Isolation of microcell hybrid clones containing retroviral vector insertions into specific human chromosomes.

Abstract

We sought an efficient means to introduce specific human chromosomes into stable interspecific hybrid cells for applications in gene mapping and studies of gene regulation. A defective amphotropic retrovirus was used to insert the gene conferring G418 resistance (neo), a dominant selectable marker, into the chromosomes of diploid human fibroblasts, and the marked chromosomes were transferred to mouse recipient cells by microcelH fusion. We recovered five microcell hybrid clones containing one or two intact human chromosomes which were identified by karyotype and marker analysis. Integration of the neo gene into a specific human chromosome in four hybrid clones was confirmed by segregation analysis or by in situ hybridization. We recovered four different human chromosomes into which the G418 resistance gene had integrated: human chromosomes 11, 14, 20, and 21. The high efficiency of retroviral vector transformation makes it possible to insert selectable markers into any mammalian chromosomes of interest.

Suppression of tumorigenicity in human ovarian cancer cell lines is controlled by a 2 cM fragment in chromosomal region 6q24-q25

“…Mouse/ human hybrid MCH262A1.D6 cells containing a human chromosome 6 were obtained from Dr Eric Stanbridge (University of California at Irvine). Hdm-18, containing an intact human chromosome 11, was obtained from Dr Keith Fournier from the Fred Hutchinson Cancer Center (Lugo et al, 1987). The human chromosome from each of these cell lines was transferred into L-M (TK 7 ) mouse cells (ATCC #CCL 1.3) before fusion with recipient human cells.…”

“…Microcell-mediated chromosome transfers of chromosomes 6 or 11q into human ovarian carcinoma cell lines were performed according to published procedures (Lugo et al, 1987) with only slight modi®cations. Brie¯y, donor cells were treated with 0.15 mg/ml colcemid (Sigma, St. Louis, MO, USA, cat #D-7385) for 48 h. The resulting micronuclei were enucleated by treatment with 2 mg/ml cytochalasin B (Sigma, cat #C6762) followed by centrifugation.…”

Delayed replication timing leads to delayed mitotic chromosome condensation and chromosomal instability of chromosome translocations

“…Donor cells were micronucleated by adding 10.0 g of colcemid per ml in DMEM plus 15% calf serum for 48 h. The micronucleate cell populations were enucleated by centrifugation in the presence of 5 g of cytochalasin B (Sigma) per ml, and the isolated microcells were fused to C2C12 recipients as described (3,4). Microcell hybrids were isolated by using cloning cylinders after 3-4 weeks of selection in medium containing 500 g of Geneticin (GIBCO) per ml.…”

A High-Resolution Physical Map of Human Chromosome 21p Using Yeast Artificial Chromosomes

“…Somatic cell hybrids WAV17 , Hdm-15 (Lugo et al 1987), and AHVI-17 (Cox and Epstein 1985) were used to select for chromosome 21 phage clones. Two clones, F14 and F32, were characterized in detail, and low-copy fragments were subcloned in plasmid pUC18.…”

“…) were detected using PCR on human genomic DNA of CEPH families 1333, 1334, 1347, or 7 unrelated individuals, and a set of somatic cell hybrids containing acrocentric chromosomes. GB3 contains chromosome 13 (Scheffer et al 1986), HDm-5 (Lugo et al 1987) and WegrothB3 (Geurts van Kessel et al 1983) chromosome 14, HorlI chromosome 15 (Heisterkamp et al 1982), and Wegroth D2 chromosome 22 (Geurts van Kessel et al 1983). The PCR reaction was carried out in a total volume of 10 µl containing ∼100 ng template DNA, 0.1 unit of Taq DNA polymerase, 5 pmoles of each primer, one of which was ␥-32 P end-labeled, 100 µM dNTP, and 1.0 mM MgCl 2 .…”