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

Technical Report · Thu Sep 01 04:00:00 EDT 1994

DOI:https://doi.org/10.2172/10179860· OSTI ID:10179860

Athwal, R S ^[1]

---

1. Temple Univ. School of Medicine, Philadelphia, PA (United States). Fels Inst. for Cancer Research and Molecular Biology

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.

• Research Organization: University of Medicine and Dentistry of New Jersey, Newark, NJ (United States)
• Sponsoring Organization: USDOE, Washington, DC (United States)
• DOE Contract Number: FG02-89ER60866
• OSTI ID: 10179860
• Report Number(s): DOE/ER/60866--4; ON: DE94017917; BR: KP0404000
• Country of Publication: United States
• Language: English

Related Subjects

550400
59 BASIC BIOLOGICAL SCIENCES
BIOLOGICAL MARKERS
DISEASE VECTORS
DNA-CLONING
GENE AMPLIFICATION
GENETIC MAPPING
GENETICS
HUMAN CHROMOSOMES
PROGRESS REPORT