The goal of our project is the construction of rodent-human somatic cell hybrids useful for physical mapping of the human genome. These properties include stability of the human component, unique derivation from a single chromosome or known chromosomal region, and the presence of a selectable marker or markers on the human chromosome. We will continue to use a system which has proved efficient in our hands: the use of a retroviral vector (SP-1) to insert a dominant selectable marker (the bacterial gene hisD ) into human lymphoblastoid lines, followed by microcell transfer of single selectable chromosomes into rodent lines. The hybrids are characterized by cytogenetic and molecular methods to define the human chromosomal material and marker insertion sites. We will also use retroviral vectors with other dominant selectable markers such as neomycin and hygromycin resistance. Monochromosomal hybrids with doubly marked human chromosomes will also be developed using a newly designed homologous recombination marker targeted for the human Ll sequences. Other methods to be tried include multiple retroviral infection of the human cell lines and transfection with another selectable marker of an already retrovirally marked hybrid line. Since some preliminary data has suggested that the insertion sites of the defective Moloney leukemia retroviral vector may not be random, we will also use viral rescue of the insertion site and in situ hybridization to analyze the insertion sites on human chromosomes.
