The long time goal of the project is to develop efficient systems for creating rodent-human somatic cell hybrids with properties designed for physical mapping of the human genome, using retroviral insertion of a dominant selectable marker into human chromosomes. These properties include stability of the human component, unique derivation from a single chromosome or portion thereof, the ability to clone the insertion site, and the presence of polylinkers containing rare cutting sites for linking macrorestriction fragments. As a first step a newly engineered defective retrovirus will be used which contains (1) the bacterial His-D gene, which allows mammalian cells to grow in the presence of histidinol, (2) an Su-lll gene in the viral LTR which will permit cloning of the insertion site, and (3) a polylinker containing Sfi 1 and Nru 1 cutting sites. Monochromosomal hybrids will be developed by microcelling of infected human lymphoblastoid lines and fusion with CHO cells. In particular a panel of hybrids containing a set of overlapping deletions of chromosome 13 will be developed by using a radiosensitive CHO line complemented by a gene on human chromosome 13. The apparently monochromosomal hybrids will be characterized by karyotypic, biochemical and molecular methods to define the human chromosome material and the retroviral insertion sites. The rare cutting sites in the polylinker inserted into the retroviral vector can be used to link macrorestriction fragments on pulse-field gels. In later phases of the project panels of human lymphoblastoid lines with known chromosomal insertion sites of the vector will be developed for use in making new hybrids. New vectors will also be used as they are developed, and by a combination of vectors and procedures which fragment chromosomes, hybrids with only small portions of a human genome will be produced.
