Monochromosomal hybrids for the analysis of the human genome. Final technical report

Abstract

The objective of this research project is to produce panels of mouse/human and/or Chinese hamster/human hybrid cell lines each harboring a single different human chromosome. The human chromosome present in rodent cell will be marked with a dominant selectable marker and maintained by selection. In these experiments human chromosomes first ``tagged`` with a selectable marker in human cells are subsequently transferred to rodent cells by microcell fusion method. Several different experimental schemes have been developed to ``tag`` human chromosomes with a selectable marker. Amphotropic retroviral vectors provide a highly efficient system to introduce selectable markers into normal diploid human cells. The integration of retroviral vector into the cell genome occurs at random by recombination at a defined nucleotide sequence in the LTRs and only a single copy of the vector integrates in a cell. This property of retroviral vectors allows to isolate a segment of the chromosomal DNA flanking the vector integration site by PCR amplification. In these studies the amphotropic retroviral vector pZIPgpt that carries a dominant selectable marker gpt, is used to tag the human chromosomes in normal diploid cells. Human DNA flanking the integrated vector is rescued by PCR amplification and cloned into a plasmid vector. Cloned human DNA is then used to probe Southern blots of DNAs from a panel of hybrid cell lines to identify the chromosome of its origin. This allows them to identify clonal human cell lines, each carrying the marker integrated into a different chromosome. Marked chromosomes are then transferred to rodent cells by MMCT.