Entrapment of metaphase chromosomes into phospholipid vesicles (lipochromosomes): Carrier potential in gene transfer

Abstract

Transfer of genes from 1 type of cultured mammalian cell to another by using isolated metaphase chromosomes has been reported with a frequency of 1/106-108 cells. Very recently a rate of 16/106 has been reported with Chinese hamster ovary cells [Spandidos, D.A. and Siminovitch]. To increase the frequency of gene transfer, metaphase chromosomes were isolated from hypoxanthine guanine phosphoribosyltransferase (HGPRT) positive cells, entrapped in liposomes, and the lipochromosomes fused 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 105 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 [constitutive heterochromatin] and Q [quinacrine] 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. Transfer of a portion of the human X chromosome has evidently occurred in these transferents. At least 3 X-linked genes have been transferred from 1 cell to another with high frequency, using metaphase chromosomes.