Our long term goal is the development and utilization of hybrid cell lines containing regions of human chromosome 9 marked with a retroviral vector for physical mapping. These hybrids can be used to produce a low resolution map of the chromosome, which ca be expanded into a detailed physical map. A newly engineered retroviral vector has been used which contains features designed for gene mapping, such as a new dominant select system, sites for rarely cutting restriction enzymes,and a tRNA suppressor gene for easy cloning of the insertion site. As an initial step, a monochromosomal microcell hybrid containing a marked human chromosome 9 was isolated. The insertion site of the retroviral vector in human chromosome 9 will be identified and isolated. Radiation hybrids will be produced which contain the region of chromosome 9 surrounding the inserted marker. These hybrids should have a stable fragment of the chromosome due to the presence of the selectable marker. Additional retroviral vectors will be constructed with different selectable markers to obtain a chromosome 9 marked in several regions with selectable markers. The location of each marker will be identified and the region of interest isolated in a rodent-human hybrid by irradiation and fusion. Overlapping radiation hybrids which contain only a small fragment of the chromosome will be derived from the same original chromosome 9. These hybrids will be characterized to delineate the region of the chromosome they contain and analyzed for chromosome 9 markers. A long range physical map of regions of chromosome 9 will be derived using pulsed field gel analysis of the radiation hybrids. There are several genetic diseases localized to human chromosome 9. Cell lines containing these regions of the chromosome in a rodent background will be a valuable source of polymorphic DNA markers. In addition, the production and analysis of hybrids from human chromosome 9 should illustrate the general utility of this approach for other chromosomes as well.
