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  • Title: Entrapment of metaphase chromosomes into phospholipid vesicles (lipochromosomes): carrier potential in gene transfer.
    Author: Mukherjee AB, Orloff S, Butler JD, Triche T, Lalley P, Schulman JD.
    Journal: Proc Natl Acad Sci U S A; 1978 Mar; 75(3):1361-5. PubMed ID: 349565.
    Abstract:
    Transfer of genes from one type of cultured mammalian cell to another by using isolated metaphase chromosomes has been reported with a frequency of one per 10(6)-10(8) cells. Very recently a rate of 16/10(6) has been reported with Chinese hamster ovary cells [Spandidos, D. A. & Siminovitch, L. (1977) Proc. Natl. Acad. Sci. USA 74, 3480-3484]. To increase the frequency of gene transfer, we isolated metaphase chromosomes from hypoxanthine guanine phosphoribosyltransferase (HGPRT) positive cells, entrapped them in liposomes, and fused the lipochromosomes with HGPRT-negative cells. Lipochromosomes were prepared with cholesterol and egg lecithin, using isolated metaphase chromosomes from a mouse-human somatic hybrid cell line (A9/HRBC2); the entire X chromosome, including the HGPRT, glucose-6-phosphate dehydrogenase, and phosphoglycerate kinase genes, is the only recognizable human genetic material retained by the hybrids. Enclosure of the chromosomes in the lipid envelope was confirmed by electron and fluorescence microscopy and differential centrifugation. These lipochromosomes were fused with HGPRT(-) mouse cells (A9) in the presence or absence of polyethylene glycol and transferents were selected in hypoxanthine/aminopterin/thymidine (HAT) medium. The frequency of transfer was at least once per 10(5) cells, a minimum 10-fold improvement over previous methods. The selected cells contained HGPRT activity similar to the amount found in the A9/HRBC2 cells. Starch gel electrophoresis verified that the observed HGPRT activity in the transferents is due to the human enzyme. Human glucose-6-phosphate dehydrogenase and phosphoglycerate kinase were also identified electrophoretically in the transferents. Karyotyping with C and Q banding did not reveal the presence of the whole human X chromosome or a visible extra fragment of a human chromosome associated with the mouse genome. The biochemical data strongly suggest, however, that transfer of a portion of the human X chromosome has occurred in these transferents. Thus, at least three X-linked genes have been transferred from one cell to another with high frequency, using metaphase chromosomes.
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