dosage imbalance for genes on distal HC21 that are not represented in Chr16. The effects on development of genes in these segments will be assessed directly and after introduction into partial trisomy 16 mice (Project III). YAC transgenic mice will also be used to evaluate the effects of dosage imbalance for the conserved region of Chr16 and HC21 around the D21S55 locus. This region is associated with many characteristic of DS in "translocation DS" individuals who exhibit triplication of a subset of HC21 genes. This region is also likely to contain the homolog of the cerebellum observed in DS. Further YAC vector development will be pursued to establish a highly efficient method for identifying genes and mapping exons based on homologous recombination with specially constructed PCR-amplified cDNA libraries; to examine the application of YACs in homologous recombination-based approaches to gene deletion and the use of this approach to creation of segmental monosomy; and to characterize YAC integration sites in mammalian cells.