Construction and characterization of a highly stable human:rodent monochromosomal hybrid panel for genetic complementation and genome mapping studies

Abstract

Humamrodent somatic cell hybrids carrying a single, intact, selectable human chromosome are valuable both for functional somatic cell genetic analysis and genome mapping procedures. Here, we describe the construction and detailed molecular cytogenetic characterization of a panel of 23 stable hybrids, representing all 22 human autosomes plus the X-chromosome. Individual normal human chromosomes have been tagged with a selectable fusion gene (Hytk) introduced into the chromosome in a small (4.2 kbp) retro viral vector. Use of the Hytk marker permits both positive and negative (“in-out”) selection to be applied to the human chromosome in any mammalian cell background. The panel includes 18 new hybrids isolated by direct microcell transfer from normal human diploid fibroblasts into mouse A9 cells.

Functional evidence for a squamous cell carcinoma mortality gene(s) on human chromosome 4

“…Human fibroblasts taken from the same patients and used as a source of normal DNA (Edington et al, 1995;Loughran et al, 1997) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) plus 20% foetal bovine serum (FBS). Mouse A92 cells containing a single copy of human chromosome 4 carrying the selectable markers for hygromycin B and G418 resistance (HYTK4; (Cuthbert et al, 1995;Lupton et al, 1991)) were grown in DMEM plus 10% FBS and 800 units/ml hygromycin B (Calbiochem Novachem (UK) Ltd.). The osteosarcoma line, 143B, was used as a control recipient owing to its inability to senesce following the introduction of a normal human chromosome 4 (Ning et al, 1991).…”

“…Microcells were prepared as described previously (Cuthbert et al, 1995) and the purified microcell pellet was then resuspended in 50 mg/ml phytoheamagglutinin in serum-free medium and applied to 70 -80% confluent recipient cells.…”

“…Higher doses of radiation caused complete fragmentation of the chromosomes. The microcells were then transferred into new A92 background cells as described previously (Cuthbert et al, 1995). The A92-chromosome hybrid cells were selected on hygromycin (800 U/ml) and then checked for dual resistance to G418 (1 mg/ml).…”

“…Metaphase chromosome spreads were prepared essentially as described by Cuthbert et al (1995). Metaphase spreads were hybridized with a chromosome 4 probe prepared from flowsorted chromosome 4 DNA and chromosome painting carried out as described previously (Cottage et al, 2001).…”

Analysis of the CAVEOLIN-1 gene at human chromosome 7q31.1 in primary tumours and tumour-derived cell lines

“…PCR assays indicated at the top of the panel were performed on DNA from the monochromosome hybrid cell line A92/Hytk7, which contains a single cytogenetically intact copy of human chromosome 7 (Cuthbert et al, 1995), YAC clone DNA, and PAC clone 162-O21 DNA. (b) A physical map of the D7S486-D7S2742 genomic interval.…”