Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases.

Cirullo, R E; Dana, Sharon L.; Wasmuth, John J.

doi:10.1128/mcb.3.5.892

Cited by 40 publications

(9 citation statements)

References 37 publications

(62 reference statements)

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“…The hybrid can be used as the donor for transfer of isolated metaphase chromosomes (22,32), or it can be gamma-irradiated to introduce chromosome breaks and then rescued by fusion with an unirradiated partner (5). Both techniques result in the transfer of acentric chromosome fragments that are retained by virtue of the selectable marker.…”

Section: Discussionmentioning

confidence: 99%

“…Such hybrid cells have been used to determine the chromosomal locations of many isozyme loci (26) and, more recently, DNA restriction fragments (33). Used in conjunction with probes for species-specific repetitive DNA, hybrid cells are advantageous starting materials for the cloning of DNA sequences known to reside on particular chromosomes (5). Hybrid cells can also be used to study the effects of trans-acting factors on the expression of tissuespecific genes (20).…”

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confidence: 99%

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

Lugo¹,

Handelin²,

Killary³

et al. 1987

Mol. Cell. Biol.

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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.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Lugo¹,

Handelin²,

Killary³

et al. 1987

Mol. Cell. Biol.

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“…The cell fusion procedure was similar to that described by Cirullo et al (1) and was adopted because it * Corresponding author. predisposes hybrids to lose hamster chromosomes rather than human chromosomes.…”

Section: Methodsmentioning

confidence: 99%

Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells.

Karentz¹,

Cleaver²

1986

Mol. Cell. Biol.

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Xeroderma pigmentosum (XP) is an inherited human disease caused by a malfunction in the dimer excision step of DNA repair. The disease is manifested as an extreme sensitivity to UV light resulting in the development of skin cancers and, in some cases, neurological disorders. At least nine complementation groups have been identified, indicating that the disease comprises a variety of mutations at several genetic loci (2, 5). The genetic basis of the physiological defect of XP has not yet been characterized. Neither genes nor gene products have been identified, and it is not known whether the deficiency is due to mutations in structural or regulatory aspects of DNA repair.Introduction of isolated DNA into XP cells by standard DNA transfection methods has thus far been unsuccessful in introducing genes that code for UV resistance, presenting a major obstacle to the cloning of genetic information that can complement the deficiency of XP (12,18). In contrast, the introduction of human DNA into repair-deficient hamster cells has resulted in the identification of DNA repair loci not represented in the XP complementation groups (4,16,21,22). We used a method of fusing X-irradiated Chinese hamster ovary (CHO) cells with XP cells so that fragments of CHO chromosomes, which appear to support DNA repair, were transferred into the human cells. This report presents the initial work of establishing stable UV-resistant, repaircompetent hybrid cell lines, which will then be used to clone hamster DNA sequences that correct the repair defect of XP cells. MATERIALS AND METHODSCell lines. Normal human fibroblasts (1508), simian virus 40-transformed normal human fibroblasts (A13-6F and GM637), simian virus 40-transformed XP group A fibroblasts (XP12RO), and Chinese hamster ovary (CHO) cells (AA8) were grown in Eagle minimal essential medium with 10% fetal calf serum.Cell fusion. The cell fusion procedure was similar to that described by Cirullo et al. (1) and was adopted because it * Corresponding author. predisposes hybrids to lose hamster chromosomes rather than human chromosomes. CHO cells were exposed to a lethal dose of X rays (50 Gy, 10 Gy/min), mixed with approximately equal numbers of unirradiated simian virus 40-transformed XP fibroblasts, and dispersed into 100-mm dishes. After 5 h at 37°C, cell fusion was mediated by exposure for 30 s to 40% polyethylene glycol 1000 and 10% dimethyl sulfoxide in minimal essential medium, followed by three rinses with 10% dimethyl sulfoxide in minimal essential medium. Complete culture medium was added after the final rinse, and cells were incubated overnight. To select for hybrids that contained functional CHO DNA repair genes and to eliminate unfused XP cells, we irradiated cells with 13 J of UV light per m2 (254 nm, 1.3 J/m2 per s) once a day for 3 consecutive days. At this dosage, XP12RO cell survival is 5 x 10-3, as determined by colony formation. Cultures were incubated for 2 to 3 weeks until colonies developed, and colonies on each plate were pooled to establish primary hybrid cell l...

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“…The fragmented genome was recovered by fusion to the cell line UCW113, a hypoxanthine phosphoribosyl transferase (HPRT)-deficient derivative of V79 Chinese hamster lung fibroblasts. The irradiation and fusion methodologies have been described previously (Cirullo et al 1983). After selection in hypoxanthine/aminopterin/ thymidine (HAT) media, 400 RH clones were isolated and transferred to 24-well cluster plates.…”

Section: Isolation Of Rhsmentioning

confidence: 99%

A Radiation Hybrid Map of Human Chromosome 5 with Integration of Cytogenetic, Genetic, and Transcript Maps

Mcpherson¹,

Apostol

Wagner-McPherson

et al. 1997

Genome Res.

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One of the major goals of the human genome project is to establish a physical map of each human chromosome with a density of sequence-tagged site (STS) markers exceeding one every 100 kb. We report here the generation of a human chromosome 5-specific radiation hybrid (RH) map that includes 556 markers. Of these markers, 132 loci are ordered with a maximum likelihood ratio of >1000:1 compared with the next most likely order. An additional 113 loci were ordered relative to these backbone markers with a maximum likelihood ratio of >10:1 but <1000:1. Together, these 245 loci form an ordered framework map for the chromosome. Using this framework, >300 more markers were localized based on two-point analysis with the ordered set. On average, there are 50 markers in common with the RH map presented here and other chromosome 5 maps included in the current whole genome cytogenetic, genetic, and physical maps. The accuracy of all the maps is evident in that there are no more than two discrepancies between any one of them and these data. All of the maps encompassing chromosome 5 complement each other providing excellent STS coverage with >2200 loci combined. The chromosome 5-specific RH map contains 20% of these independent loci. In addition, our RH map contains STSs derived from clones suitable for fluorescent in situ hybridization, allowing alignment to the cytogenetic map. Together, these maps will assist in the assembly of sequence-ready contigs and will aid in the identification of disease loci on chromosome 5 by positional cloning and positional candidate approaches.[The STS sequences described in this paper have been submitted to dbSTS under accession nos. G15666–G15715 andG16049–G16063. A complete map of human chromosome 5 is available as an on-line supplement at http://www.cshl.org/gr.]

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Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases.

Cited by 40 publications

References 37 publications

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

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

Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells.

A Radiation Hybrid Map of Human Chromosome 5 with Integration of Cytogenetic, Genetic, and Transcript Maps

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